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PSD-95 MAGUK scaffolding protein antibody

RRID:AB_2292909

Antibody ID

AB_2292909

Target Antigen

PSD95 human, mouse, rat

Proper Citation

(UC Davis/NIH NeuroMab Facility Cat# 75-028, RRID:AB_2292909)

Clonality

monoclonal antibody

Comments

Originating manufacturer of this product. The following antibodies were determined to be duplicates and consolidated by curator on 2/2019: AB_2307331, AB_2292909.

Clone ID

k28/43

Host Organism

mouse

Vendor

UC Davis/NIH NeuroMab Facility Go To Vendor

Cat Num

75-028

Publications that use this research resource

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

  • Vuong CK
  • Neuron
  • 2018 Apr 4

Literature context: H NeuroMab Facility Cat#75-028; RRID:AB_2292909 Guinea pig anti-Synaptophysin1


Abstract:

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Funding information:
  • NIDDK NIH HHS - DK094311(United States)
  • NIGMS NIH HHS - R01 GM114463()
  • NIGMS NIH HHS - T32 GM007185()
  • NIMH NIH HHS - R01 MH060919()
  • NIMH NIH HHS - R21 MH101684()
  • NINDS NIH HHS - F31 NS093923()

Directional selectivity of afferent neurons in zebrafish neuromasts is regulated by Emx2 in presynaptic hair cells.

  • Ji YR
  • Elife
  • 2018 Apr 19

Literature context: 86 (1:500; NeuroMab, Davis, CA, RRID:AB_2292909). To detect F-actin, we stained


Abstract:

The orientation of hair bundles on top of sensory hair cells (HCs) in neuromasts of the lateral line system allows fish to detect direction of water flow. Each neuromast shows hair bundles arranged in two opposing directions and each afferent neuron innervates only HCs of the same orientation. Previously, we showed that this opposition is established by expression of Emx2 in half of the HCs, where it mediates hair bundle reversal (Jiang et al., 2017). Here, we show that Emx2 also regulates neuronal selection: afferent neurons innervate either Emx2-positive or negative HCs. In emx2 knockout and gain-of-function neuromasts, all HCs are unidirectional and the innervation patterns and physiological responses of the afferent neurons are dependent on the presence or absence of Emx2. Our results indicate that Emx2 mediates the directional selectivity of neuromasts by two distinct processes: regulating hair bundle orientation in HCs and selecting afferent neuronal targets.

Funding information:
  • National Institute on Deafness and Other Communication Disorders - Intramural Research Program Grant 1ZIADC000021()
  • National Institute on Deafness and Other Communication Disorders - Intramural Research Program Grant 1ZIADC000085-01()
  • NIAID NIH HHS - R01 AI052310(United States)

Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca2+-Permeable AMPA Receptors.

  • Sanderson JL
  • J. Neurosci.
  • 2018 Mar 14

Literature context: ntibodies (NeuroMab, 75-028; RRID:AB_2292909; 1:1000) in 1% BSA followed by


Abstract:

Neuronal information processing requires multiple forms of synaptic plasticity mediated by NMDARs and AMPA-type glutamate receptors (AMPARs). These plasticity mechanisms include long-term potentiation (LTP) and long-term depression (LTD), which are Hebbian, homosynaptic mechanisms locally regulating synaptic strength of specific inputs, and homeostatic synaptic scaling, which is a heterosynaptic mechanism globally regulating synaptic strength across all inputs. In many cases, LTP and homeostatic scaling regulate AMPAR subunit composition to increase synaptic strength via incorporation of Ca2+-permeable receptors (CP-AMPAR) containing GluA1, but lacking GluA2, subunits. Previous work by our group and others demonstrated that anchoring of the kinase PKA and the phosphatase calcineurin (CaN) to A-kinase anchoring protein (AKAP) 150 play opposing roles in regulation of GluA1 Ser845 phosphorylation and CP-AMPAR synaptic incorporation during hippocampal LTP and LTD. Here, using both male and female knock-in mice that are deficient in PKA or CaN anchoring, we show that AKAP150-anchored PKA and CaN also play novel roles in controlling CP-AMPAR synaptic incorporation during homeostatic plasticity in hippocampal neurons. We found that genetic disruption of AKAP-PKA anchoring prevented increases in Ser845 phosphorylation and CP-AMPAR synaptic recruitment during rapid homeostatic synaptic scaling-up induced by combined blockade of action potential firing and NMDAR activity. In contrast, genetic disruption of AKAP-CaN anchoring resulted in basal increases in Ser845 phosphorylation and CP-AMPAR synaptic activity that blocked subsequent scaling-up by preventing additional CP-AMPAR recruitment. Thus, the balanced, opposing phospho-regulation provided by AKAP-anchored PKA and CaN is essential for control of both Hebbian and homeostatic plasticity mechanisms that require CP-AMPARs.SIGNIFICANCE STATEMENT Neuronal circuit function is shaped by multiple forms of activity-dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts strength of individual synapses and homeostatic plasticity that adjusts overall strength of all synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR regulation by the kinase PKA and phosphatase CaN coanchored to the scaffold protein AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic strength during homeostatic plasticity. These novel findings significantly expand our understanding of homeostatic plasticity mechanisms and further emphasize how intertwined they are with LTP and LTD.

Funding information:
  • NIAID NIH HHS - 2T32AI007414(United States)

Early structural and functional plasticity alterations in a susceptibility period of DYT1 dystonia mouse striatum.

  • Maltese M
  • Elife
  • 2018 Mar 5

Literature context: 28/43) - catalog number 75-028; RRID:AB_2292909 dilution 1:2000 in I-Block


Abstract:

The onset of abnormal movements in DYT1 dystonia is between childhood and adolescence, although it is unclear why clinical manifestations appear during this developmental period. Plasticity at corticostriatal synapses is critically involved in motor memory. In the Tor1a+/Δgag DYT1 dystonia mouse model, long-term potentiation (LTP) appeared prematurely in a critical developmental window in striatal spiny neurons (SPNs), while long-term depression (LTD) was never recorded. Analysis of dendritic spines showed an increase of both spine width and mature mushroom spines in Tor1a+/Δgag neurons, paralleled by an enhanced AMPA receptor (AMPAR) accumulation. BDNF regulates AMPAR expression during development. Accordingly, both proBDNF and BDNF levels were significantly higher in Tor1a+/Δgag mice. Consistently, antagonism of BDNF rescued synaptic plasticity deficits and AMPA currents. Our findings demonstrate that early loss of functional and structural synaptic homeostasis represents a unique endophenotypic trait during striatal maturation, promoting the appearance of clinical manifestations in mutation carriers.

Funding information:
  • Dystonia Medical Research Foundation - 2017()
  • Ministero dell'Istruzione, dell'Università e della Ricerca - PRIN 2010-2011()
  • NHLBI NIH HHS - R01 HL084498-01A2(United States)

Alterations in mGlu5 receptor expression and function in the striatum in a rat depression model.

  • Mao LM
  • J. Neurochem.
  • 2018 Jan 18

Literature context: ic density protein 95 (PSD-95) (RRID:AB_2292909; UC Davis/NIH NeuroMab Facility


Abstract:

Major depressive disorder is a common form of mental illness. Many brain regions are implicated in the pathophysiology and symptomatology of depression. Among key brain areas is the striatum that controls reward and mood and is involved in the development of core depression-like behavior in animal models of depression. While molecular mechanisms in this region underlying depression-related behavior are poorly understood, the glutamatergic input to the striatum is believed to play a role. In this study, we investigated changes in metabotropic glutamate (mGlu) receptor expression and signaling in the striatum of adult rats in response to prolonged (10-12 weeks) social isolation, a pre-validated animal paradigm modeling depression in adulthood. We found that mGlu5 receptor protein levels in the striatum were increased in rats that showed typical depression- and anxiety-like behavior after chronic social isolation. This increase in mGlu5 receptor expression was seen in both subdivisions of the striatum, the nucleus accumbens and caudate putamen. At subcellular and subsynaptic levels, mGlu5 receptor expression was elevated in surface membranes at synaptic sites. In striatal neurons, the mGlu5-associated phosphoinositide signaling pathway was augmented in its efficacy after prolonged social isolation. These data indicate that the mGlu5 receptor is a sensitive substrate of depression. Adulthood social isolation leads to the up-regulation of mGlu5 receptor expression and function in striatal neurons.

Funding information:
  • NIDA NIH HHS - R01 DA010355()
  • NIGMS NIH HHS - GM57089(United States)
  • NIMH NIH HHS - R01 MH061469()

Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins.

  • Smith KR
  • J. Neurosci.
  • 2017 Nov 15

Literature context: : mouse anti-PSD-95 monoclonal (RRID:AB_2292909; Neuromab), mouse anti-GFP mono


Abstract:

Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by CDH10 within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions.SIGNIFICANCE STATEMENT The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.

Loss of SynDIG1 Reduces Excitatory Synapse Maturation But Not Formation In Vivo.

  • Chenaux G
  • eNeuro
  • 2017 Oct 31

Literature context: NeuroMab (RRID:AB_2292909)], synapto


Abstract:

Modification of the strength of excitatory synaptic connections is a fundamental mechanism by which neural circuits are refined during development and learning. Synapse Differentiation Induced Gene 1 (SynDIG1) has been shown to play a key role in regulating synaptic strength in vitro. Here, we investigated the role of SynDIG1 in vivo in mice with a disruption of the SynDIG1 gene rather than use an alternate loxP-flanked conditional mutant that we find retains a partial protein product. The gene-trap insertion with a reporter cassette mutant mice shows that the SynDIG1 promoter is active during embryogenesis in the retina with some activity in the brain, and postnatally in the mouse hippocampus, cortex, hindbrain, and spinal cord. Ultrastructural analysis of the hippocampal CA1 region shows a decrease in the average PSD length of synapses and a decrease in the number of synapses with a mature phenotype. Intriguingly, the total synapse number appears to be increased in SynDIG1 mutant mice. Electrophysiological analyses show a decrease in AMPA and NMDA receptor function in SynDIG1-deficient hippocampal neurons. Glutamate stimulation of individual dendritic spines in hippocampal slices from SynDIG1-deficient mice reveals increased short-term structural plasticity. Notably, the overall levels of PSD-95 or glutamate receptors enriched in postsynaptic biochemical fractions remain unaltered; however, activity-dependent synapse development is strongly compromised upon the loss of SynDIG1, supporting its importance for excitatory synapse maturation. Together, these data are consistent with a model in which SynDIG1 regulates the maturation of excitatory synapse structure and function in the mouse hippocampus in vivo.

Funding information:
  • NIMH NIH HHS - R01 MH104638(United States)

Histone Hypervariants H2A.Z.1 and H2A.Z.2 Play Independent and Context-Specific Roles in Neuronal Activity-Induced Transcription of Arc/Arg3.1 and Other Immediate Early Genes.

  • Dunn CJ
  • eNeuro
  • 2017 Aug 31

Literature context: Incorporated, 75-028; RRID:AB_2292909), rabbit anti-SH3 and multiple


Abstract:

The histone variant H2A.Z is an essential and conserved regulator of eukaryotic gene transcription. However, the exact role of this histone in the transcriptional process remains perplexing. In vertebrates, H2A.Z has two hypervariants, H2A.Z.1 and H2A.Z.2, that have almost identical sequences except for three amino acid residues. Due to such similarity, functional specificity of these hypervariants in neurobiological processes, if any, remain largely unknown. In this study with dissociated rat cortical neurons, we asked if H2A.Z hypervariants have distinct functions in regulating basal and activity-induced gene transcription. Hypervariant-specific RNAi and microarray analyses revealed that H2A.Z.1 and H2A.Z.2 regulate basal expression of largely nonoverlapping gene sets, including genes that code for several synaptic proteins. In response to neuronal activity, rapid transcription of our model gene Arc is impaired by depletion of H2A.Z.2, but not H2A.Z.1. This impairment is partially rescued by codepletion of the H2A.Z chaperone, ANP32E. In contrast, under a different context (after 48 h of tetrodotoxin, TTX), rapid transcription of Arc is impaired by depletion of either hypervariant. Such context-dependent roles of H2A.Z hypervariants, as revealed by our multiplexed gene expression assays, are also evident with several other immediate early genes, where regulatory roles of these hypervariants vary from gene to gene under different conditions. Together, our data suggest that H2A.Z hypervariants have context-specific roles that complement each other to mediate activity-induced neuronal gene transcription.

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses.

  • Sai K
  • J. Comp. Neurol.
  • 2017 Aug 15

Literature context: ne K28/43 RRID:AB_2292909    


Abstract:

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects.

  • Shaqura M
  • Neuropharmacology
  • 2017 Jul 11

Literature context: 75-028, # RRID:AB_2292909 1:1.000


Abstract:

Glucocorticoids were long believed to primarily function through cytosolic glucocorticoid receptor (GR) activation and subsequent classical genomic pathways. Recently, however, evidence has emerged that suggests the presence of rapid non-genomic GR-dependent signaling pathways within the brain, though their existence in spinal and peripheral nociceptive neurons remains elusive. In this paper, we aim to systemically identify GR within the spinal cord and periphery, to verify their putative membrane location and to characterize possible G protein coupling and pain modulating properties. Double immunofluorescence confocal microscopy revealed that GR predominantly localized in peripheral peptidergic and non-peptidergic nociceptive C- and Aδ-neurons and existed only marginally in myelinated mechanoreceptive and proprioreceptive neurons. Within the spinal cord, GR predominantly localized in incoming presynaptic nociceptive neurons, in pre- and postsynaptic structures of the dorsal horn, as well as in microglia. GR saturation binding revealed that these receptors are linked to the cell membrane of sensory neurons and, upon activation, they trigger membrane targeted [35S]GTPγS binding, indicating G protein coupling to a putative receptor. Importantly, subcutaneous dexamethasone immediately and dose-dependently attenuated acute nociceptive behavior elicited in an animal model of formalin-induced pain hypersensitivity compared to naive rats. Overall, this study provides firm evidence for a novel neuronal mechanism of GR agonists that is rapid, non-genomic, dependent on membrane binding and G protein coupling, and acutely modulates nociceptive behavior, thus unraveling a yet unconsidered mechanism of pain relief.

Funding information:
  • NINDS NIH HHS - T32 NS061764(United States)
  • NINDS NIH HHS - U01 NS090595(United States)

Membrane-associated guanylate kinase scaffolds organize a horizontal cell synaptic complex restricted to invaginating contacts with photoreceptors.

  • Vila A
  • J. Comp. Neurol.
  • 2017 Mar 1

Literature context: . 75-028; RRID:AB_2292909) blotted a


Abstract:

Synaptic processes and plasticity of synapses are mediated by large suites of proteins. In most cases, many of these proteins are tethered together by synaptic scaffold proteins. Scaffold proteins have a large number and typically a variety of protein interaction domains that allow many different proteins to be assembled into functional complexes. Because each scaffold protein has a different set of protein interaction domains and a unique set of interacting partners, the presence of synaptic scaffolds can provide insight into the molecular mechanisms that regulate synaptic processes. In studies of rabbit retina, we found SAP102 and Chapsyn110 selectively localized in the tips of B-type horizontal cell processes, where they contact cone and rod photoreceptors. We further identified some known SAP102 binding partners, kainate receptor GluR6/7 and inward rectifier potassium channel Kir2.1, closely associated with SAP102 in photoreceptor invaginations. The kainate receptor occupies a position distinct from that of the majority of AMPA receptors that dominate the horizontal cell postsynaptic response. GluR6/7 and Kir2.1 presumably are involved in synaptic processes that govern cell-to-cell communication and could both contribute in different ways to synaptic currents that mediate feedback signaling. Notably, we failed to find evidence for the presence of Cx57 or Cx59 that might be involved in ephaptic feedback signaling in this complex. The presence of SAP102 and its binding partners in both cone and rod invaginating synapses suggests that whatever mechanism is supported by this protein complex is present in both types of photoreceptors. J. Comp. Neurol. 525:850-867, 2017. © 2016 Wiley Periodicals, Inc.

GRASP1 Regulates Synaptic Plasticity and Learning through Endosomal Recycling of AMPA Receptors.

  • Chiu SL
  • Neuron
  • 2017 Mar 22

Literature context: # 75-028, RRID:AB_2307331 Mouse anti


Abstract:

Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders.

Funding information:
  • NINDS NIH HHS - R01 NS036715()
  • NINDS NIH HHS - R01 NS073854()

Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

  • Lee WH
  • Elife
  • 2016 Nov 15

Literature context: # 75-028, RRID:AB_2292909, Davis, CA


Abstract:

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.

  • Loh KH
  • Cell
  • 2016 Aug 25

Literature context: t# 75-028 RRID:AB_2292909 Mouse anti


Abstract:

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

Erratum to: Rectocutaneous fistula with transmigration of the suture: a rare delayed complication of vault fixation with the sacrospinous ligament.

  • Kadam PD
  • Int Urogynecol J
  • 2016 Mar 25

Literature context:


Abstract:

There was an oversight in the Authorship of a recent Images in Urogynecology article titled: Rectocutaneous fistula with transmigration of the suture: a rare delayed complication of vault fixation with the sacrospinous ligament (DOI 10.1007/ s00192-015-2823-5). We would like to include Adj A/P Han How Chuan’s name in the list of authors. Adj A/P Han is a Senior Consultant and Department Head of Urogynaecology at the KK Hospital for Women and Children, Singapore.

Funding information:
  • NHGRI NIH HHS - R01HG005855(United States)

Casein kinase 2 phosphorylates GluA1 and regulates its surface expression.

  • Lussier MP
  • Eur. J. Neurosci.
  • 2014 Dec 18

Literature context:


Abstract:

Controlling the density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at synapses is essential for regulating the strength of excitatory neurotransmission. In particular, the phosphorylation of AMPARs is important for defining both synaptic expression and intracellular routing of receptors. Phosphorylation is a post-translational modification known to regulate many cellular events and the C-termini of glutamate receptors are important targets. Recently, the first intracellular loop1 region of the GluA1 subunit of AMPARs was reported to regulate synaptic targeting through phosphorylation of S567 by Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Intriguingly, the loop1 region of all four AMPAR subunits contains many putative phosphorylation sites (S/T/Y), leaving the possibility that other kinases may regulate AMPAR surface expression via phosphorylation of the loop regions. To explore this hypothesis, we used in vitro phosphorylation assays with a small panel of purified kinases and found that casein kinase 2 (CK2) phosphorylates the GluA1 and GluA2 loop1 regions, but not GluA3 or GluA4. Interestingly, when we reduced the endogenous expression of CK2 using a specific short hairpin RNA against the regulatory subunit CK2β, we detected a reduction of GluA1 surface expression, whereas GluA2 was unchanged. Furthermore, we identified S579 of GluA1 as a substrate of CK2, and the expression of GluA1 phosphodeficient mutants in hippocampal neurons displayed reduced surface expression. Therefore, our study identifies CK2 as a regulator of GluA1 surface expression by phosphorylating the intracellular loop1 region.

Funding information:
  • NINDS NIH HHS - NS-23805(United States)

Regulation of neuronal gene expression and survival by basal NMDA receptor activity: a role for histone deacetylase 4.

  • Chen Y
  • J. Neurosci.
  • 2014 Nov 12

Literature context: #75-028 (RRID:AB_2292909), NeuroMab


Abstract:

Neuronal gene expression is modulated by activity via calcium-permeable receptors such as NMDA receptors (NMDARs). While gene expression changes downstream of evoked NMDAR activity have been well studied, much less is known about gene expression changes that occur under conditions of basal neuronal activity. In mouse dissociated hippocampal neuronal cultures, we found that a broad NMDAR antagonist, AP5, induced robust gene expression changes under basal activity, but subtype-specific antagonists did not. While some of the gene expression changes are also known to be downstream of stimulated NMDAR activity, others appear specific to basal NMDAR activity. The genes altered by AP5 treatment of basal cultures were enriched for pathways related to class IIa histone deacetylases (HDACs), apoptosis, and synapse-related signaling. Specifically, AP5 altered the expression of all three class IIa HDACs that are highly expressed in the brain, HDAC4, HDAC5, and HDAC9, and also induced nuclear accumulation of HDAC4. HDAC4 knockdown abolished a subset of the gene expression changes induced by AP5, and led to neuronal death under long-term tetrodotoxin or AP5 treatment in rat hippocampal organotypic slice cultures. These data suggest that basal, but not evoked, NMDAR activity regulates gene expression in part through HDAC4, and, that HDAC4 has neuroprotective functions under conditions of low NMDAR activity.

Funding information:
  • NIA NIH HHS - AG012609(United States)

Ablation of ErbB4 from excitatory neurons leads to reduced dendritic spine density in mouse prefrontal cortex.

  • Cooper MA
  • J. Comp. Neurol.
  • 2014 Oct 1

Literature context: # 75-028, RRID:AB_2292909) was used


Abstract:

Dendritic spine loss is observed in many psychiatric disorders, including schizophrenia, and likely contributes to the altered sense of reality, disruption of working memory, and attention deficits that characterize these disorders. ErbB4, a member of the EGF family of receptor tyrosine kinases, is genetically associated with schizophrenia, suggesting that alterations in ErbB4 function contribute to the disease pathology. Additionally, ErbB4 functions in synaptic plasticity, leading us to hypothesize that disruption of ErbB4 signaling may affect dendritic spine development. We show that dendritic spine density is reduced in the dorsomedial prefrontal cortex of ErbB4 conditional whole-brain knockout mice. We find that ErbB4 localizes to dendritic spines of excitatory neurons in cortical neuronal cultures and is present in synaptic plasma membrane preparations. Finally, we demonstrate that selective ablation of ErbB4 from excitatory neurons leads to a decrease in the proportion of mature spines and an overall reduction in dendritic spine density in the prefrontal cortex of weanling (P21) mice that persists at 2 months of age. These results suggest that ErbB4 signaling in excitatory pyramidal cells is critical for the proper formation and maintenance of dendritic spines in excitatory pyramidal cells.

Funding information:
  • NINDS NIH HHS - R44 NS074540(United States)

DSCAM localization and function at the mouse cone synapse.

  • de Andrade GB
  • J. Comp. Neurol.
  • 2014 Aug 1

Literature context:


Abstract:

The Down syndrome cell adhesion molecule (DSCAM) is required for regulation of cell number, soma spacing, and cell type-specific dendrite avoidance in many types of retinal ganglion and amacrine cells. In this study we assay the organization of cells making up the outer plexiform layer of the retina in the absence of Dscam. Some types of OFF bipolar cells, type 3b and type 4 bipolar cells, had defects in dendrite arborization in the Dscam mutant retina, whereas other cell types appeared similar to wild type. The cone synapses that these cells project their dendrites to were intact, as visualized by electron microscopy, and had a distribution and density that was not significantly different from that of wild type. The spacing of type 3b bipolar cell dendrites was further analyzed by Voronoi domain analysis, density recovery profiling (DRP) analysis, and nearest neighbor analysis. Spacing was found to be significantly different when wild-type and mutant type 3b bipolar cell dendrites were compared. Defects in arborization of these bipolar cells could not be attributed to the disorganization of inner plexiform layer cells that occurs in the Dscam mutant retina or an increase in cell number, as they arborized when Dscam was targeted in retinal ganglion cells only or in the bax null retina. Localization of DSCAM was assayed and the protein was localized near to cone synapses in mouse, macaque, and ground squirrel retinas. DSCAM protein was detected in several types of bipolar cells, including type 3b and type 4 bipolar cells.

Funding information:
  • NINDS NIH HHS - R56 NS094589(United States)

Down-regulation of endogenous KLHL1 decreases voltage-gated calcium current density.

  • Perissinotti PP
  • Cell Calcium
  • 2014 May 5

Literature context:


Abstract:

The actin-binding protein Kelch-like 1 (KLHL1) can modulate voltage-gated calcium channels in vitro. KLHL1 interacts with actin and with the pore-forming subunits of Cav2.1 and CaV3.2 calcium channels, resulting in up-regulation of P/Q and T-type current density. Here we tested whether endogenous KLHL1 modulates voltage gated calcium currents in cultured hippocampal neurons by down-regulating the expression of KLHL1 via adenoviral delivery of shRNA targeted against KLHL1 (shKLHL1). Control adenoviruses did not affect any of the neuronal properties measured, yet down-regulation of KLHL1 resulted in HVA current densities ~68% smaller and LVA current densities 44% smaller than uninfected controls, with a concomitant reduction in α(1A) and α(1H) protein levels. Biophysical analysis and western blot experiments suggest Ca(V)3.1 and 3.3 currents are also present in shKLHL1-infected neurons. Synapsin I levels, miniature postsynaptic current frequency, and excitatory and inhibitory synapse number were reduced in KLHL1 knockdown. This study corroborates the physiological role of KLHL1 as a calcium channel modulator and demonstrates a novel, presynaptic role.

Funding information:
  • Medical Research Council - G0800034(United Kingdom)
  • NCI NIH HHS - CA133346(United States)

Dynamic changes in hair cell ribbon synapse induced by loss of spiral ganglion neurons in mice.

  • Yuan Y
  • Chin. Med. J.
  • 2014 May 14

Literature context:


Abstract:

BACKGROUND: Previous studies have suggested that primary degeneration of hair cells causes secondary degeneration of spiral ganglion neurons (SGNs), but the effect of SGN degeneration on hair cells has not been studied. In the adult mouse inner ear ouabain can selectively and permanently induce the degeneration of type 1 SGNs while leaving type 2 SGNs, efferent fibers, and sensory hair cells relatively intact. This study aimed to investigate the dynamic changes in hair cell ribbon synapse induced by loss of SGNs using ouabain application to the round window niche of adult mice. METHODS: In the analysis, 24 CBA/CAJ mice aged 8-10 weeks, were used, of which 6 normal mice were used as the control group. After ouabain application in the round window niche 6 times in an hour, ABR threshold shifts at least 30 dB in the three experimental groups which had six mice for 1-week group, six for 1-month group, and six for 3-month group. All 24 animals underwent function test at 1 week and then immunostaining at 1 week, 1 month, and 3 months. RESULTS: The loss of neurons was followed by degeneration of postsynaptic specializations at the afferent synapse with hair cells. One week after ouabain treatment, the nerve endings of type 1 SGNs and postsynaptic densities, as measured by Na/K ATPase and PSD-95, were affected but not entirely missing, but their partial loss had consequences for synaptic ribbons that form the presynaptic specialization at the synapse between hair cells and primary afferent neurons. Ribbon numbers in inner hair cells decreased (some of them broken and the ribbon number much decreased), and the arrangement of the synaptic ribbons had undergone a dynamic reorganization: ribbons with or without associated postsynaptic densities moved from their normal location in the basal membrane of the cell to a more apical location and the neural endings alone were also found at more apical locations without associated ribbons. After 1 month, when the neural postsynaptic densities had completed their degeneration, most ribbons were lost and the remaining ribbons had no contact with postsynaptic densities; after 3 months, the ribbon synapses were gone except for an occasional remnant of a CtBP2-positive vesicle. Hair cells were intact other than the loss of ribbons (based on immunohistochemistry and DPOAE). CONCLUSION: These findings define the effect of SGN loss on the precise spatiotemporal size and location of ribbons and the time course of synaptic degeneration and provide a model for studying plasticity and regeneration.

Funding information:
  • NCRR NIH HHS - 1 U41 RR 01685(United States)
  • NINDS NIH HHS - R56 NS028721(United States)

Neural differentiation of pluripotent cells in 3D alginate-based cultures.

  • Bozza A
  • Biomaterials
  • 2014 May 25

Literature context:


Abstract:

Biomaterial-supported culture methods, allowing for directed three-dimensional differentiation of stem cells are an alternative to canonical two-dimensional cell cultures. In this paper, we evaluate the suitability of alginate for three-dimensional cultures to enhance differentiation of mouse embryonic stem cells (mESCs) towards neural lineages. We tested whether encapsulation of mESCs within alginate beads could support and/or enhance neural differentiation with respect to two-dimensional cultures. We encapsulated cells in beads of alginate with or without modification by fibronectin (Fn) or hyaluronic acid (HA). Gene expression analysis showed that cells grown in alginate and alginate-HA present increased differentiation toward neural lineages with respect to the two-dimensional control and to Fn group. Immunocytochemistry analyses confirmed these results, further showing terminal differentiation of neurons as seen by the expression of synaptic markers and markers of different neuronal subtypes. Our data show that alginate, alone or modified, is a suitable biomaterial to promote in vitro differentiation of pluripotent cells toward neural fates.

Funding information:
  • NHLBI NIH HHS - HL107147(United States)

Cortical synaptic NMDA receptor deficits in α7 nicotinic acetylcholine receptor gene deletion models: implications for neuropsychiatric diseases.

  • Lin H
  • Neurobiol. Dis.
  • 2014 Mar 20

Literature context:


Abstract:

Microdeletion of the human CHRNA7 gene (α7 nicotinic acetylcholine receptor, nAChR) as well as dysfunction in N-methyl-d-aspartate receptors (NMDARs) have been associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia. However, the pathophysiological roles of synaptic vs. extrasynaptic NMDARs and their interactions with α7 nAChRs in cortical dysfunction remain largely uncharacterized. Using a combination of in vivo and in vitro models, we demonstrate that α7 nAChR gene deletion leads to specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in mouse cortex. α7 nAChR null mice had decreased cortical NMDAR expression and glutamatergic synapse formation during postnatal development. Similar reductions in NMDAR expression and glutamatergic synapse formation were revealed in cortical cultures lacking α7 nAChRs. Interestingly, synaptic, but not extrasynaptic, NMDAR currents were specifically diminished in cultured cortical pyramidal neurons as well as in acute prefrontal cortical slices of α7 nAChR null mice. Moreover, d-serine responsive synaptic NMDAR-mediated currents and levels of the d-serine synthetic enzyme serine racemase were both reduced in α7 nAChR null cortical pyramidal neurons. Our findings thus identify specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in α7 nAChR gene deletion models of cortical dysfunction, thereby implicating α7 nAChR-mediated control of synaptic NMDARs and serine racemase/d-serine pathways in cortical dysfunction underlying many neuropsychiatric and neurodevelopmental disorders, particularly those associated with deletion of human CHRNA7.

Funding information:
  • NCRR NIH HHS - 3P41RR024851-02S1(United States)

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes.

  • Schwenk BM
  • EMBO J.
  • 2014 Mar 3

Literature context:


Abstract:

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP-43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule-associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over-expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant-negative Rab7-interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.

Funding information:
  • NHGRI NIH HHS - P50HG004233(United States)

EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloid-β oligomers.

  • Vargas LM
  • PLoS ONE
  • 2014 Mar 24

Literature context:


Abstract:

The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-β oligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.

  • Yuan Y
  • J. Assoc. Res. Otolaryngol.
  • 2014 Feb 27

Literature context:


Abstract:

Ouabain application to the round window can selectively destroy type-I spiral ganglion cells, producing an animal model of auditory neuropathy. To assess the long-term effects of this deafferentation on synaptic organization in the organ of Corti and cochlear nucleus, and to ask whether surviving cochlear neurons show any post-injury plasticity in the adult, we quantified the peripheral and central synapses of type-I neurons at posttreatment times ranging from 1 to 3 months. Measures of normal DPOAEs and greatly reduced auditory brainstem responses (ABRs) confirmed the neuropathy phenotype. Counts of presynaptic ribbons and postsynaptic glutamate receptor patches in the inner hair cell area decreased with post-exposure time, as did counts of cochlear nerve terminals in the cochlear nucleus. Although these counts provided no evidence of new synapse formation via branching from surviving neurons, the regular appearance of ectopic neurons in the inner hair cell area suggested that neurite extension is not uncommon. Correlations between pathophysiology and histopathology showed that ABR thresholds are very insensitive to even massive neural degeneration, whereas the amplitude of ABR wave 1 is a better metric of synaptic degeneration.

Funding information:
  • NIGMS NIH HHS - GM19351(United States)

In vivo activation of Wnt signaling pathway enhances cognitive function of adult mice and reverses cognitive deficits in an Alzheimer's disease model.

  • Vargas JY
  • J. Neurosci.
  • 2014 Feb 5

Literature context:


Abstract:

The role of the Wnt signaling pathway during synaptic development has been well established. In the adult brain, different components of Wnt signaling are expressed, but little is known about its role in mature synapses. Emerging in vitro studies have implicated Wnt signaling in synaptic plasticity. Furthermore, activation of Wnt signaling has shown to protect against amyloid-β-induced synaptic impairment. The present study provides the first evidence that in vivo activation of Wnt signaling improves episodic memory, increases excitatory synaptic transmission, and enhances long-term potentiation in adult wild-type mice. Moreover, the activation of Wnt signaling also rescues memory loss and improves synaptic dysfunction in APP/PS1-transgenic mice that model the amyloid pathology of Alzheimer's diseases. These findings indicate that Wnt signaling modulates cognitive function in the adult brain and could be a novel promising target for Alzheimer's disease therapy.

Funding information:
  • NEI NIH HHS - EY04067(United States)

Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain.

  • Dagley LF
  • Proteomics
  • 2014 Feb 4

Literature context:


Abstract:

Despite major advances in neuroscience, a comprehensive understanding of the structural and functional components of the adult brain compartments remains to be fully elucidated at a quantitative molecular level. Indeed, over half of the soluble- and membrane-annotated proteins are currently unmapped within online digital brain atlases. In this study, two complementary approaches were used to assess the unique repertoire of proteins enriched within select regions of the adult mouse CNS, including the brain stem, cerebellum, and remaining brain hemispheres. Of the 1200 proteins visualized by 2D-DIGE, approximately 150 (including cytosolic and membrane proteins) were found to exhibit statistically significant changes in relative abundance thus representing putative region-specific brain markers. In addition to using a high-precision (18) O-labeling strategy for the quantitative LC-MS/MS mapping of membrane proteins isolated from myelin-enriched fractions, we have identified over 1000 proteins that have yet to be described in any other mammalian myelin proteome. A comparison of our myelin proteome was made to an existing transcriptome database containing mRNA abundance profiles during oligodendrocyte differentiation and has confirmed statistically significant abundance changes for ∼500 of these newly mapped proteins, thus revealing new roles in oligodendrocyte and myelin biology. These data offer a resource for the neuroscience community studying the molecular basis for specialized neuronal activities in the CNS and myelin-related disorders. The MS proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000327 (http://proteomecentral.proteomexchange.org/dataset/PXD000327).

Funding information:
  • NINDS NIH HHS - R01-NS060120(United States)

C-terminal interactors of the AMPA receptor auxiliary subunit Shisa9.

  • Karataeva AR
  • PLoS ONE
  • 2014 Feb 5

Literature context:


Abstract:

Shisa9 (initially named CKAMP44) has been identified as auxiliary subunit of the AMPA-type glutamate receptors and was shown to modulate its physiological properties. Shisa9 is a type-I transmembrane protein and contains a C-terminal PDZ domain that potentially interacts with cytosolic proteins. In this study, we performed a yeast two-hybrid screening that yielded eight PDZ domain-containing interactors of Shisa9, which were independently validated. The identified interactors are known scaffolding proteins residing in the neuronal postsynaptic density. To test whether C-terminal scaffolding interactions of Shisa9 affect synaptic AMPA receptor function in the hippocampus, we disrupted these interactions using a Shisa9 C-terminal mimetic peptide. In the absence of scaffolding interactions of Shisa9, glutamatergic AMPA receptor-mediated synaptic currents in the lateral perforant path of the mouse hippocampus had a faster decay time, and paired-pulse facilitation was reduced. Furthermore, disruption of the PDZ interactions between Shisa9 and its binding partners affected hippocampal network activity. Taken together, our data identifies novel interaction partners of Shisa9, and shows that the C-terminal interactions of Shisa9 through its PDZ domain interaction motif are important for AMPA receptor synaptic and network functions.

Funding information:
  • NINDS NIH HHS - 4R00NS057944-03(United States)

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses.

  • Bemben MA
  • Nat. Neurosci.
  • 2014 Jan 26

Literature context:


Abstract:

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

Funding information:
  • NIDA NIH HHS - DA003910(United States)

The novel caspase-3 substrate Gap43 is involved in AMPA receptor endocytosis and long-term depression.

  • Han MH
  • Mol. Cell Proteomics
  • 2013 Dec 9

Literature context:


Abstract:

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.

Inhibition of amyloid beta-induced synaptotoxicity by a pentapeptide derived from the glycine zipper region of the neurotoxic peptide.

  • Peters C
  • Neurobiol. Aging
  • 2013 Dec 20

Literature context:


Abstract:

A major characteristic of Alzheimer's disease is the presence of amyloid beta (Aβ) oligomers and aggregates in the brain. Aβ oligomers interact with the neuronal membrane inducing perforations, causing an influx of calcium ions and increasing the release of synaptic vesicles that leads to a delayed synaptic failure by vesicle depletion. Here, we identified a neuroprotective pentapeptide anti-Aβ compound having the sequence of the glycine zipper region of the C-terminal of Aβ (G33LMVG37). Docking and Förster resonance energy transfer experiments showed that G33LMVG37 interacts with Aβ at the C-terminal region, which is important for Aβ association and insertion into the lipid membrane. Furthermore, this pentapeptide interfered with Aβ aggregation, association, and perforation of the plasma membrane. The synaptotoxicity induced by Aβ after acute and chronic applications were abolished by G33LMVG37. These results provide a novel rationale for drug development against Alzheimer's disease.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • NIAID NIH HHS - U01AI068635-01(United States)

Peptidylglycine α-amidating monooxygenase heterozygosity alters brain copper handling with region specificity.

  • Gaier ED
  • J. Neurochem.
  • 2013 Dec 3

Literature context:


Abstract:

Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.

Funding information:
  • NICHD NIH HHS - HD40035(United States)

PRICKLE1 interaction with SYNAPSIN I reveals a role in autism spectrum disorders.

  • Paemka L
  • PLoS ONE
  • 2013 Dec 6

Literature context:


Abstract:

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Funding information:
  • NIGMS NIH HHS - R01 GM093123(United States)

Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus.

  • Raissi AJ
  • Mol. Cell. Neurosci.
  • 2013 Nov 26

Literature context:


Abstract:

While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.

Funding information:
  • NCI NIH HHS - 5 T32 CA106196-05(United States)

SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties.

  • Han K
  • Nature
  • 2013 Nov 7

Literature context:


Abstract:

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

Funding information:
  • Medical Research Council - G0800578(United Kingdom)

Netrin-1 promotes excitatory synaptogenesis between cortical neurons by initiating synapse assembly.

  • Goldman JS
  • J. Neurosci.
  • 2013 Oct 30

Literature context:


Abstract:

Netrin-1 is a secreted protein that directs long-range axon guidance during early stages of neural circuit formation and continues to be expressed in the mammalian forebrain during the postnatal period of peak synapse formation. Here we demonstrate a synaptogenic function of netrin-1 in rat and mouse cortical neurons and investigate the underlying mechanism. We report that netrin-1 and its receptor DCC are widely expressed by neurons in the developing mammalian cortex during synapse formation and are enriched at synapses in vivo. We detect DCC protein distributed along the axons and dendrites of cultured cortical neurons and provide evidence that newly translated netrin-1 is selectively transported to dendrites. Using gain and loss of function manipulations, we demonstrate that netrin-1 increases the number and strength of excitatory synapses made between developing cortical neurons. We show that netrin-1 increases the complexity of axon and dendrite arbors, thereby increasing the probability of contact. At sites of contact, netrin-1 promotes adhesion, while locally enriching and reorganizing the underlying actin cytoskeleton through Src family kinase signaling and m-Tor-dependent protein translation to locally cluster presynaptic and postsynaptic proteins. Finally, we demonstrate using whole-cell patch-clamp electrophysiology that netrin-1 increases the frequency and amplitude of mEPSCs recorded from cortical pyramidal neurons. These findings identify netrin-1 as a synapse-enriched protein that promotes synaptogenesis between mammalian cortical neurons.

Funding information:
  • NCI NIH HHS - CA06927(United States)

Synapse maturation by activity-dependent ectodomain shedding of SIRPα.

  • Toth AB
  • Nat. Neurosci.
  • 2013 Oct 26

Literature context:


Abstract:

Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of signal regulatory protein-α (SIRPα) mediates presynaptic maturation. Two target-derived molecules, fibroblast growth factor 22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain in turn promotes the maturation of the presynaptic terminal. This process involves calcium/calmodulin-dependent protein kinase, matrix metalloproteinases and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has an impact on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses.

Shank3 deficiency induces NMDA receptor hypofunction via an actin-dependent mechanism.

  • Duffney LJ
  • J. Neurosci.
  • 2013 Oct 2

Literature context:


Abstract:

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.

Funding information:
  • Canadian Institutes of Health Research - FRN 82940(Canada)

Antihypertensive drug Valsartan promotes dendritic spine density by altering AMPA receptor trafficking.

  • Sohn YI
  • Biochem. Biophys. Res. Commun.
  • 2013 Oct 4

Literature context:


Abstract:

Recent studies demonstrated that the antihypertensive drug Valsartan improved spatial and episodic memory in mouse models of Alzheimer's Disease (AD) and human subjects with hypertension. However, the molecular mechanism by which Valsartan can regulate cognitive function is still unknown. Here, we investigated the effect of Valsartan on dendritic spine formation in primary hippocampal neurons, which is correlated with learning and memory. Interestingly, we found that Valsartan promotes spinogenesis in developing and mature neurons. In addition, we found that Valsartan increases the puncta number of PSD-95 and trends toward an increase in the puncta number of synaptophysin. Moreover, Valsartan increased the cell surface levels of AMPA receptors and selectively altered the levels of spinogenesis-related proteins, including CaMKIIα and phospho-CDK5. These data suggest that Valsartan may promote spinogenesis by enhancing AMPA receptor trafficking and synaptic plasticity signaling.

Funding information:
  • NHLBI NIH HHS - U01 HL66678(United States)

Sortilin-related receptor SORCS3 is a postsynaptic modulator of synaptic depression and fear extinction.

  • Breiderhoff T
  • PLoS ONE
  • 2013 Sep 26

Literature context:


Abstract:

SORCS3 is an orphan receptor of the VPS10P domain receptor family, a group of sorting and signaling receptors central to many pathways in control of neuronal viability and function. SORCS3 is highly expressed in the CA1 region of the hippocampus, but the relevance of this receptor for hippocampal activity remained absolutely unclear. Here, we show that SORCS3 localizes to the postsynaptic density and that loss of receptor activity in gene-targeted mice abrogates NMDA receptor-dependent and -independent forms of long-term depression (LTD). Consistent with a loss of synaptic retraction, SORCS3-deficient mice suffer from deficits in behavioral activities associated with hippocampal LTD, particularly from an accelerated extinction of fear memory. A possible molecular mechanism for SORCS3 in synaptic depression was suggested by targeted proteomics approaches that identified the ability of SORCS3 to functionally interact with PICK1, an adaptor that sorts glutamate receptors at the postsynapse. Faulty localization of PICK1 in SORCS3-deficient neurons argues for altered glutamate receptor trafficking as the cause of altered synaptic plasticity in the SORCS3-deficient mouse model. In conclusion, our studies have identified a novel function for VPS10P domain receptors in control of synaptic depression and suggest SORCS3 as a novel factor modulating aversive memory extinction.

Funding information:
  • NIGMS NIH HHS - R01GM093217(United States)

Differential roles of postsynaptic density-93 isoforms in regulating synaptic transmission.

  • Krüger JM
  • J. Neurosci.
  • 2013 Sep 25

Literature context:


Abstract:

In the postsynaptic density of glutamatergic synapses, the discs large (DLG)-membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins coordinates a multiplicity of signaling pathways to maintain and regulate synaptic transmission. Postsynaptic density-93 (PSD-93) is the most variable paralog in this family; it exists in six different N-terminal isoforms. Probably because of the structural and functional variability of these isoforms, the synaptic role of PSD-93 remains controversial. To accurately characterize the synaptic role of PSD-93, we quantified the expression of all six isoforms in the mouse hippocampus and examined them individually in hippocampal synapses. Using molecular manipulations, including overexpression, gene knockdown, PSD-93 knock-out mice combined with biochemical assays, and slice electrophysiology both in rat and mice, we demonstrate that PSD-93 is required at different developmental synaptic states to maintain the strength of excitatory synaptic transmission. This strength is differentially regulated by the six isoforms of PSD-93, including regulations of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-active and inactive synapses, and activity-dependent modulations. Collectively, these results demonstrate that alternative combinations of N-terminal PSD-93 isoforms and DLG-MAGUK paralogs can fine-tune signaling scaffolds to adjust synaptic needs to regulate synaptic transmission.

Funding information:
  • NIGMS NIH HHS - P01 GM061354(United States)

Molecular mechanisms underlying activity-dependent AMPA receptor cycling in retinal ganglion cells.

  • Casimiro TM
  • Mol. Cell. Neurosci.
  • 2013 Sep 4

Literature context:


Abstract:

On retinal ganglion cells (RGCs) transmit light encoded information to the brain and receive excitatory input from On cone bipolar cells (CBPs). The synaptic CBP input onto On RGCs is mediated by AMPA-type glutamate receptors (AMPARs) that include both those lacking a GluA2 subunit, and are therefore permeable to Ca(2+), and those that possess at least one GluA2 subunit and are Ca(2+)-impermeable. We have previously demonstrated in electrophysiological studies that periods of low synaptic activity, brought about by housing animals in darkness, enhance the proportion of GluA2-lacking AMPARs at the On CBP-On RGC synapse by mobilizing surface GluA2 containing receptors into a receptor pool that rapidly cycles in and out of the membrane. AMPAR cycling induction by reduced synaptic activity takes several hours. This delay suggests that changes in expression of proteins which regulate AMPAR trafficking may mediate the altered mobility of GluA2 AMPARs in RGCs. In this study, we test the hypothesis that AMPAR trafficking proteins couple synaptic activity to AMPAR cycling in RGCs. Immunocytochemical and biochemical analyses confirmed that darkness decreases surface GluA2 in RGCs and changed the expression levels of three proteins associated with GluA2 trafficking. GRIP was decreased, while PICK1 and Arc were increased. Knockdown of GRIP with siRNA elevated constitutive AMPAR cycling, mimicking effects of reduced synaptic activity, while knockdown of PICK1 and Arc blocked increases in constitutive GluA2 trafficking. Our results support a role for correlated, activity-driven changes in multiple AMPAR trafficking proteins that modulate GluA2 cycling which can in turn affect synaptic AMPAR composition in RGCs.

Funding information:
  • NCRR NIH HHS - U13 RR017436-05(United States)

Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation.

  • Czöndör K
  • Nat Commun
  • 2013 Aug 12

Literature context:


Abstract:

Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1β-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1β dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.

DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development.

  • Lin L
  • Nat Commun
  • 2013 Aug 5

Literature context:


Abstract:

Dipeptidyl-peptidase 6 is an auxiliary subunit of Kv4-mediated A-type K(+) channels that, in addition to enhancing channel surface expression, potently accelerates their kinetics. The dipeptidyl-peptidase 6 gene has been associated with a number of human central nervous system disorders including autism spectrum disorders and schizophrenia. Here we employ knockdown and genetic deletion of dipeptidyl-peptidase 6 to reveal its importance for the formation and stability of dendritic filopodia during early neuronal development. We find that the hippocampal neurons lacking dipeptidyl-peptidase 6 show a sparser dendritic branching pattern along with fewer spines throughout development and into adulthood. In electrophysiological and imaging experiments, we show that these deficits lead to fewer functional synapses and occur independently of the potassium channel subunit Kv4.2. We report that dipeptidyl-peptidase 6 interacts with a filopodia-associated myosin as well as with fibronectin in the extracellular matrix. dipeptidyl-peptidase 6 therefore has an unexpected but important role in cell adhesion and motility, impacting the hippocampal synaptic development and function.

Funding information:
  • NCRR NIH HHS - P20 RR-016463(United States)

A Ca2+-dependent mechanism of neuronal survival mediated by the microtubule-associated protein p600.

  • Belzil C
  • J. Biol. Chem.
  • 2013 Aug 23

Literature context:


Abstract:

In acute and chronic neurodegeneration, Ca(2+) mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca(2+) influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca(2+) release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca(2+)-induced death pathways through regulation of Ca(2+) homeostasis and signaling.

Funding information:
  • NINDS NIH HHS - R01 NS054814-05(United States)

Synaptic state-dependent functional interplay between postsynaptic density-95 and synapse-associated protein 102.

  • Bonnet SA
  • J. Neurosci.
  • 2013 Aug 14

Literature context:


Abstract:

Activity-dependent regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is the basis for establishing differences in synaptic weights among individual synapses during developmental and experience-dependent synaptic plasticity. Synaptic signaling scaffolds of the Discs large (DLG)-membrane-associated guanylate kinase (MAGUK) protein family regulate these processes by tethering signaling proteins to receptor complexes. Using a molecular replacement strategy with RNAi-mediated knockdown in rat and mouse hippocampal organotypic slice cultures, a postsynaptic density-95 (PSD-95) knock-out mouse line and electrophysiological analysis, our current study identified a functional interplay between two paralogs, PSD-95 and synapse-associated protein 102 (SAP102) to regulate synaptic AMPARs. During synaptic development, the SAP102 protein levels normally plateau but double if PSD-95 expression is prevented during synaptogenesis. For an autonomous function of PSD-95 in regulating synaptic AMPARs, in addition to the previously demonstrated N-terminal multimerization and the first two PDZ (PSD-95, Dlg1, zona occludens-1) domains, the PDZ3 and guanylate kinase domains were required. The Src homology 3 domain was dispensable for the PSD-95-autonomous regulation of basal synaptic transmission. However, it mediated the functional interaction with SAP102 of PSD-95 mutants to enhance AMPARs. These results depict a protein domain-based multifunctional aspect of PSD-95 in regulating excitatory synaptic transmission and unveil a novel form of domain-based interplay between signaling scaffolds of the DLG-MAGUK family.

Funding information:
  • Wellcome Trust - 077046(United Kingdom)

Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression.

  • Nelson CD
  • J. Neurosci.
  • 2013 Jul 17

Literature context:


Abstract:

Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β. In organotypic rat hippocampal slices, we find that a nonphosphorylatable PSD-95 mutant (T19A) tagged with photoactivatable green fluorescent protein (PAGFP) shows enhanced stability in dendritic spines versus wild-type PSD-95, whereas the phosphomimetic mutant (PSD-95-T19D) is more readily dispersed. Further, overexpression of PSD-95-T19A, but not WT-PSD-95, impairs AMPA receptor internalization and the induction of LTD. These data indicate that phosphorylation on T19 by GSK-3β destabilizes PSD-95 within the PSD and is a critical step for AMPA receptor mobilization and LTD.

Funding information:
  • NEI NIH HHS - N01-EY-5-0007(United States)

Assembly of synapses: biomimetic assays to control neurexin/neuroligin interactions at the neuronal surface.

  • Mondin M
  • Curr Protoc Neurosci
  • 2013 Jul 15

Literature context:


Abstract:

The role of adhesion molecules in the assembly of synapses in the nervous system is an important issue. To characterize the role of neurexin/neuroligin adhesion complexes in synapse differentiation, various imaging assays can be performed in primary hippocampal cultures. First, to temporally control contact formation, biomimetic assays can be performed using microspheres coated with purified neurexin or with antibody clusters that aggregate neurexin. These models are combined with live fluorescence imaging to study the dynamics of accumulation of post-synaptic components, including scaffolding molecules and glutamate receptors. To demonstrate that AMPA receptors can be recruited to nascent neurexin/neuroligin contacts through lateral diffusion, the mobility of AMPA receptors in the neuronal membrane is monitored by tracking individual quantum dots (QDs) conjugated to antibodies against AMPA receptors. Experiments monitoring the attachment and detachment of Nrx-coated QDs to measure the rates of neurexin/neuroligin interaction can also be performed. Each of these assays is detailed in this unit.

Funding information:
  • PHS HHS - 84620(United States)

CHP1-mediated NHE1 biosynthetic maturation is required for Purkinje cell axon homeostasis.

  • Liu Y
  • J. Neurosci.
  • 2013 Jul 31

Literature context:


Abstract:

Axon degeneration is a critical pathological feature of many neurodegenerative diseases. Misregulation of local axonal ion homeostasis has been recognized as an important yet understudied mechanism for axon degeneration. Here we report a chemically induced, recessive mouse mutation, vacillator (vac), which causes ataxia and concomitant axon degeneration of cerebellar Purkinje cells. By positional cloning, we identified vac as a point mutation in the calcineurin-like EF hand protein 1 (Chp1) gene that resulted in the production of mutant CHP1 isoforms with an amino acid substitution in a functional EF-hand domain or a truncation of this motif by aberrant splicing and significantly reduced protein levels. CHP1 has been previously shown to interact with the sodium hydrogen exchanger 1 (NHE1), a major regulator of intracellular pH. We demonstrated that CHP1 assists in the full glycosylation of NHE1 that is necessary for the membrane localization of this transporter and that truncated isoforms of CHP1 were defective in stimulating NHE1 biosynthetic maturation. Consistent with this, membrane localization of NHE1 at axon terminals was greatly reduced in Chp1-deficient Purkinje cells before axon degeneration. Furthermore, genetic ablation of Nhe1 also resulted in Purkinje cell axon degeneration, pinpointing the functional convergence of the two proteins. Our findings clearly demonstrate that the polarized presynaptic localization of NHE/CHP1 is an important feature of neuronal axons and that selective disruption of NHE1-mediated proton homeostasis in axons can lead to degeneration, suggesting that local regulation of pH is pivotal for axon survival.

Funding information:
  • Autism Speaks - AS2077(United States)

Dapper antagonist of catenin-1 cooperates with Dishevelled-1 during postsynaptic development in mouse forebrain GABAergic interneurons.

  • Arguello A
  • PLoS ONE
  • 2013 Jul 4

Literature context:


Abstract:

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.

Funding information:
  • PHS HHS - 04-2085(United States)

Eps8 controls dendritic spine density and synaptic plasticity through its actin-capping activity.

  • Menna E
  • EMBO J.
  • 2013 Jun 12

Literature context:


Abstract:

Actin-based remodelling underlies spine structural changes occurring during synaptic plasticity, the process that constantly reshapes the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation. A positive correlation exists between spine shape and synaptic strength and, consistently, abnormalities in spine number and morphology have been described in a number of neurological disorders. In the present study, we demonstrate that the actin-regulating protein, Eps8, is recruited to the spine head during chemically induced long-term potentiation in culture and that inhibition of its actin-capping activity impairs spine enlargement and plasticity. Accordingly, mice lacking Eps8 display immature spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Additionally, we found that reduction in the levels of Eps8 occurs in brains of patients affected by autism compared to controls. Our data reveal the key role of Eps8 actin-capping activity in spine morphogenesis and plasticity and indicate that reductions in actin-capping proteins may characterize forms of intellectual disabilities associated with spine defects.

Funding information:
  • NCI NIH HHS - U24 CA126546(United States)

Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1.

  • Giannone G
  • Cell Rep
  • 2013 Jun 27

Literature context:


Abstract:

Adhesion between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1) induces early recruitment of the postsynaptic density protein 95 (PSD-95) scaffold; however, the associated signaling mechanisms are unknown. To dissociate the effects of ligand binding and receptor multimerization, we compared conditions in which Nlg1 in neurons was bound to Nrx1β or nonactivating HA antibodies. Time-lapse imaging, fluorescence recovery after photobleaching, and single-particle tracking demonstrated that in addition to aggregating Nlg1, Nrx1β binding stimulates the interaction between Nlg1 and PSD-95. Phosphotyrosine immunoblots and pull-down of gephyrin by Nlg1 peptides in vitro showed that Nlg1 can be phosphorylated at a unique tyrosine (Y782), preventing gephyrin binding. Expression of Nlg1 point mutants in neurons indicated that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and accordingly the formation of inhibitory and excitatory synapses. We propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.

Funding information:
  • NIGMS NIH HHS - P41 GM104603(United States)
  • NINDS NIH HHS - NS080108(United States)

mSYD1A, a mammalian synapse-defective-1 protein, regulates synaptogenic signaling and vesicle docking.

  • Wentzel C
  • Neuron
  • 2013 Jun 19

Literature context:


Abstract:

Structure and function of presynaptic terminals are critical for the transmission and processing of neuronal signals. Trans-synaptic signaling systems instruct the differentiation and function of presynaptic release sites, but their downstream mediators are only beginning to be understood. Here, we identify the intracellular mSYD1A (mouse Synapse-Defective-1A) as a regulator of presynaptic function in mice. mSYD1A forms a complex with presynaptic receptor tyrosine phosphatases and controls tethering of synaptic vesicles at synapses. mSYD1A function relies on an intrinsically disordered domain that interacts with multiple structurally unrelated binding partners, including the active zone protein liprin-α2 and nsec1/munc18-1. In mSYD1A knockout mice, synapses assemble in normal numbers but there is a significant reduction in synaptic vesicle docking at the active zone and an impairment of synaptic transmission. Thus, mSYD1A is a regulator of presynaptic release sites at central synapses.

A tetra(ethylene glycol) derivative of benzothiazole aniline enhances Ras-mediated spinogenesis.

  • Megill A
  • J. Neurosci.
  • 2013 May 29

Literature context:


Abstract:

The tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG4, is a novel amyloid-binding small molecule that can penetrate the blood-brain barrier and protect cells from Aβ-induced toxicity. However, the effects of Aβ-targeting molecules on other cellular processes, including those that modulate synaptic plasticity, remain unknown. We report here that BTA-EG4 decreases Aβ levels, alters cell surface expression of amyloid precursor protein (APP), and improves memory in wild-type mice. Interestingly, the BTA-EG4-mediated behavioral improvement is not correlated with LTP, but with increased spinogenesis. The higher dendritic spine density reflects an increase in the number of functional synapses as determined by increased miniature EPSC (mEPSC) frequency without changes in presynaptic parameters or postsynaptic mEPSC amplitude. Additionally, BTA-EG4 requires APP to regulate dendritic spine density through a Ras signaling-dependent mechanism. Thus, BTA-EG4 may provide broad therapeutic benefits for improving neuronal and cognitive function, and may have implications in neurodegenerative disease therapy.

Funding information:
  • NHGRI NIH HHS - R01HG005058(United States)

A Myosin Va mutant mouse with disruptions in glutamate synaptic development and mature plasticity in visual cortex.

  • Yoshii A
  • J. Neurosci.
  • 2013 May 8

Literature context:


Abstract:

Myosin Va (MyoVa) mediates F-actin-based vesicular transport toward the plasma membrane and is found at neuronal postsynaptic densities (PSDs), but the role of MyoVa in synaptic development and function is largely unknown. Here, in studies using the dominant-negative MyoVa neurological mutant mouse Flailer, we find that MyoVa plays an essential role in activity-dependent delivery of PSD-95 and other critical PSD molecules to synapses and in endocytosis of AMPA-type glutamate receptors (AMPAR) in the dendrites of CNS neurons. MyoVa is known to carry a complex containing the major scaffolding proteins of the mature PSD, PSD-95, SAPAP1/GKAP, Shank, and Homer to dendritic spine synapses. In Flailer, neurons show abnormal dendritic shaft localization of PSD-95, stargazin, dynamin3, AMPARs and abnormal spine morphology. Flailer neurons also have abnormally high AMPAR miniature current frequencies and spontaneous AMPAR currents that are more frequent and larger than in wild-type while numbers of NMDAR containing synapses remain normal. The AMPAR abnormalities are consistent with a severely disrupted developmental regulation of long-term depression that we find in cortical Flailer neurons. Thus MyoVa plays a fundamentally important role both in localizing mature glutamate synapses to spines and in organizing the synapse for normal function. For this reason Flailer mice will be valuable in further dissecting the role of MyoVa in normal synaptic and circuit refinement and also in studies of neurological and neuropsychiatric diseases where disruptions of normal glutamate synapses are frequently observed.

Neuroligin1 drives synaptic and behavioral maturation through intracellular interactions.

  • Hoy JL
  • J. Neurosci.
  • 2013 May 29

Literature context:


Abstract:

In vitro studies suggest that the intracellular C terminus of Neuroligin1 (NL1) could play a central role in the maturation of excitatory synapses. However, it is unknown how this activity affects synapses in vivo, and whether it may impact the development of complex behaviors. To determine how NL1 influences the state of glutamatergic synapses in vivo, we compared the synaptic and behavioral phenotypes of mice overexpressing a full-length version of NL1 (NL1FL) with mice overexpressing a version missing part of the intracellular domain (NL1ΔC). We show that overexpression of full-length NL1 yielded an increase in the proportion of synapses with mature characteristics and impaired learning and flexibility. In contrast, the overexpression of NL1ΔC increased the number of excitatory postsynaptic structures and led to enhanced flexibility in mnemonic and social behaviors. Transient overexpression of NL1FL revealed that elevated levels are not necessary to maintain synaptic and behavioral states altered earlier in development. In contrast, overexpression of NL1FL in the fully mature adult was able to impair normal learning behavior after 1 month of expression. These results provide the first evidence that NL1 significantly impacts key developmental processes that permanently shape circuit function and behavior, as well as the function of fully developed neural circuits. Overall, these manipulations of NL1 function illuminate the significance of NL1 intracellular signaling in vivo, and enhance our understanding of the factors that gate the maturation of glutamatergic synapses and complex behavior. This has significant implications for our ability to address disorders such as autism spectrum disorders.

Funding information:
  • Wellcome Trust - WT077008(United Kingdom)

PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95: quantitative characterization of interactions.

  • Møller TC
  • PLoS ONE
  • 2013 May 21

Literature context:


Abstract:

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present a quantitative characterization of the kinetics and affinity of interactions between GPCRs and one of the best characterized PDZ scaffold proteins, postsynaptic density protein 95 (PSD-95), using fluorescence polarization (FP) and surface plasmon resonance (SPR). By comparing these in vitro findings with colocalization of the full-length proteins in cells and with previous studies, we suggest that the range of relevant interactions might extend to interactions with K i = 450 µM in the in vitro assays. Within this range, we identify novel PSD-95 interactions with the chemokine receptor CXCR2, the neuropeptide Y receptor Y2, and four of the somatostatin receptors (SSTRs). The interaction with SSTR1 was further investigated in mouse hippocampal neurons, where we found a clear colocalization between the endogenously expressed proteins, indicating a potential for further investigation of the role of this interaction. The approach can easily be transferred to other receptors and scaffold proteins and this could help accelerate the discovery and quantitative characterization of GPCR-PDZ interactions.

Funding information:
  • NINDS NIH HHS - NS35224(United States)
  • NLM NIH HHS - 1R01LM010185-01(United States)

Integrin α3 is required for late postnatal stability of dendrite arbors, dendritic spines and synapses, and mouse behavior.

  • Kerrisk ME
  • J. Neurosci.
  • 2013 Apr 17

Literature context:


Abstract:

Most dendrite branches and a large fraction of dendritic spines in the adult rodent forebrain are stable for extended periods of time. Destabilization of these structures compromises brain function and is a major contributing factor to psychiatric and neurodegenerative diseases. Integrins are a class of transmembrane extracellular matrix receptors that function as αβ heterodimers and activate signaling cascades regulating the actin cytoskeleton. Here we identify integrin α3 as a key mediator of neuronal stability. Dendrites, dendritic spines, and synapses develop normally in mice with selective loss of integrin α3 in excitatory forebrain neurons, reaching their mature sizes and densities through postnatal day 21 (P21). However, by P42, integrin α3 mutant mice exhibit significant reductions in hippocampal dendrite arbor size and complexity, loss of dendritic spine and synapse densities, and impairments in hippocampal-dependent behavior. Furthermore, gene-dosage experiments demonstrate that integrin α3 interacts functionally with the Arg nonreceptor tyrosine kinase to activate p190RhoGAP, which inhibits RhoA GTPase and regulates hippocampal dendrite and synapse stability and mouse behavior. Together, our data support a fundamental role for integrin α3 in regulating dendrite arbor stability, synapse maintenance, and proper hippocampal function. In addition, these results provide a biochemical and structural explanation for the defects in long-term potentiation, learning, and memory reported previously in mice lacking integrin α3.

Sumoylated MEF2A coordinately eliminates orphan presynaptic sites and promotes maturation of presynaptic boutons.

  • Yamada T
  • J. Neurosci.
  • 2013 Mar 13

Literature context:


Abstract:

Presynaptic differentiation of axons plays a fundamental role in the establishment of neuronal connectivity. However, the mechanisms that govern presynaptic differentiation in the brain remain largely to be elucidated. We report that knockdown of the transcription factor MEF2A in primary neurons and importantly in the rat cerebellar cortex in vivo robustly increases the density of orphan presynaptic sites. Remarkably, the sumoylated transcriptional repressor form of MEF2A drives the suppression of orphan presynaptic sites. We also identify the gene encoding synaptotagmin 1 (Syt1), which acts locally at presynaptic sites, as a direct repressed target gene of sumoylated MEF2A in neurons, and demonstrate that repression of Syt1 mediates MEF2A-dependent elimination of orphan presynaptic sites. Finally, we uncover a role for the MEF2A-induced elimination of orphan presynaptic sites in the accumulation of presynaptic material at large maturing presynaptic boutons. Collectively, these findings define sumoylated MEF2A and Syt1 as components of a novel cell-intrinsic mechanism that orchestrates presynaptic differentiation in the mammalian brain. Our study has important implications for understanding neuronal connectivity in brain development and disease.

Funding information:
  • Intramural NIH HHS - (United States)

Postsynaptic density scaffold SAP102 regulates cortical synapse development through EphB and PAK signaling pathway.

  • Murata Y
  • J. Neurosci.
  • 2013 Mar 13

Literature context:


Abstract:

Membrane-associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93, and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor (AMPAR) localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 coimmunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation, and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Last, p21-activated kinases (PAKs) were downregulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development.

The kinase activity of EphA4 mediates homeostatic scaling-down of synaptic strength via activation of Cdk5.

  • Peng YR
  • Neuropharmacology
  • 2013 Feb 12

Literature context:


Abstract:

Neurons within a network have the ability to homeostatically scale-down their excitatory synaptic strength under conditions of persistent neuronal activity elevation, a process pivotal to neural circuit stability. How this homeostatic regulation is achieved at the molecular level in developing neural circuits, which face gradually elevated neuronal activity as part of circuit wiring, is not well-understood. Using dissociated hippocampal neuronal cultures, we identified a critical and cell autonomous role for the receptor tyrosine kinase EphA4 in mediating activity-induced homeostatic down-regulation of excitatory synaptic strength. Reducing the endogenous level of EphA4 in individual neurons by RNAi effectively blocked activity-induced scaling-down of excitatory synaptic strength, while co-transfection of RNAi resistant EphA4 rescued this effect. Furthermore, interfering with EphA4 forward signaling using EphA4-Fc blocked activity-induced homeostatic synaptic scaling-down, while direct activation of EphA4 with its ligand EphrinA1 weakened excitatory synaptic strength. Up- or down-regulating EphA4 function in individual neurons also did not affect the density of excitatory synapses. The kinase activities of EphA4 and its downstream effector Cdk5 were both required for homeostatic synaptic scaling, as overexpression of EphA4 with constitutively active kinase activity reduced excitatory synaptic strength, while interfering with either the kinase activity of EphA4 or Cdk5 blocked activity-induced synaptic scaling. Consistently, the activities of EphA4 and Cdk5 increased significantly during global and persistent activity elevation. Together, our work demonstrated that the kinase activity of EphA4, via activation of downstream Cdk5 activity, mediates the scaling-down of excitatory synaptic strength under conditions of global activity elevation.

RNA-binding protein Sam68 controls synapse number and local β-actin mRNA metabolism in dendrites.

  • Klein ME
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2013 Feb 19

Literature context:


Abstract:

Proper synaptic function requires the spatial and temporal compartmentalization of RNA metabolism via transacting RNA-binding proteins (RBPs). Loss of RBP activity leads to abnormal posttranscriptional regulation and results in diverse neurological disorders with underlying deficits in synaptic morphology and transmission. Functional loss of the 68-kDa RBP Src associated in mitosis (Sam68) is associated with the pathogenesis of the neurological disorder fragile X tremor/ataxia syndrome. Sam68 binds to the mRNA of β-actin (actb), an integral cytoskeletal component of dendritic spines. We show that Sam68 knockdown or disruption of the binding between Sam68 and its actb mRNA cargo in primary hippocampal cultures decreases the amount of actb mRNA in the synaptodendritic compartment and results in fewer dendritic spines. Consistent with these observations, we find that Sam68-KO mice have reduced levels of actb mRNA associated with synaptic polysomes and diminished levels of synaptic actb protein, suggesting that Sam68 promotes the translation of actb mRNA at synapses in vivo. Moreover, genetic knockout of Sam68 or acute knockdown in vivo results in fewer excitatory synapses in the hippocampal formation as assessed morphologically and functionally. Therefore, we propose that Sam68 regulates synapse number in a cell-autonomous manner through control of postsynaptic actb mRNA metabolism. Our research identifies a role for Sam68 in synaptodendritic posttranscriptional regulation of actb and may provide insight into the pathophysiology of fragile X tremor/ataxia syndrome.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/G022623/1(United Kingdom)

Peroxisome proliferators reduce spatial memory impairment, synaptic failure, and neurodegeneration in brains of a double transgenic mice model of Alzheimer's disease.

  • Inestrosa NC
  • J. Alzheimers Dis.
  • 2013 Jan 23

Literature context:


Abstract:

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, accumulation of the amyloid-β peptide (Aβ), increase of oxidative stress, and synaptic alterations. The scavenging of reactive oxygen species through their matrix enzyme catalase is one of the most recognized functions of peroxisomes. The induction of peroxisome proliferation is attained through different mechanisms by a set of structurally diverse molecules called peroxisome proliferators. In the present work, a double transgenic mouse model of AD that co-expresses a mutant human amyloid-β protein precursor (AβPPswe) and presenilin 1 without exon 9 (PS1dE9) was utilized in order to assess the effect of peroxisomal proliferation on Aβ neurotoxicity in vivo. Mice were tested for spatial memory and their brains analyzed by cytochemical, electrophysiological, and biochemical methods. We report here that peroxisomal proliferation significantly reduces (i) memory impairment, found in this model of AD; (ii) Aβ burden and plaque-associated acetylcholinesterase activity; (iii) neuroinflammation, measured by the extent of astrogliosis and microgliosis; and (iv) the decrease in postsynaptic proteins, while promoting synaptic plasticity in the form of long-term potentiation. We concluded that peroxisomal proliferation reduces various AD neuropathological markers and peroxisome proliferators may be considered as potential therapeutic agents against the disease.

Funding information:
  • Canadian Institutes of Health Research - JNM-90959(Canada)

Eye opening and PSD95 are required for long-term potentiation in developing superior colliculus.

  • Zhao JP
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2013 Jan 8

Literature context:


Abstract:

The only major glutamate receptor membrane-associated guanylate kinase scaffolds expressed in the young superficial superior colliculus (SC) are synapse-associated protein 102 (SAP102) and postsynaptic density protein 95 (PSD95). In this, as in all visual brain regions examined, synaptic PSD95 increases rapidly following simultaneous eyelid opening (EO). We show that EO and PSD95 are necessary for SC NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and this LTP is eliminated or reinstated by manipulating EO. PSD95 knockdown (KD) in vivo blocks this LTP, but not long-term depression, and reduces frequencies of miniature AMPA receptor and NMDAR currents with no change in presynaptic release. Furthermore, miniature NMDAR currents after PSD95 KD show an activity-triggered calcineurin sensitivity that is normally only found in the pre-EO period when SAP102 binds mixed GluN2A/GluN2B NMDARs. These data indicate that young SC LTP arises from PSD95 unsilencing of silent synapses, that unsilencing is labile in young brain, and that even though SAP102 and PSD95 can bind the same NMDARs, only PSD95 enables SC synaptic maturation.

Funding information:
  • NIGMS NIH HHS - GM070064(United States)

Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons.

  • Loriol C
  • Biol. Cell
  • 2013 Jan 2

Literature context:


Abstract:

BACKGROUND INFORMATION: Sumoylation is a key post-translational modification by which the Small Ubiquitin-like MOdifier (SUMO) polypeptide is covalently attached to specific lysine residues of substrate proteins through a specific enzymatic pathway. Although sumoylation participates in the regulation of nuclear homeostasis, the sumoylation machinery is also expressed outside of the nucleus where little is still known regarding its non-nuclear functions, particularly in the Central Nervous System (CNS). We recently reported that the sumoylation process is developmentally regulated in the rat CNS. RESULTS: Here, we demonstrate that there is an activity-dependent redistribution of endogenous sumoylation enzymes in hippocampal neurons. By performing biochemical and immunocytochemical experiments on primary cultures of rat hippocampal neurons, we show that sumoylation and desumoylation enzymes are differentially redistributed in and out of synapses upon neuronal stimulation. This enzymatic redistribution in response to a neuronal depolarisation results in the transient decrease of sumoylated protein substrates at synapses. CONCLUSIONS: Taken together, our data identify an activity-dependent regulation of the sumoylation machinery in neurons that directly impacts on synaptic sumoylation levels. This process may provide a mechanism for neurons to adapt their physiological responses to changes occurring during neuronal activation.

Funding information:
  • NHGRI NIH HHS - R01-HG-004885(United States)
  • NIAMS NIH HHS - AR059311(United States)

Developmental up-regulation of vesicular glutamate transporter-1 promotes neocortical presynaptic terminal development.

  • Berry CT
  • PLoS ONE
  • 2012 Dec 11

Literature context:


Abstract:

Presynaptic terminal formation is a complex process that requires assembly of proteins responsible for synaptic transmission at sites of axo-dendritic contact. Accumulation of presynaptic proteins at developing terminals is facilitated by glutamate receptor activation. Glutamate is loaded into synaptic vesicles for release via the vesicular glutamate transporters VGLUT1 and VGLUT2. During postnatal development there is a switch from predominantly VGLUT2 expression to high VGLUT1 and low VGLUT2, raising the question of whether the developmental increase in VGLUT1 is important for presynaptic development. Here, we addressed this question using confocal microscopy and quantitative immunocytochemistry in primary cultures of rat neocortical neurons. First, in order to understand the extent to which the developmental switch from VGLUT2 to VGLUT1 occurs through an increase in VGLUT1 at individual presynaptic terminals or through addition of VGLUT1-positive presynaptic terminals, we examined the spatio-temporal dynamics of VGLUT1 and VGLUT2 expression. Between 5 and 12 days in culture, the percentage of presynaptic terminals that expressed VGLUT1 increased during synapse formation, as did expression of VGLUT1 at individual terminals. A subset of VGLUT1-positive terminals also expressed VGLUT2, which decreased at these terminals. At individual terminals, the increase in VGLUT1 correlated with greater accumulation of other synaptic vesicle proteins, such as synapsin and synaptophysin. When the developmental increase in VGLUT1 was prevented using VGLUT1-shRNA, the density of presynaptic terminals and accumulation of synapsin and synaptophysin at terminals were decreased. Since VGLUT1 knock-down was limited to a small number of neurons, the observed effects were cell-autonomous and independent of changes in overall network activity. These results demonstrate that up-regulation of VGLUT1 is important for development of presynaptic terminals in the cortex.

Funding information:
  • PHS HHS - NIH-NINDS R37-31146(United States)

GKAP orchestrates activity-dependent postsynaptic protein remodeling and homeostatic scaling.

  • Shin SM
  • Nat. Neurosci.
  • 2012 Dec 28

Literature context:


Abstract:

How does chronic activity modulation lead to global remodeling of proteins at synapses and synaptic scaling? Here we report that guanylate kinase-associated protein (GKAP; also known as SAPAP), a scaffolding molecule linking NMDA receptor-PSD-95 to Shank-Homer complexes, acts in these processes. Overexcitation removes GKAP from synapses via the ubiquitin-proteasome system, whereas inactivity induces synaptic accumulation of GKAP in rat hippocampal neurons. Bidirectional changes in synaptic GKAP amounts are controlled by specific CaMKII isoforms coupled to different Ca(2+) channels. CaMKIIα activated by the NMDA receptor phosphorylates GKAP Ser54 to induce polyubiquitination of GKAP. In contrast, CaMKIIβ activation via L-type voltage-dependent calcium channels promotes GKAP recruitment by phosphorylating GKAP Ser340 and Ser384, which uncouples GKAP from myosin Va motor complex. Overexpressing GKAP turnover mutants not only hampers activity-dependent remodeling of PSD-95 and Shank but also blocks bidirectional synaptic scaling. Therefore, activity-dependent turnover of PSD proteins orchestrated by GKAP is critical for homeostatic plasticity.

Funding information:
  • NHGRI NIH HHS - P41HG004118(United States)

Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin.

  • Shipman SL
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2012 Nov 20

Literature context:


Abstract:

The transsynaptic complex of neuroligin (NLGN) and neurexin forms a physical connection between pre- and postsynaptic neurons that occurs early in the course of new synapse assembly. Both neuroligin and neurexin have, indeed, been proposed to exhibit active, instructive roles in the formation of synapses. However, the process by which these instructive roles play out during synaptogenesis is not well understood. Here, we examine one aspect of postsynaptic neuroligin with regard to its synaptogenic properties: its basal state as a constitutive dimer. We show that dimerization is required for the synaptogenic properties of neuroligin and likely serves to induce presynaptic differentiation via a transsynaptic clustering of neurexin. Further, we introduce chemically inducible, exogenous dimerization domains to the neuroligin molecule, effectively bestowing chemical control of neuroligin dimerization. This allows us to identify the acute requirements of neuroligin dimerization by chemically manipulating the monomeric-to-dimeric conversion of neuroligin. Based on the results of the inducible dimerization experiments, we propose a model in which dimerized neuroligin induces the mechanical clustering of presynaptic molecules as part of a requisite step in the coordinated assembly of a chemical synapse.

Funding information:
  • NIMH NIH HHS - 5R01MH070370(United States)

PSD-95 expression controls L-DOPA dyskinesia through dopamine D1 receptor trafficking.

  • Porras G
  • J. Clin. Invest.
  • 2012 Nov 1

Literature context:


Abstract:

L-DOPA-induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson's disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson's patients.

Funding information:
  • NIAAA NIH HHS - AA05524(United States)

Microtubule plus-end tracking protein CLASP2 regulates neuronal polarity and synaptic function.

  • Beffert U
  • J. Neurosci.
  • 2012 Oct 3

Literature context:


Abstract:

Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The short-hairpin RNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length, whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number, and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between microtubule stabilization and synapse formation and activity.

5-hydroxytryptamine 2C receptors tonically augment synaptic currents in the nucleus tractus solitarii.

  • Austgen JR
  • J. Neurophysiol.
  • 2012 Oct 16

Literature context:


Abstract:

The nucleus tractus solitarii (nTS) is the primary termination and integration point for visceral afferents in the brain stem. Afferent glutamate release and its efficacy on postsynaptic activity within this nucleus are modulated by additional neuromodulators and transmitters, including serotonin (5-HT) acting through its receptors. The 5-HT(2) receptors in the medulla modulate the cardiorespiratory system and autonomic reflexes, but the distribution of the 5-HT(2C) receptor and the role of these receptors during synaptic transmission in the nTS remain largely unknown. In the present study, we examined the distribution of 5-HT(2C) receptors in the nTS and their role in modulating excitatory postsynaptic currents (EPSCs) in monosynaptic nTS neurons in the horizontal brain stem slice. Real-time RT-PCR and immunohistochemistry identified 5-HT(2C) receptor message and protein in the nTS and suggested postsynaptic localization. In nTS neurons innervated by general visceral afferents, 5-HT(2C) receptor activation increased solitary tract (TS)-EPSC amplitude and input resistance and depolarized membrane potential. Conversely, 5-HT(2C) receptor blockade reduced TS-EPSC and miniature EPSC amplitude, as well as input resistance, and hyperpolarized membrane potential. Synaptic parameters in nTS neurons that receive sensory input from carotid body chemoafferents were also attenuated by 5-HT(2C) receptor blockade. Taken together, these data suggest that 5-HT(2C) receptors in the nTS are located postsynaptically and augment excitatory neurotransmission.

AKAP150-anchored calcineurin regulates synaptic plasticity by limiting synaptic incorporation of Ca2+-permeable AMPA receptors.

  • Sanderson JL
  • J. Neurosci.
  • 2012 Oct 24

Literature context:


Abstract:

AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca(2+) influx. However, a small number of Ca(2+)-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca(2+) signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca(2+)-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca(2+)-permeable AMPARs both basally and during LTP and LTD.

Funding information:
  • NIGMS NIH HHS - GM088076(United States)

Selective decline of synaptic protein levels in the frontal cortex of female mice deficient in the extracellular metalloproteinase ADAMTS1.

  • Howell MD
  • PLoS ONE
  • 2012 Oct 16

Literature context:


Abstract:

The chondroitin sulfate-bearing proteoglycans, also known as lecticans, are a major component of the extracellular matrix (ECM) in the central nervous system and regulate neural plasticity. Growing evidence indicates that endogenous, extracellular metalloproteinases that cleave lecticans mediate neural plasticity by altering the structure of ECM aggregates. The bulk of this in vivo data examined the matrix metalloproteinases, but another metalloproteinase family that cleaves lecticans, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), modulates structural plasticity in vitro, although few in vivo studies have tested this concept. Thus, the purpose of this study was to examine the neurological phenotype of a mouse deficient in ADAMTS1. Adamts1 mRNA was absent in the ADAMTS1 null mouse frontal cortex, but there was no change in the abundance or proteolytic processing of the prominent lecticans brevican and versican V2. However, there was a marked increase in the perinatal lectican neurocan in juvenile ADAMTS1 null female frontal cortex. More prominently, there were declines in synaptic protein levels in the ADAMTS1 null female, but not male, frontal cortex beginning at postnatal day 28. These synaptic marker declines did not affect learning or memory in the adult female ADAMTS1 null mice when tested with the radial-arm water maze. These results indicate that in vivo Adamts1 knockout leads to sexual dimorphism in frontal cortex synaptic protein levels. Since changes in lectican abundance and proteolytic processing did not accompany the synaptic protein declines, ADAMTS1 may play a nonproteolytic role in regulating neural plasticity.

Funding information:
  • NIGMS NIH HHS - GM59507(United States)

Afadin is required for maintenance of dendritic structure and excitatory tone.

  • Srivastava DP
  • J. Biol. Chem.
  • 2012 Oct 19

Literature context:


Abstract:

The dendritic field of a neuron, which is determined by both dendritic architecture and synaptic strength, defines the synaptic input of a cell. Once established, a neuron's dendritic field is thought to remain relatively stable throughout a cell's lifetime. Perturbations in a dendritic structure or excitatory tone of a cell and thus its dendritic field are cellular alterations thought to be correlated with a number of psychiatric disorders. Although several proteins are known to regulate the development of dendritic arborization, much less is known about the mechanisms that maintain dendritic morphology and synaptic strength. In this study, we find that afadin, a component of N-cadherin·β-catenin·α-N-catenin adhesion complexes, is required for the maintenance of established dendritic arborization and synapse number. We further demonstrate that afadin directly interacts with AMPA receptors and that loss of this protein reduces the surface expression of GluA1- and GluA2-AMPA receptor subunits. Collectively, these data suggest that afadin is required for the maintenance of dendritic structure and excitatory tone.

Funding information:
  • Wellcome Trust - 085532(United Kingdom)

CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons.

  • Ricciardi S
  • Nat. Cell Biol.
  • 2012 Sep 5

Literature context:


Abstract:

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

Funding information:
  • Wellcome Trust - 076113(United Kingdom)

Chronic ethanol up-regulates the synaptic expression of the nuclear translational regulatory protein AIDA-1 in primary hippocampal neurons.

  • Mulholland PJ
  • Alcohol
  • 2012 Sep 3

Literature context:


Abstract:

Recent studies have identified synaptic proteins that undergo synapse-to-nucleus translocation in response to neuronal activity that modulate protein synthesis. One such translational regulatory protein of the postsynaptic density (PSD) is AIDA-1d, which binds to PSD-95 via its C-terminus. Activation of synaptic NMDA receptors induces the cleavage of AIDA-1d, and the N-terminus is then shuttled to nuclear Cajal bodies where it plays a role in regulating global protein synthesis. Chronic ethanol exposure has been shown to increase the synaptic clustering of NMDA receptors and PSD-95. Here, we tested the hypothesis that AIDA-1d regulates chronic ethanol-induced increases in synaptic NMDA receptor expression. As reported, we found that AIDA-1 was highly enriched in dendritic spines and co-localized with PSD-95. Acute NMDA treatment increased AIDA-1 colocalization with p80 coilin, a marker of Cajal bodies. Chronic treatment (4 day) of cultures with ethanol (25-100 mM) or with the NMDA receptor antagonist AP-V (50 μM) enhanced the clustering of AIDA-1 at synaptic sites. However, chronic ethanol treatment (50 mM) in the presence of the NMDA receptor agonist NMDA (2.5 μM) prevented this increase. Surprisingly, PSD-95 did not seem to play a role in the synaptic distribution of AIDA-1 as this distribution was not affected by declustering PSD-95 from synapses in response to inhibition of palmitoylation. We found that lentiviral knockdown of AIDA-1d did not affect protein expression levels of NMDA receptor subunits GluN1, GluN1 C2', or GluN2B. The results of this study demonstrate that synaptic AIDA-1 expression is enhanced by chronic ethanol exposure that can be prevented by concurrent stimulation of NMDA receptors. In addition, we found that the association of AIDA-1 with PSD-95 is not required for its localization to the PSD. Moreover, we found that AIDA-1 does not regulate protein expression levels or alternative splicing of the GluN1 subunit of NMDA receptors.

Funding information:
  • NIDA NIH HHS - R01DA030304(United States)

Social, communication, and cortical structural impairments in Epac2-deficient mice.

  • Srivastava DP
  • J. Neurosci.
  • 2012 Aug 22

Literature context:


Abstract:

Deficits in social and communication behaviors are common features of a number of neurodevelopmental disorders. However, the molecular and cellular substrates of these higher order brain functions are not well understood. Here we report that specific alterations in social and communication behaviors in mice occur as a result of loss of the EPAC2 gene, which encodes a protein kinase A-independent cAMP target. Epac2-deficient mice exhibited robust deficits in social interactions and ultrasonic vocalizations, but displayed normal olfaction, working and reference memory, motor abilities, anxiety, and repetitive behaviors. Epac2-deficient mice displayed abnormal columnar organization in the anterior cingulate cortex, a region implicated in social behavior in humans, but not in somatosensory cortex. In vivo two-photon imaging revealed reduced dendritic spine motility and density on cortical neurons in Epac2-deficient mice, indicating deficits at the synaptic level. Together, these findings provide novel insight into the molecular and cellular substrates of social and communication behavior.

Funding information:
  • NIGMS NIH HHS - R01-GM083871(United States)

MeCP2 is critical for maintaining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain.

  • Nguyen MV
  • J. Neurosci.
  • 2012 Jul 18

Literature context:


Abstract:

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects approximately one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/β, AMPA, and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

Funding information:
  • PHS HHS - 107-360(United States)

Activity maintains structural plasticity of mossy fiber terminals in the hippocampus.

  • Chierzi S
  • Mol. Cell. Neurosci.
  • 2012 Jul 13

Literature context:


Abstract:

Neural activity plays an important role in organizing and optimizing neural circuits during development and in the mature nervous system. However, the cellular events that underlie this process still remain to be fully understood. In this study, we investigated the role of neural activity in regulating the structural plasticity of presynaptic terminals in the hippocampal formation. We designed a virus to drive the Drosophila Allatostatin receptor in individual dentate granule neurons to suppress activity of complex mossy fiber terminals 'on-demand' in organotypic slices and used time-lapse confocal imaging to determine the impact on presynaptic remodeling. We found that activity played an important role in maintaining the structural plasticity of the core region of the mossy fiber terminal (MFT) that synapses onto CA3 pyramidal cell thorny excrescences but was not essential for the motility of terminal filopodial extensions that contact local inhibitory neurons. Short-term suppression of activity did not have an impact on the size of the MFT, however, longer-term suppression reduced the overall size of the MFT. Remarkably, global blockade of activity with tetrodotoxin (TTX) interfered with the ability of single cell activity deprivation to slow down terminal dynamics suggesting that differences in activity levels among neighboring synapses promote synaptic remodeling events. The results from our studies indicate that neural activity plays an important role in maintaining structural plasticity of presynaptic compartments in the central nervous system and provide new insight into the time-frame during which activity can affect the morphology of synaptic connections.

Funding information:
  • NHGRI NIH HHS - U54 HG006493(United States)

Early formation of GABAergic synapses governs the development of adult-born neurons in the olfactory bulb.

  • Pallotto M
  • J. Neurosci.
  • 2012 Jun 27

Literature context:


Abstract:

In mammals, olfactory bulb granule cells (GCs) are generated throughout life in the subventricular zone. GABAergic inputs onto newborn neurons likely regulate their maturation, but the details of this process remain still elusive. Here, we investigated the differentiation, synaptic integration, and survival of adult-born GCs when their afferent GABAergic inputs are challenged by conditional gene targeting. Migrating GC precursors were targeted with Cre-eGFP-expressing lentiviral vectors in mice with a floxed gene encoding the GABA(A) receptor α2-subunit (i.e., Gabra2). Ablation of the α2-subunit did not affect GC survival but dramatically delayed their maturation. We found a reduction in postsynaptic α2-subunit and gephyrin clusters accompanied by a decrease in the frequency and amplitude of GABAergic postsynaptic currents beginning ∼14 d post-injection (dpi). In addition, mutant cells exhibited altered dendritic branching and spine density. Spine loss appeared with mislocation of glutamatergic synapses on dendritic shafts and a reduction of spontaneous glutamatergic postsynaptic currents, underscoring the relevance of afferent GABAergic transmission for a proper synaptic integration of newborn GCs. To test the role of GABAergic signaling during much early stages of GC maturation, we used a genetic strategy to selectively inactivate Gabra2 in precursor cells of the subventricular zone. In these mice, labeling of newborn GCs with eGFP lentiviruses revealed similar morphological alterations as seen on delayed Gabra2 inactivation in migrating neuroblasts, with reduced dendritic branching and spine density at 7 dpi. Collectively, these results emphasize the critical role of GABAergic synaptic signaling for structural maturation of adult-born GCs and formation of glutamatergic synapses.

Funding information:
  • NIGMS NIH HHS - GM089662(United States)

β2-Adrenergic receptor supports prolonged theta tetanus-induced LTP.

  • Qian H
  • J. Neurophysiol.
  • 2012 May 16

Literature context:


Abstract:

The widespread noradrenergic innervation in the brain promotes arousal and learning by molecular mechanisms that remain largely undefined. Recent work shows that the β(2)-adrenergic receptor (β(2)AR) is linked to the AMPA-type glutamate receptor subunit GluA1 via stargazin and PSD-95 (Joiner ML, Lise MF, Yuen EY, Kam AY, Zhang M, Hall DD, Malik ZA, Qian H, Chen Y, Ulrich JD, Burette AC, Weinberg RJ, Law PY, El-Husseini A, Yan Z, Hell JW. EMBO J 29: 482-495, 2010). We now demonstrate that the β(2)AR plays a prominent role in long-term potentiation (LTP) induced by a train of 900 stimuli at 5 Hz (prolonged theta-tetanus-LTP, or PTT-LTP) in the hippocampal CA1 region in mice, which requires simultaneous β-adrenergic stimulation. Although PTT-LTP was impaired in hippocampal slices from β(1)AR and β(2)AR knockout (KO) mice, only β(2)AR-selective stimulation with salbutamol supported this PTT-LTP in wild-type (WT) slices, whereas β(1)AR-selective stimulation with dobutamine (+ prazosin) did not. Furthermore, only the β(2)AR-selective antagonist ICI-118551 and not the β(1)AR-selective antagonist CGP-20712 inhibited PTT-LTP and phosphorylation of GluA1 on its PKA site S845 in WT slices. Our analysis of S845A knockin (KI) mice indicates that this phosphorylation is relevant for PTT-LTP. These results identify the β(2)AR-S845 signaling pathway as a prominent regulator of synaptic plasticity.

Funding information:
  • Wellcome Trust - 087618/Z/08/Z(United Kingdom)

Palmitoylation of A-kinase anchoring protein 79/150 regulates dendritic endosomal targeting and synaptic plasticity mechanisms.

  • Keith DJ
  • J. Neurosci.
  • 2012 May 23

Literature context:


Abstract:

NMDA receptor-dependent long-term potentiation (LTP) and depression (LTD) are forms of synaptic plasticity underlying learning and memory that are expressed through increases and decreases, respectively, in dendritic spine size and AMPA receptor (AMPAR) phosphorylation and postsynaptic localization. The A-kinase anchoring protein 79/150 (AKAP79/150) signaling scaffold regulates AMPAR phosphorylation, channel activity, and endosomal trafficking associated with LTP and LTD. AKAP79/150 is targeted to dendritic spine plasma membranes by an N-terminal polybasic domain that binds phosphoinositide lipids, F-actin, and cadherin cell adhesion molecules. However, we do not understand how regulation of AKAP targeting controls AMPAR endosomal trafficking. Here, we report that palmitoylation of the AKAP N-terminal polybasic domain targets it to postsynaptic lipid rafts and dendritic recycling endosomes. AKAP palmitoylation was regulated by seizure activity in vivo and LTP/LTD plasticity-inducing stimuli in cultured rat hippocampal neurons. With chemical LTP induction, we observed AKAP79 dendritic spine recruitment that required palmityolation and Rab11-regulated endosome recycling coincident with spine enlargement and AMPAR surface delivery. Importantly, a palmitoylation-deficient AKAP79 mutant impaired regulation of spine size, endosome recycling, AMPAR trafficking, and synaptic potentiation. These findings emphasize the emerging importance of palmitoylation in controlling synaptic function and reveal novel roles for the AKAP79/150 signaling complex in dendritic endosomes.

Funding information:
  • NCRR NIH HHS - P40-RR-17072(United States)

WIP is a negative regulator of neuronal maturation and synaptic activity.

  • Franco A
  • Cereb. Cortex
  • 2012 May 18

Literature context:


Abstract:

Wiskott-Aldrich syndrome protein (WASP) -interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP(-)(/-) hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP(-)(/-) neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity.

Funding information:
  • NIDDK NIH HHS - R24 DK090962(United States)

S-SCAM/MAGI-2 is an essential synaptic scaffolding molecule for the GluA2-containing maintenance pool of AMPA receptors.

  • Danielson E
  • J. Neurosci.
  • 2012 May 16

Literature context:


Abstract:

Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved despite protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that the synaptic scaffolding molecule (S-SCAM; also called membrane-associated guanylate kinase inverted-2 and atrophin interacting protein-1) plays the critical role of maintaining synaptic strength. Increasing S-SCAM levels in rat hippocampal neurons led to specific increases in the surface AMPAR levels, enhanced AMPAR-mediated synaptic transmission, and enlargement of dendritic spines, without significantly effecting GluN levels or NMDA receptor (NMDAR) EPSC. Conversely, decreasing S-SCAM levels by RNA interference-mediated knockdown caused the loss of synaptic AMPARs, which was followed by a severe reduction in the dendritic spine density. Importantly, S-SCAM regulated synaptic AMPAR levels in a manner, dependent on GluA2 not GluA1, sensitive to N-ethylmaleimide-sensitive fusion protein interaction, and independent of activity. Further, S-SCAM increased surface AMPAR levels in the absence of PSD-95, while PSD-95 was dependent on S-SCAM to increase surface AMPAR levels. Finally, S-SCAM overexpression hampered NMDA-induced internalization of AMPARs and prevented the induction of long term-depression, while S-SCAM knockdown did not. Together, these results suggest that S-SCAM is an essential AMPAR scaffolding molecule for the GluA2-containing pool of AMPARs, which are involved in the constitutive pathway of maintaining synaptic strength.

Funding information:
  • NIMH NIH HHS - MH059490(United States)

Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex.

  • Kononenko NL
  • Hippocampus
  • 2012 Apr 22

Literature context:


Abstract:

Navigation is mediated by a network of brain areas, and research has focused on the head-direction system in the presubiculum (PrS), the grid cell containing medial entorhinal cortex (EC) (MEC) and place cells in the hippocampus. Less research addressed the interactions of the retrosplenial cortex (RSC) and the navigational system, although it is well established that damage to the RSC leads to navigational deficits. We previously showed that RSC provides a dense input to deep layers of MEC and to superficial layers of PrS. In this study we use confocal microscopical analysis and show that the dense projection from the caudal part of the ventral retrosplenial granular cortex targets neurons in Layer III of PrS, which provide input to superficial layers of MEC. Our high resolution anatomical data indicate that sparsely spiny pyramidal neurons in Layer III of PrS that originate projections to Layer III of MEC are the main target of these retrosplenial projections. Retrosplenial axonal boutons were found to equally contact spines and shafts of basal dendrites in Layer III, but contacts on shafts are more prominent close to the soma, indicating the potential for efficient synaptic transfer. These observations suggest that neurons in Layer III of PrS have an important role in mediating RSC contributions to navigation.

Funding information:
  • NIDA NIH HHS - R21 DA030118(United States)
  • NIDDK NIH HHS - R01 DK079005-04(United States)

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging.

  • Végh MJ
  • J. Proteome Res.
  • 2012 Mar 2

Literature context:


Abstract:

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.

Funding information:
  • NIH HHS - P51 OD011133(United States)

Facilitation of neocortical presynaptic terminal development by NMDA receptor activation.

  • Sceniak MP
  • Neural Dev
  • 2012 Feb 16

Literature context:


Abstract:

BACKGROUND: Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. RESULTS: Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR) activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. CONCLUSIONS: Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

Funding information:
  • NIDA NIH HHS - DA019114(United States)
  • NINDS NIH HHS - R01-NS082266(United States)

Altered synaptic marker abundance in the hippocampal stratum oriens of Ts65Dn mice is associated with exuberant expression of versican.

  • Howell MD
  • ASN Neuro
  • 2012 Feb 8

Literature context:


Abstract:

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.

Funding information:
  • NIDDK NIH HHS - R01 DK103723(United States)

A cis-complex of NB-2/contactin-5 with amyloid precursor-like protein 1 is localized on the presynaptic membrane.

  • Shimoda Y
  • Neurosci. Lett.
  • 2012 Feb 29

Literature context:


Abstract:

NB-2/contactin-5 plays an important role in synapse formation in the developing auditory system of rodents. In this study, to further elucidate the molecular role of NB-2 in synapse formation, we examined the interaction between NB-2 and amyloid precursor-like protein 1 (APLP1), as well as their possible co-localization at the synapse. Pull-down assays and cell surface binding assays demonstrated that NB-2 interacts with APLP1. Furthermore, the protein expression profile of APLP1 in western blots was similar to that of NB-2, and localization of APLP1 mRNA partially overlapped that of NB-2 mRNA. In cultured hippocampal neurons, immunofluorescence signals for both NB-2 and APLP1 overlapped with synapsin, a presynaptic marker. Biochemical analysis showed that both NB-2 and APLP1 were enriched in the presynaptic fraction. These results indicate that NB-2 forms a cis-complex with APLP1 on the presynaptic membrane.

Funding information:
  • NIGMS NIH HHS - R01 GM087517(United States)
  • NIMH NIH HHS - R56 MH081152(United States)

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology.

  • Berkel S
  • Hum. Mol. Genet.
  • 2012 Jan 15

Literature context:


Abstract:

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD.

Funding information:
  • NCI NIH HHS - 7R01CA137098(United States)
  • NHLBI NIH HHS - HL062571(United States)

The X-linked intellectual disability protein IL1RAPL1 regulates excitatory synapse formation by binding PTPδ and RhoGAP2.

  • Valnegri P
  • Hum. Mol. Genet.
  • 2011 Dec 15

Literature context:


Abstract:

Mutations of the Interleukin-1-receptor accessory protein like 1 (IL1RAPL1) gene are associated with cognitive impairment ranging from non-syndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of IL1/Toll receptors, which is localized at excitatory synapses and interacts with PSD-95. We previously showed that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun N-terminal kinase activity and PSD-95 phosphorylation. Here, we show that the IgG-like extracellular domains of IL1RAPL1 induce excitatory pre-synapse formation by interacting with protein tyrosine phosphatase delta (PTPδ). We also found that IL1RAPL1 TIR domains interact with RhoGAP2, which is localized at the excitatory post-synaptic density. More interestingly, the IL1RAPL1/PTPδ complex recruits RhoGAP2 at excitatory synapses to induce dendritic spine formation. We also found that the IL1RAPL1 paralog, IL1RAPL2, interacts with PTPδ and induces excitatory synapse and dendritic spine formation. The interaction of the IL1RAPL1 family of proteins with PTPδ and RhoGAP2 reveals a pathophysiological mechanism of cognitive impairment associated with a novel type of trans-synaptic signaling that regulates excitatory synapse and dendritic spine formation.

Funding information:
  • NINDS NIH HHS - NS 20498(United States)

Postsynaptic density-95 scaffolding of Shaker-type K⁺ channels in smooth muscle cells regulates the diameter of cerebral arteries.

  • Joseph BK
  • J. Physiol. (Lond.)
  • 2011 Nov 1

Literature context:


Abstract:

Postsynaptic density-95 (PSD95) is a 95 kDa scaffolding molecule in the brain that clusters postsynaptic proteins including ion channels, receptors, enzymes and other signalling partners required for normal cognition. The voltage-gated, Shaker-type K(+) (K(V)1) channel is one key binding partner of PSD95 scaffolds in neurons. However, K(V)1 channels composed of α1.2 and α1.5 pore-forming subunits also are expressed in the vascular smooth muscle cells (cVSMCs) of the cerebral circulation, although the identity of their molecular scaffolds is unknown. Since α1.2 contains a binding motif for PSD95, we explored the possibility that cVSMCs express PSD95 as a scaffold to promote K(V)1 channel expression and cerebral vasodilatation. Cerebral arteries from Sprague-Dawley rats were isolated for analysis of PSD95 and K(V)1 channel proteins. PSD95 was detected in cVSMCs and it co-immunoprecipitated and co-localized with the pore-forming α1.2 subunit of the K(V)1 channel. Antisense-mediated knockdown of PSD95 profoundly reduced K(V)1 channel expression and suppressed K(V)1 current in patch-clamped cVSMCs. Loss of PSD95 also depolarized cVSMCs in pressurized cerebral arteries and induced a strong constriction associated with a loss of functional K(V)1 channels. Our findings provide initial evidence that PSD95 is expressed in cVSMCs, and the K(V)1 channel is one of its important binding partners. PSD95 appears to function as a critical 'dilator' scaffold in cerebral arteries by increasing the number of functional K(V)1 channels at the plasma membrane.

Funding information:
  • NEI NIH HHS - R01 EY015128(United States)

PKA/AKAP1 and PP2A/Bβ2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics.

  • Dickey AS
  • J. Neurosci.
  • 2011 Nov 2

Literature context:


Abstract:

Mitochondrial shape is determined by fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by dynamin-related protein 1 (Drp1), a large GTPase of the dynamin family that is highly expressed in neurons and regulated by various posttranslational modifications, including phosphorylation. We report here that reversible phosphorylation of Drp1 at a conserved Ser residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase (PP2A/Bβ2) impacts dendrite and synapse development in cultured rat hippocampal neurons. PKA/AKAP1-mediated phosphorylation of Drp1 at Ser656 increased mitochondrial length and dendrite occupancy, enhancing dendritic outgrowth but paradoxically decreasing synapse number and density. Opposing PKA/AKAP1, PP2A/Bβ2-mediated dephosphorylation of Drp1 at Ser656 fragmented and depolarized mitochondria and depleted them from dendrites, stunting dendritic outgrowth but augmenting synapse formation. Raising and lowering intracellular calcium reproduced the effects of dephospho-Drp1 and phospho-Drp1on dendrite and synapse development, respectively, while boosting mitochondrial membrane potential with l-carnitine-fostered dendrite at the expense of synapse formation without altering mitochondrial size or distribution. Thus, outer mitochondrial PKA and PP2A regulate neuronal development by inhibiting and promoting mitochondrial division, respectively. We propose that the bioenergetic state of mitochondria, rather than their localization or shape per se, is the key effector of Drp1, altering calcium homeostasis to modulate neuronal morphology and connectivity.

Funding information:
  • NINDS NIH HHS - R01 NS078214(United States)

Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses.

  • Verpelli C
  • J. Biol. Chem.
  • 2011 Oct 7

Literature context:


Abstract:

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

Funding information:
  • NHGRI NIH HHS - R01-HG004885(United States)

Biphasic effects of copper on neurotransmission in rat hippocampal neurons.

  • Peters C
  • J. Neurochem.
  • 2011 Oct 12

Literature context:


Abstract:

The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5 μM; copper: EC50 2.9 ± 0.2 μM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).

Funding information:
  • NIGMS NIH HHS - R01 GM056834(United States)

Neurexin-neuroligin adhesions capture surface-diffusing AMPA receptors through PSD-95 scaffolds.

  • Mondin M
  • J. Neurosci.
  • 2011 Sep 21

Literature context:


Abstract:

The mechanisms governing the recruitment of functional glutamate receptors at nascent excitatory postsynapses following initial axon-dendrite contact remain unclear. We examined here the ability of neurexin/neuroligin adhesions to mobilize AMPA-type glutamate receptors (AMPARs) at postsynapses through a diffusion/trap process involving the scaffold molecule PSD-95. Using single nanoparticle tracking in primary rat and mouse hippocampal neurons overexpressing or lacking neuroligin-1 (Nlg1), a striking inverse correlation was found between AMPAR diffusion and Nlg1 expression level. The use of Nlg1 mutants and inhibitory RNAs against PSD-95 demonstrated that this effect depended on intact Nlg1/PSD-95 interactions. Furthermore, functional AMPARs were recruited within 1 h at nascent Nlg1/PSD-95 clusters assembled by neurexin-1β multimers, a process requiring AMPAR membrane diffusion. Triggering novel neurexin/neuroligin adhesions also caused a depletion of PSD-95 from native synapses and a drop in AMPAR miniature EPSCs, indicating a competitive mechanism. Finally, both AMPAR level at synapses and AMPAR-dependent synaptic transmission were diminished in hippocampal slices from newborn Nlg1 knock-out mice, confirming an important role of Nlg1 in driving AMPARs to nascent synapses. Together, these data reveal a mechanism by which membrane-diffusing AMPARs can be rapidly trapped at PSD-95 scaffolds assembled at nascent neurexin/neuroligin adhesions, in competition with existing synapses.

Funding information:
  • NIDA NIH HHS - R01 DA001457(United States)
  • NIMH NIH HHS - U54 MH084690(United States)

Glutamatergic plasticity in medial prefrontal cortex and ventral tegmental area following extended-access cocaine self-administration.

  • Ghasemzadeh MB
  • Brain Res.
  • 2011 Sep 21

Literature context:


Abstract:

Glutamate signaling in prefrontal cortex and ventral tegmental area plays an important role in the molecular and behavioral plasticity associated with addiction to drugs of abuse. The current study investigated the expression and postsynaptic density redistribution of glutamate receptors and synaptic scaffolding proteins in dorsomedial and ventromedial prefrontal cortex and ventral tegmental area after cocaine self-administration. After 14 days of extended-access (6h/day) cocaine self-administration, rats were exposed to one of three withdrawal regimen for 10 days. Animals either stayed in home cages (Home), returned to self-administration boxes with the levers withdrawn (Box), or underwent extinction training (Extinction). Extinction training was associated with significant glutamatergic plasticity. In dorsomedial prefrontal cortex of the Extinction group, there was an increase in postsynaptic density GluR1, PSD95, and actin proteins; while postsynaptic density mGluR5 protein decreased and there was no change in NMDAR1, Homer1b/c, or PICK1 proteins. These changes were not observed in ventromedial prefrontal cortex or ventral tegmental area. In ventral tegmental area, Extinction training reversed the decreased postsynaptic density NMDAR1 protein in the Home and Box withdrawal groups. These data suggest that extinction of drug seeking is associated with selective glutamatergic plasticity in prefrontal cortex and ventral tegmental area that include modulation of receptor trafficking to postsynaptic density.

Funding information:
  • NINDS NIH HHS - R01 NS014841(United States)

Dendritic EGFP-STIM1 activation after type I metabotropic glutamate and muscarinic acetylcholine receptor stimulation in hippocampal neuron.

  • Ng AN
  • J. Neurosci. Res.
  • 2011 Aug 3

Literature context:


Abstract:

Several signaling pathways in neurons engage the endoplasmic reticulum (ER) calcium store by triggering calcium release. After release, ER calcium levels must be restored. In many non-neuronal cell types, this is mediated by store-operated calcium entry (SOCE), a cellular homeostatic mechanism that activates specialized store-operated calcium channels (SOC). Although much evidence supports the existence of SOCE in neurons, its importance has been difficult to determine because of the abundance of calcium channels expressed and the lack of SOC-specific pharmacological agents. We have explored the function of the SOCE-inducing protein STIM1 in neurons. In EGFP-STIM1-expressing hippocampal neurons, the sarco- and endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin caused rapid aggregation (i.e., activation) of STIM1 in soma and dendrites. Upon STIM1 activation by thapsigargin, a dramatic reduction in STIM1 mobility was detected by fluorescence recovery after photobleaching (FRAP). By triggering release of ER calcium with 3,5-dihydroxyphenylglycine (DHPG) or carbachol (Cch), agonists of type I metabotropic glutamate receptors (mGluR) and muscarinic acetylcholine receptors (mAChR), respectively, STIM1 was activated, and calcium entry (likely to represent SOCE) occurred in dendrites. It is therefore possible that neuronal SOCE is activated by physiological stimuli, some of which may alter the postsynaptic calcium signaling properties.

Funding information:
  • NCI NIH HHS - CA06927(United States)
  • NICHD NIH HHS - T32-HD046387(United States)

Synaptic autoregulation by metalloproteases and γ-secretase.

  • Restituito S
  • J. Neurosci.
  • 2011 Aug 24

Literature context:


Abstract:

The proteolytic machinery comprising metalloproteases and γ-secretase, an intramembrane aspartyl protease involved in Alzheimer's disease, cleaves several substrates in addition to the extensively studied amyloid precursor protein. Some of these substrates, such as N-cadherin, are synaptic proteins involved in synapse remodeling and maintenance. Here we show, in rats and mice, that metalloproteases and γ-secretase are physiologic regulators of synapses. Both proteases are synaptic, with γ-secretase tethered at the synapse by δ-catenin, a synaptic scaffolding protein that also binds to N-cadherin and, through scaffolds, to AMPA receptor and a metalloprotease. Activity-dependent proteolysis by metalloproteases and γ-secretase takes place at both sides of the synapse, with the metalloprotease cleavage being NMDA receptor-dependent. This proteolysis decreases levels of synaptic proteins and diminishes synaptic transmission. Our results suggest that activity-dependent substrate cleavage by synaptic metalloproteases and γ-secretase modifies synaptic transmission, providing a novel form of synaptic autoregulation.

Funding information:
  • NIMH NIH HHS - R01 MH059950(United States)

TrkB and protein kinase Mζ regulate synaptic localization of PSD-95 in developing cortex.

  • Yoshii A
  • J. Neurosci.
  • 2011 Aug 17

Literature context:


Abstract:

Postsynaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We showed previously that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also, PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely, signaling through phospholipase Cγ and the brain-specific PKC variant protein kinase M ζ (PKMζ). We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by overexpressing ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as aging brains.

Funding information:
  • NIDA NIH HHS - R01 DA033150(United States)

Cyclin-dependent kinase 5 regulates PSD-95 ubiquitination in neurons.

  • Bianchetta MJ
  • J. Neurosci.
  • 2011 Aug 17

Literature context:


Abstract:

Cyclin-dependent kinase 5 (Cdk5) and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer's, learning and memory, and synapse maturation and plasticity. However, the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the ubiquitin E3 ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a nonproteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiquitination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis.

Funding information:
  • NIDDK NIH HHS - R01 DK089113(United States)

Rivastigmine lowers Aβ and increases sAPPα levels, which parallel elevated synaptic markers and metabolic activity in degenerating primary rat neurons.

  • Bailey JA
  • PLoS ONE
  • 2011 Jul 29

Literature context:


Abstract:

Overproduction of amyloid-β (Aβ) protein in the brain has been hypothesized as the primary toxic insult that, via numerous mechanisms, produces cognitive deficits in Alzheimer's disease (AD). Cholinesterase inhibition is a primary strategy for treatment of AD, and specific compounds of this class have previously been demonstrated to influence Aβ precursor protein (APP) processing and Aβ production. However, little information is available on the effects of rivastigmine, a dual acetylcholinesterase and butyrylcholinesterase inhibitor, on APP processing. As this drug is currently used to treat AD, characterization of its various activities is important to optimize its clinical utility. We have previously shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures. Given previous reports on the effects of APP and Aβ on synapses, regulation of APP processing represents a plausible mechanism for the synaptic effects of rivastigmine. To test this hypothesis, we treated degenerating primary cultures with rivastigmine and measured secreted APP (sAPP) and Aβ. Rivastigmine treatment increased metabolic activity in these cultured cells, and elevated APP secretion. Analysis of the two major forms of APP secreted by these cultures, attributed to neurons or glia based on molecular weight showed that rivastigmine treatment significantly increased neuronal relative to glial secreted APP. Furthermore, rivastigmine treatment increased α-secretase cleaved sAPPα and decreased Aβ secretion, suggesting a therapeutic mechanism wherein rivastigmine alters the relative activities of the secretase pathways. Assessment of sAPP levels in rodent CSF following once daily rivastigmine administration for 21 days confirmed that elevated levels of APP in cell culture translated in vivo. Taken together, rivastigmine treatment enhances neuronal sAPP and shifts APP processing toward the α-secretase pathway in degenerating neuronal cultures, which mirrors the trend of synaptic proteins, and metabolic activity.

Funding information:
  • NIGMS NIH HHS - GM41624(United States)

EphA4 expression promotes network activity and spine maturation in cortical neuronal cultures.

  • Clifford MA
  • Neural Dev
  • 2011 May 4

Literature context:


Abstract:

BACKGROUND: Neurons form specific connections with targets via synapses and patterns of synaptic connectivity dictate neural function. During development, intrinsic neuronal specification and environmental factors guide both initial formation of synapses and strength of resulting connections. Once synapses form, non-evoked, spontaneous activity serves to modulate connections, strengthening some and eliminating others. Molecules that mediate intercellular communication are particularly important in synaptic refinement. Here, we characterize the influences of EphA4, a transmembrane signaling molecule, on neural connectivity. RESULTS: Using multi-electrode array analysis on in vitro cultures, we confirmed that cortical neurons mature and generate spontaneous circuit activity as cells differentiate, with activity growing both stronger and more patterned over time. When EphA4 was over-expressed in a subset of neurons in these cultures, network activity was enhanced: bursts were longer and were composed of more spikes than in control-transfected cultures. To characterize the cellular basis of this effect, dendritic spines, the major excitatory input site on neurons, were examined on transfected neurons in vitro. Strikingly, while spine number and density were similar between conditions, cortical neurons with elevated levels of EphA4 had significantly more mature spines, fewer immature spines, and elevated colocalization with a mature synaptic marker. CONCLUSIONS: These results demonstrate that experimental elevation of EphA4 promotes network activity in vitro, supporting spine maturation, producing more functional synaptic pairings, and promoting more active circuitry.

Funding information:
  • NINDS NIH HHS - 1-R01 NS054814-05(United States)

Membrane palmitoylated proteins regulate trafficking and processing of nectins.

  • Dudak A
  • Eur. J. Cell Biol.
  • 2011 May 11

Literature context:


Abstract:

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.

Funding information:
  • NCRR NIH HHS - R01 RR024031(United States)

Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism.

  • Bangash MA
  • Cell
  • 2011 May 27

Literature context:


Abstract:

We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the wild-type (WT) gene product and results in >90% reduction of Shank3 at synapses. This "gain-of-function" phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of postsynaptic density proteins, spine morphology, and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder.

Funding information:
  • NIMH NIH HHS - P50 MH071616(United States)

A requirement for nuclear factor-kappaB in developmental and plasticity-associated synaptogenesis.

  • Boersma MC
  • J. Neurosci.
  • 2011 Apr 6

Literature context:


Abstract:

Structural plasticity of dendritic spines and synapses is a fundamental mechanism governing neuronal circuits and may form an enduring basis for information storage in the brain. We find that the p65 subunit of the nuclear factor-κB (NF-κB) transcription factor, which is required for learning and memory, controls excitatory synapse and dendritic spine formation and morphology in murine hippocampal neurons. Endogenous NF-κB activity is elevated by excitatory transmission during periods of rapid spine and synapse development. During in vitro synaptogenesis, NF-κB enhances dendritic spine and excitatory synapse density and loss of endogenous p65 decreases spine density and spine head volume. Cell-autonomous function of NF-κB within the postsynaptic neuron is sufficient to regulate the formation of both presynaptic and postsynaptic elements. During synapse development in vivo, loss of NF-κB similarly reduces spine density and also diminishes the amplitude of synaptic responses. In contrast, after developmental synaptogenesis has plateaued, endogenous NF-κB activity is low and p65 deficiency no longer attenuates basal spine density. Instead, NF-κB in mature neurons is activated by stimuli that induce demand for new synapses, including estrogen and short-term bicuculline, and is essential for upregulating spine density in response to these stimuli. p65 is enriched in dendritic spines making local protein-protein interactions possible; however, the effects of NF-κB on spine density require transcription and the NF-κB-dependent regulation of PSD-95, a critical postsynaptic component. Collectively, our data define a distinct role for NF-κB in imparting transcriptional regulation required for the induction of changes to, but not maintenance of, excitatory synapse and spine density.

Funding information:
  • NEI NIH HHS - R01 EY013315(United States)

The maturation of photoreceptors in the avian retina is stimulated by thyroid hormone.

  • Fischer AJ
  • Neuroscience
  • 2011 Mar 31

Literature context:


Abstract:

During retinal development, the cell-fate of photoreceptors is committed long before maturation, which entails the expression of opsins and functional transduction of light. The mechanisms that delay the maturation of photoreceptors remain unknown. We have recently reported that immature photoreceptors express the LIM domain transcription factors Islet2 and Lim3, as well as the cell-surface glycoprotein axonin1 [Fischer et al., (2008a) J Comp Neurol 506:584-603]. As the photoreceptors mature to form outer segments and express photopigments, the expression of the Islet2, Lim3 and axonin1 is diminished. The purpose of this study was to investigate whether thyroid hormone (TH) influences the maturation of photoreceptors. We studied the maturation of photoreceptors across the gradient of maturity that exists in far peripheral regions of the post-natal chicken retina [Ghai et al., (2008) Brain Res 1192:76-89]. We found that intraocular injections of TH down-regulated Islet2, Lim3 and axonin1 in photoreceptors in far peripheral regions of the retina. By contrast, TH stimulated the up-regulation of red-green opsin, violet opsin, rhodopsin and calbindin in photoreceptors. We found a correlation between the onset of RLIM (RING finger LIM-domain binding protein) and down-regulation of Islet2 and Lim3 in maturing photoreceptors; RLIM is known to interfere with the transcriptional activity of LIM-domain transcription factors. We conclude that TH stimulates the maturation of photoreceptors in the avian retina. We propose that TH inhibits the expression of Islet2 and Lim3, which thereby permits photoreceptor maturation and the onset of photopigment-expression.

Funding information:
  • NCI NIH HHS - CA-85129(United States)

Selective pharmacogenetic inhibition of mammalian target of Rapamycin complex I (mTORC1) blocks long-term synaptic plasticity and memory storage.

  • Stoica L
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2011 Mar 1

Literature context:


Abstract:

Both the formation of long-term memory (LTM) and late-long-term potentiation (L-LTP), which is thought to represent the cellular model of learning and memory, require de novo protein synthesis. The mammalian target of Rapamycin (mTOR) complex I (mTORC1) integrates information from various synaptic inputs and its best characterized function is the regulation of translation. Although initial studies have shown that rapamycin reduces L-LTP and partially blocks LTM, recent genetic and pharmacological evidence indicating that mTORC1 promotes L-LTP and LTM is controversial. Thus, the role of mTORC1 in L-LTP and LTM is unclear. To selectively inhibit mTORC1 activity in the adult brain, we used a "pharmacogenetic" approach that relies on the synergistic action of a drug (rapamycin) and a genetic manipulation (mTOR heterozygotes, mTOR(+/-) mice) on the same target (mTORC1). Although L-LTP and LTM are normal in mTOR(+/-) mice, application of a low concentration of rapamycin-one that is subthreshold for WT mice-prevented L-LTP and LTM only in mTOR(+/-) mice. Furthermore, we found that mTORC1-mediated translational control is required for memory reconsolidation. We provide here direct genetic evidence supporting the role of mTORC1 in L-LTP and behavioral memory.

Funding information:
  • Biotechnology and Biological Sciences Research Council - (United Kingdom)
  • PHS HHS - 04-2085(United States)

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse.

  • Pacey LK
  • Mol. Cell. Neurosci.
  • 2011 Mar 28

Literature context:


Abstract:

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA(B) receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS.

Funding information:
  • NCRR NIH HHS - RR19895(United States)

Transgenic conversion of omega-6 into omega-3 fatty acids in a mouse model of Parkinson's disease.

  • Bousquet M
  • J. Lipid Res.
  • 2011 Feb 17

Literature context:


Abstract:

We have recently identified a neuroprotective role for omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a toxin-induced mouse model of Parkinson's disease (PD). Combined with epidemiological data, these observations suggest that low n-3 PUFA intake is a modifiable environmental risk factor for PD. In order to strengthen these preclinical findings as prerequisite to clinical trials, we further investigated the neuroprotective role of n-3 PUFAs in Fat-1 mice, a transgenic model expressing an n-3 fatty acid desaturase converting n-6 PUFAs into n-3 PUFAs. Here, we report that the expression of the fat-1 transgene increased cortical n-3:n-6 PUFA ratio (+28%), but to a lesser extent than dietary supplementation (92%). Such a limited endogenous production of n-3 PUFAs in the Fat-1 mouse was insufficient to confer neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity as assessed by dopamine levels, tyrosine hydroxylase (TH)-positive neurons and fibers, as well as nigral Nurr1 and dopamine transporter (DAT) mRNA expression. Nevertheless, higher cortical docosahexaenoic acid (DHA) concentrations were positively correlated with markers of nigral dopaminergic neurons such as the number of TH-positive cells, in addition to Nurr1 and DAT mRNA levels. These associations are consistent with the protective role of DHA in a mouse model of PD. Taken together, these data suggest that dietary intake of a preformed DHA supplement is more effective in reaching the brain and achieving neuroprotection in an animal model of PD.

Funding information:
  • Intramural NIH HHS - ZIA DK015600-18(United States)

The Toll-like receptor-3 agonist polyinosinic:polycytidylic acid triggers nigrostriatal dopaminergic degeneration.

  • Deleidi M
  • J. Neurosci.
  • 2010 Dec 1

Literature context:


Abstract:

In Parkinson's disease (PD), loss of striatal dopaminergic (DA) terminals and degeneration of DA neurons in the substantia nigra (SN) are associated with glial reactions. Such inflammatory processes are commonly considered an epiphenomenon of neuronal degeneration. However, there is increasing recognition of the role of neuroinflammation as an initiation factor of DA neuron degeneration. To investigate this issue, we established a new model of brain inflammation by injecting the Toll-like receptor 3 (TLR-3) agonist polyinosinic:polycytidylic acid [poly(I:C)] in the SN of adult rats. Poly(I:C) injection induced a sustained inflammatory reaction in the SN and in the dorsolateral striatum. Significant changes were detected in proteins relevant to synaptic transmission and axonal transport. In addition, cytoplasmic mislocalization of neuronal TAR DNA binding protein TDP-43 was observed. Poly(I:C) injection increased the susceptibility of midbrain DA neurons to a subsequent neurotoxic trigger (low-dose 6-hydroxydopamine). Systemic delivery of interleukin-1 receptor antagonist protected SN DA neurons exposed to combined poly(I:C) induced inflammatory and neurotoxic oxidative stress. These data indicate that viral-like neuroinflammation induces predegenerative changes in the DA system, which lowers the set point toward neuronal dysfunction and degeneration. New powerful neuroprotective therapies for PD might be considered by targeting critical inflammatory mechanisms, including cytokine-induced neurotoxicity.

Funding information:
  • NHGRI NIH HHS - 5P41HG002273-09(United States)

Alternative splicing of neuroligin and its protein distribution in the outer plexiform layer of the chicken retina.

  • Wahlin KJ
  • J. Comp. Neurol.
  • 2010 Dec 15

Literature context:


Abstract:

Although synaptogenesis within the retina is obviously essential for vision, mechanisms responsible for the initiation and maintenance of retinal synapses are poorly understood. In addition to its scientific interest, understanding retinal synapse formation is becoming clinically relevant with ongoing efforts to develop transplantation-based approaches for the treatment of retinal degenerative disease. To extend our understanding, we have focused on the chick model system and have studied the neuroligin family of neuronal adhesion factors that has been shown to participate in synapse assembly in the brain. We identified chicken orthologs of neuroligins 1, -3, and -4, but could find no evidence of neuroligin 2. We investigated temporal and spatial patterns of mRNA and protein expression during development using standard polymerase chain reaction (RT-PCR), quantitative PCR (QPCR), laser-capture microdissection (LCM), and confocal microscopy. At the mRNA level, neuroligins were detected at the earliest period tested, embryonic day (ED)5, which precedes the period of inner retina synaptogenesis. Significant alternative splicing was observed through development. While neuroligin gene products were generally detected in the inner retina, low levels of neuroligin 1 mRNA were also detected in the photoreceptor layer. Neuroligin 3 and -4 transcripts, on the other hand, were only detected in the inner retina. At retinal synapses neuroligin 1 protein was detected in the inner plexiform layer, but its highest levels were detected in the outer plexiform layer on the tips of horizontal cell dendrites. This work lays the groundwork for future studies on the functional roles of the neuroligins within the retina.

Funding information:
  • Austrian Science Fund FWF - P 19467(Austria)

GABAergic complex basket formations in the human neocortex.

  • Blazquez-Llorca L
  • J. Comp. Neurol.
  • 2010 Dec 15

Literature context:


Abstract:

Certain GABAergic interneurons in the cerebral cortex, basket cells, establish multiple connections with cell bodies that typically outline the somata and proximal dendrites of pyramidal cells. During studies into the distribution of the vesicular GABA transporter (VGAT) in the human cerebral cortex, we were struck by the presence of a very dense, pericellular arrangement of multiple VGAT-immunoreactive (-ir) terminals in certain cortical areas. We called these terminals "Complex basket formations" (Cbk-formations) to distinguish them from the simpler and more typical pericellular GABAergic innervations of most cortical neurons. Here we examined the distribution of these VGAT-ir Cbk-formations in various cortical areas, including the somatosensory (area 3b), visual (areas 17 and 18), motor (area 4), associative frontal (dorsolateral areas 9, 10, 45, 46, and orbital areas 11, 12, 13, 14, 47), associative temporal (areas 20, 21, 22, and 38), and limbic cingulate areas (areas 24, 32). Furthermore, we used dual or triple staining techniques to study the chemical nature of the innervated cells. We found that VGAT-ir Cbk-formations were most frequently found in area 4 followed by areas 3b, 13, and 18. In addition, they were mostly observed in layer III, except in area 17, where they were most dense in layer IV. We also found that 70% of the innervated neurons were pyramidal cells, while the remaining 30% were multipolar cells. Most of these multipolar cells expressed the calcium-binding protein parvalbumin and the lectin Vicia villosa agglutinin.

Funding information:
  • NCRR NIH HHS - R01RR025342(United States)
  • NEI NIH HHS - EY1765(United States)

Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation.

  • Brown MT
  • PLoS ONE
  • 2010 Dec 31

Literature context:


Abstract:

BACKGROUND: Addictive drugs have in common that they cause surges in dopamine (DA) concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA). Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs) at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine) cause similar changes through their effects on the mesolimbic DA system. METHODOLOGY/PRINCIPAL FINDINGS: We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT) is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine. CONCLUSIONS/SIGNIFICANCE: We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.

Funding information:
  • NHGRI NIH HHS - RC2 HG005597-01(United States)

Array tomography: immunostaining and antibody elution.

  • Micheva KD
  • Cold Spring Harb Protoc
  • 2010 Nov 1

Literature context:


Abstract:

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are prepared for imaging by tagging with primary antibodies against specific cellular targets, followed by labeling with fluorescent secondary antibodies. Alternatively, fluorescent proteins that have been introduced into the tissue before dissection can be used.

Funding information:
  • NHGRI NIH HHS - T32 HG003284(United States)

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.

  • Micheva KD
  • Neuron
  • 2010 Nov 18

Literature context:


Abstract:

A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Funding information:
  • NICHD NIH HHS - R01 HD046236-06(United States)

Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation.

  • Evers DM
  • Nat. Neurosci.
  • 2010 Oct 29

Literature context:


Abstract:

Trafficking of AMPA receptors (AMPARs) is important for many forms of synaptic plasticity. However, the link between activity and resulting synaptic alterations is not fully understood. We identified a direct interaction between N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase involved in membrane fusion events and stabilization of surface AMPARs, and Polo-like kinase- 2 (Plk2), an activity-inducible kinase that homeostatically decreases excitatory synapse number and strength. Plk2 disrupted the interaction of NSF with the GluA2 subunit of AMPARs, promoting extensive loss of surface GluA2 in rat hippocampal neurons, greater association of GluA2 with adaptor proteins PICK1 and GRIP1, and decreased synaptic AMPAR current. Plk2 engagement of NSF, but not Plk2 kinase activity, was required for this mechanism and occurred through a motif in the Plk2 protein that was independent of the canonical polo box interaction sites. These data reveal that heightened synaptic activity, acting through Plk2, leads to homeostatic decreases in surface AMPAR expression via the direct dissociation of NSF from GluA2.

Funding information:
  • NIDDK NIH HHS - U01 DK070200-01(United States)

MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood.

  • Needleman LA
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2010 Sep 28

Literature context:


Abstract:

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined-during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

Funding information:
  • NHGRI NIH HHS - U01-HG004866(United States)

CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin.

  • Opazo P
  • Neuron
  • 2010 Jul 29

Literature context:


Abstract:

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using single-particle tracking of AMPARs, we show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilization requires both phosphorylation of the auxiliary subunit Stargazin and its binding to PDZ domain scaffolds. It does not depend on the PDZ binding domain of GluA1 AMPAR subunit nor its phosphorylation at Ser831. Finally, CaMKII-dependent AMPAR immobilization regulates short-term plasticity. Thus, NMDA-dependent Ca(2+) influx in the post-synapse triggers a CaMKII- and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic function.

Funding information:
  • Wellcome Trust - 1R24OD011883-01(United Kingdom)

Wnt-5a modulates recycling of functional GABAA receptors on hippocampal neurons.

  • Cuitino L
  • J. Neurosci.
  • 2010 Jun 23

Literature context:


Abstract:

GABA(A) receptors (GABA(A)-Rs) play a significant role in mediating fast synaptic inhibition and it is the main inhibitory receptor in the CNS. The role of Wnt signaling in coordinating synapse structure and function in the mature CNS is poorly understood. In previous studies we found that Wnt ligands can modulate excitatory synapses through remodeling both presynaptic and postsynaptic regions. In this current study we provide evidence for the effect of Wnt-5a on postsynaptic GABA(A)-Rs. We observed that Wnt-5a induces surface expression and maintenance of this receptor in the neuronal membrane. The evoked IPSC recordings in rat hippocampal slice indicate that Wnt-5a can regulates postsynaptically the hippocampal inhibitory synapses. We found also that Wnt-5a: (a) induces the insertion and clustering of GABA(A)-Rs in the membrane; (b) increases the amplitude of GABA-currents due exclusively to postsynaptic mechanisms; (c) does not affect the endocytic process, but increases the receptor recycling. Finally, all these effects on the GABA(A)-Rs are mediated by the activation of calcium/calmodulin-dependent kinase II (CaMKII). Therefore, we postulate that Wnt-5a, by activation of CaMKII, induces the recycling of functional GABA(A)-Rs on the mature hippocampal neurons.

Funding information:
  • NIDA NIH HHS - DA-03977(United States)
  • NIGMS NIH HHS - R01 GM62917(United States)

Identification and validation of novel spinophilin-associated proteins in rodent striatum using an enhanced ex vivo shotgun proteomics approach.

  • Baucum AJ
  • Mol. Cell Proteomics
  • 2010 Jun 3

Literature context:


Abstract:

Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.

Huntingtin-associated protein-1 deficiency in orexin-producing neurons impairs neuronal process extension and leads to abnormal behavior in mice.

  • Lin YF
  • J. Biol. Chem.
  • 2010 May 21

Literature context:


Abstract:

Huntingtin-associated protein-1 (Hap1) is a neuronal protein that associates with huntingtin, the Huntington disease protein. Although Hap1 and huntingtin are known to be involved in intracellular trafficking, whether and how the impairment of Hap1-associated trafficking leads to neurological pathology and symptoms remain to be seen. As Hap1 is enriched in neuronal cells in the brain, addressing this issue is important in defining the role of defective intracellular trafficking in the selective neuropathology associated with Hap1 dysfunction. Here, we find that Hap1 is abundantly expressed in orexin (hypocretin)-producing neurons (orexin neurons), which are distinctly distributed in the hypothalamus and play an important role in the regulation of feeding and behavior. We created conditional Hap1 knock-out mice to selectively deplete Hap1 in orexin neurons via the Cre-loxP system. These mice show process fragmentation of orexin neurons and reductions in food intake, body weight, and locomotor activity. Sucrose density gradient fractionation reveals that loss of Hap1 in the mouse brain also reduces the distribution of trafficking protein complexes and cargo proteins in the fractions that are enriched in synaptosomes. These results suggest that Hap1 is critical for the transport of multiple proteins to the nerve terminals to maintain the integrity of neuronal processes and that selective disruption of the processes of orexin neurons can cause abnormal feeding and locomotor activity.

Panel of synaptic protein ELISAs for evaluating neurological phenotype.

  • Gottschall PE
  • Exp Brain Res
  • 2010 Apr 16

Literature context:


Abstract:

The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 microg protein for PSD-95 to 0.23 microg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.

Dact1 is a postsynaptic protein required for dendrite, spine, and excitatory synapse development in the mouse forebrain.

  • Okerlund ND
  • J. Neurosci.
  • 2010 Mar 24

Literature context:


Abstract:

Dact1 (Dapper/Frodo), an intracellular phosphoprotein that binds Dishevelled, catenins, and other signaling proteins, is expressed in the developing and mature mammalian CNS, but its function there is unknown. Dact1 colocalized with synaptic markers and partitioned to postsynaptic fractions from cultured mouse forebrain neurons. Hippocampal neurons from Dact1 knock-out mice had simpler dendritic arbors and fewer spines than hippocampal neurons from wild-type littermates. This correlated with reductions in excitatory synapses and miniature EPSCs, whereas inhibitory synapses were not affected. Loss of Dact1 resulted in a decrease in activated Rac, and recombinant expression of either Dact1 or constitutively active Rac, but not Rho or Cdc42, rescued dendrite and spine phenotypes in Dact1 mutant neurons. Our findings suggest that, during neuronal differentiation, Dact1 plays a critical role in a molecular pathway promoting Rac activity underlying the elaboration of dendrites and the establishment of spines and excitatory synapses.

Activation of Wnt signaling by lithium and rosiglitazone reduced spatial memory impairment and neurodegeneration in brains of an APPswe/PSEN1DeltaE9 mouse model of Alzheimer's disease.

  • Toledo EM
  • Mol. Psychiatry
  • 2010 Mar 19

Literature context:


Abstract:

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, accumulation of the amyloid-beta-peptide (Abeta) and synaptic alterations. Treatment with lithium has been shown to provide neuroprotection against several insults, including protection against Abeta neurotoxicity in vitro. Rosiglitazone, a peroxisome proliferator activated receptor-gamma agonist, has been shown to attenuate Abeta-peptide neurotoxic effects, including the inflammatory response of microglia and astrocytes. Both types of drugs activate Wnt signaling, a pathway that has been shown to be related to AD. In this study, a double transgenic mouse model, which coexpresses APPswe and the exon 9 deletion of the presenilin 1 (PSEN1) gene, was used to examine, in vivo, the effect of lithium and rosiglitazone on Abeta neurotoxicity. Mice were tested for spatial memory, and their brain samples were used for histochemical and biochemical analysis. In this study, we report that both drugs significantly reduced (1) spatial memory impairment induced by amyloid burden; (2) Abeta aggregates and Abeta oligomers; and (3) astrocytic and microglia activation. They also prevented changes in presynaptic and postsynaptic marker proteins. Finally, both drugs activate Wnt signaling shown by the increase in beta-catenin and by the inhibition of the glycogen synthase kinase-3beta. We conclude that lithium and rosiglitazone, possibly by the activation of the Wnt signaling pathway, reduce various AD neuropathological markers and may be considered as potential therapeutic agents against the disease.

Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1.

  • Hayashi-Takagi A
  • Nat. Neurosci.
  • 2010 Mar 18

Literature context:


Abstract:

Synaptic spines are dynamic structures that regulate neuronal responsiveness and plasticity. We examined the role of the schizophrenia risk factor DISC1 in the maintenance of spine morphology and function. We found that DISC1 anchored Kalirin-7 (Kal-7), regulating access of Kal-7 to Rac1 and controlling the duration and intensity of Rac1 activation in response to NMDA receptor activation in both cortical cultures and rat brain in vivo. These results explain why Rac1 and its activator (Kal-7) serve as important mediators of spine enlargement and why constitutive Rac1 activation decreases spine size. This mechanism likely underlies disturbances in glutamatergic neurotransmission that have been frequently reported in schizophrenia that can lead to alteration of dendritic spines with consequential major pathological changes in brain function. Furthermore, the concept of a signalosome involving disease-associated factors, such as DISC1 and glutamate, may well contribute to the multifactorial and polygenetic characteristics of schizophrenia.

Distribution of RGS9-2 in neurons of the mouse striatum.

  • Mancuso JJ
  • J. Neurochem.
  • 2010 Feb 20

Literature context:


Abstract:

Regulators of G protein signaling (RGS) proteins negatively modulate G protein-coupled receptor (GPCR) signaling activity by accelerating G protein hydrolysis of GTP, hastening pathway shutoff. A wealth of data from cell culture experiments using exogenously expressed proteins indicates that RGS9 and other RGS proteins have the potential to down-regulate a significant number of pathways. We have used an array of biochemical and tissue staining techniques to examine the subcellular localization and membrane binding characteristics of endogenous RGS9-2 and known binding partners in rodent striatum and tissue homogenates. A small fraction of RGS9-2 is present in the soluble cytoplasmic fraction, whereas the majority is present primarily associated with the plasma membrane and structures insoluble in non-ionic detergents that efficiently extract the vast majority of its binding partners, R7BP and G(beta5). It is specifically excluded from the cell nucleus in mouse striatal tissue. In cultured striatal neurons, RGS9-2 is found at extrasynaptic sites primarily along the dendritic shaft near the spine neck. Heterogeneity in RGS9-2 detergent solubility along with its unique subcellular localization suggests that its mechanism of membrane anchoring and localization is complex and likely involves additional proteins beside R7BP. An important nuclear function for RGS9-2 seems unlikely.

ADAM22, a Kv1 channel-interacting protein, recruits membrane-associated guanylate kinases to juxtaparanodes of myelinated axons.

  • Ogawa Y
  • J. Neurosci.
  • 2010 Jan 20

Literature context:


Abstract:

Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes.

A postsynaptic signaling pathway that may account for the cognitive defect due to IL1RAPL1 mutation.

  • Pavlowsky A
  • Curr. Biol.
  • 2010 Jan 26

Literature context:


Abstract:

BACKGROUND: Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene mutations are associated with cognitive impairment ranging from nonsyndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of Toll/IL-1 receptors, whose expression in the brain is upregulated by neuronal activity. Currently, very little is known about the function of this protein. We previously showed that IL1RAPL1 interacts with the neuronal calcium sensor NCS-1 and that it regulates voltage-gated calcium channel activity in PC12 cells. RESULTS: Here we show that IL1RAPL1 is present in dendritic spine where it interacts with PSD-95, a major component of excitatory postsynaptic compartment. Using gain- and loss-of-function experiments in neurons, we demonstrated that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun terminal kinase (JNK) activity and PSD-95 phosphorylation. Mice carrying a null mutation of the mouse Il1rapl1 gene show a reduction of both dendritic spine density and excitatory synapses in the CA1 region of the hippocampus. These structural abnormalities are associated with specific deficits in hippocampal long-term synaptic plasticity. CONCLUSION: The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.

SynDIG1: an activity-regulated, AMPA- receptor-interacting transmembrane protein that regulates excitatory synapse development.

  • Kalashnikova E
  • Neuron
  • 2010 Jan 14

Literature context:


Abstract:

During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.

A novel somatostatin-immunoreactive mossy fiber pathway associated with HSP25-immunoreactive purkinje cell stripes in the mouse cerebellum.

  • Armstrong CL
  • J. Comp. Neurol.
  • 2009 Dec 1

Literature context:


Abstract:

Somatostatin 28 immunoreactivity (Sst28-ir) identifies a specific subset of mossy fiber terminals in the adult mouse cerebellum. By using double-labeling immunohistochemistry, we determined that Sst28-ir is associated with presynaptic mossy fiber terminal rosettes, and not Purkinje cells, Golgi cells, or unipolar brush cells. Sst28-ir mossy fibers are restricted to the central zone (lobules VI/VII) and nodular zone (lobules IX, X) of the vermis, and the paraflocculus and flocculus. Within each transverse zone the mossy fiber terminal fields form a reproducible array of parasagittal stripes. The boundaries of Sst28-ir stripes align with a specific array of Purkinje cell stripes revealed by using immunocytochemistry for the small heat shock protein HSP25. In the cerebellum of the homozygous weaver mouse, in which a subpopulation of HSP25-ir Purkinje cells are located ectopically, the corresponding Sst28-ir mossy fiber projection is also ectopic, suggesting a role for a specific Purkinje cell subset in afferent pattern formation. Likewise, in the scrambler mutant mouse, Sst28-ir mossy fibers show a very close association with HSP25-ir Purkinje cell clusters. HSP25 itself does not appear to be critical for normal patterning, however: in the KJR mouse, which does not express cerebellar HSP25, Sst28 expression appears to be normal. Likewise, the Purkinje cell patterning antigens zebrin II and HSP25 are expressed normally in both Sst- and Sst-receptor knockout mice, suggesting that somatostatinergic transmission is not necessary for Purkinje cell stripe formation.

A coordinated local translational control point at the synapse involving relief from silencing and MOV10 degradation.

  • Banerjee S
  • Neuron
  • 2009 Dec 24

Literature context:


Abstract:

Persistent changes in synaptic strength are locally regulated by both protein degradation and synthesis; however, the coordination of these opposing limbs is poorly understood. Here, we found that the RISC protein MOV10 was present at synapses and was rapidly degraded by the proteasome in an NMDA-receptor-mediated activity-dependent manner. We designed a translational trap to capture those mRNAs whose spatiotemporal translation is regulated by MOV10. When MOV10 was suppressed, a set of mRNAs--including alpha-CaMKII, Limk1, and the depalmitoylating enzyme lysophospholipase1 (Lypla1)--selectively entered the polysome compartment. We also observed that Lypla1 mRNA is associated with the brain-enriched microRNA miR-138. Using a photoconvertible translation reporter, Kaede, we analyzed the activity-dependent protein synthesis driven by Lypla1 and alpha-CaMKII 3'UTRs. We established this protein synthesis to be MOV10 and proteasome dependent. These results suggest a unifying picture of a local translational regulatory mechanism during synaptic plasticity.

Early synapse formation in developing interneurons of the adult olfactory bulb.

  • Panzanelli P
  • J. Neurosci.
  • 2009 Dec 2

Literature context:


Abstract:

New olfactory bulb granule cells (GCs) are GABAergic interneurons continuously arising from neuronal progenitors and integrating into preexisting bulbar circuits. They receive both GABAergic and glutamatergic synaptic inputs from olfactory bulb intrinsic neurons and centrifugal afferents. Here, we investigated the spatiotemporal dynamic of newborn GC synaptogenesis in adult mouse olfactory bulb. First, we established that GABAergic synapses onto mature GC dendrites contain the GABA(A) receptor alpha2 subunit along with the postsynaptic scaffolding protein gephyrin. Next, we characterized morphologically and electrophysiologically the development of GABAergic and glutamatergic inputs onto newborn GCs labeled with eGFP (enhanced green fluorescent protein) using lentiviral vectors. Already when reaching the GC layer (GCL), at 3 d post-vector injection (dpi), newborn GCs exhibited tiny voltage-dependent sodium currents and received functional GABAergic and glutamatergic synapses, recognized immunohistochemically by apposition of specific presynaptic and postsynaptic markers. Thereafter, GABAergic and glutamatergic synaptic contacts increased differentially in the GCL, and at 7 dpi, PSD-95 clusters outnumbered gephyrin clusters. Thus, the weight of GABAergic input was predominant at early stages of GC maturation, but not later. Newborn GC dendrites first reached the external plexiform layer at 4 dpi, where they received functional GABAergic contacts at 5 dpi. Reciprocal synapses initially were formed on GC dendritic shafts, where they might contribute to spine formation. Their presence was confirmed ultrastructurally at 7 dpi. Together, our findings unravel rapid synaptic integration of newborn GCs in adult mouse olfactory bulb, with GABAergic and glutamatergic influences being established proximally before formation of output synapses by apical GC dendrites onto mitral/tufted cells.

Funding information:
  • Wellcome Trust - (United Kingdom)

Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function.

  • Varela-Nallar L
  • Neural Dev
  • 2009 Nov 2

Literature context:


Abstract:

BACKGROUND: The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. RESULTS: We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. CONCLUSION: Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

Toponomics analysis of drug-induced changes in arachidonic acid-dependent signaling pathways during spinal nociceptive processing.

  • Linke B
  • J. Proteome Res.
  • 2009 Oct 2

Literature context:


Abstract:

Multi-Epitope-Ligand-Carthography (MELC) allows consecutive immunohistochemical visualization of up to 100 proteins on the same tissue sample. Subsequent biomathematical analysis of these images allows a quantitative description of changes in protein networks. We used the MELC technology to study the effect of the nonopioid analgesic drug dipyrone on protein network profiles associated with arachidonic acid-dependent signaling pathways. MELC analysis with 31 different fluorescence-labeled tags was used to compare the effect of dipyrone on protein networks in spinal cords of mice with zymosan-induced hyperalgesia, a common model for inflammatory pain. We found that the number of motifs which describe the colocalization of 5-lipoxygenase (5-LO) or 12-LO with other proteins increased disproportionally after dipyrone treatment. Activation of 5-LO and 12-LO induces their translocation to membrane compartments which was also reflected by MELC results. Although no changes in 5-LO or 12-LO expression were seen by Western blot analysis or by immunohistochemistry in spinal cords of dipyrone-treated mice, the activation of both enzymes was verified by determining LO-products. Spinal amounts of 5(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are generated by 5-LO and 12-LO, respectively, were significantly increased in spinal cords of dipyrone-treated animals. In primary spinal cord neurons, dipyrone selectively and dose-dependently increased 5(S)-(HETE) and 12(S)-HETE synthesis. Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identify mechanisms of drug actions.

Arrested maturation of excitatory synapses in autosomal dominant lateral temporal lobe epilepsy.

  • Zhou YD
  • Nat. Med.
  • 2009 Oct 8

Literature context:


Abstract:

A subset of central glutamatergic synapses are coordinately pruned and matured by unresolved mechanisms during postnatal development. We report that the human epilepsy gene LGI1, encoding leucine-rich, glioma-inactivated protein-1 and mutated in autosomal dominant lateral temporal lobe epilepsy (ADLTE), mediates this process in hippocampus. We created transgenic mice either expressing a truncated mutant LGI1 (835delC) found in ADLTE or overexpressing a wild-type LGI1. We discovered that the normal postnatal maturation of presynaptic and postsynaptic functions was arrested by the 835delC mutant LGI1, and contrastingly, was magnified by excess wild-type LGI1. Concurrently, mutant LGI1 inhibited dendritic pruning and increased the spine density to markedly increase excitatory synaptic transmission. Inhibitory transmission, by contrast, was unaffected. Furthermore, mutant LGI1 promoted epileptiform discharge in vitro and kindling epileptogenesis in vivo with partial gamma-aminobutyric acid(A) (GABA(A)) receptor blockade. Thus, LGI1 represents a human gene mutated to promote epilepsy through impaired postnatal development of glutamatergic circuits.

Contribution of the neural cell recognition molecule NB-3 to synapse formation between parallel fibers and Purkinje cells in mouse.

  • Sakurai K
  • Dev Neurobiol
  • 2009 Oct 15

Literature context:


Abstract:

The neural cell recognition molecule NB-3, also referred to as contactin-6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB-3 to cerebellar development, focusing on lobule 3 where NB-3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG-1, L1, and NB-3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB-3-immunoreactive zone did not overlap with TAG-1-immunoreactive zone. By contrast, the L1-immunoreactive zone overlapped with both the TAG-1- and NB-3-immunoreactive zones. NB-3-positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB-3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB-3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB-3 knockout mice relative to wild-type mice at P5 to P10. In parallel with these findings, caspase-dependent cell death was significantly increased in the NB- 3-deficient cerebellum at P15. Collectively, our results indicate that NB-3 deficiency affects synapse formation during postnatal cerebellar development.

SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway.

  • Jeyifous O
  • Nat. Neurosci.
  • 2009 Aug 28

Literature context:


Abstract:

Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.

  • Hahn CG
  • PLoS ONE
  • 2009 Apr 16

Literature context:


Abstract:

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

Neuroadaptations in the cellular and postsynaptic group 1 metabotropic glutamate receptor mGluR5 and Homer proteins following extinction of cocaine self-administration.

  • Ghasemzadeh MB
  • Neurosci. Lett.
  • 2009 Mar 13

Literature context:


Abstract:

This study examined the role of group1 metabotropic glutamate receptor mGluR5 and associated postsynaptic scaffolding protein Homer1b/c in behavioral plasticity after three withdrawal treatments from cocaine self-administration. Rats self-administered cocaine or saline for 14 days followed by a withdrawal period during which rats underwent extinction training, remained in their home cages, or were placed in the self-administration chambers in the absence of extinction. Subsequently, the tissue level and distribution of proteins in the synaptosomal fraction associated with the postsynaptic density were examined. Cocaine self-administration followed by home cage exposure reduced the mGluR5 protein in nucleus accumbens (NA) shell and dorsolateral striatum. While extinction training reduced mGluR5 protein in NAshell, NAcore and dorsolateral striatum did not display any change. The scaffolding protein PSD95 increased in NAcore of the extinguished animals. Extinction of drug seeking was associated with a significant decrease in the synaptosomal mGluR5 protein in NAshell and an increase in dorsolateral striatum, while that of NAcore was not modified. Interestingly, both Homer1b/c and PSD95 scaffolding proteins were decreased in the synaptosomal fraction after extinction training in NAshell but not NAcore. Extinguished drug-seeking behavior was also associated with an increase in the synaptosomal actin proteins in dorsolateral striatum. Therefore, extinction of cocaine seeking is associated with neuroadaptations in mGluR5 expression and distribution that are region-specific and consist of extinction-induced reversal of cocaine-induced adaptations as well as emergent extinction-induced alterations. Concurrent plasticity in the scaffolding proteins further suggests that mGluR5 receptor neuroadaptations may have implications for synaptic function.

Functional tetrodotoxin-resistant Na(+) channels are expressed presynaptically in rat dorsal root ganglia neurons.

  • Medvedeva YV
  • Neuroscience
  • 2009 Mar 17

Literature context:


Abstract:

The tetrodotoxin-resistant (TTX-R) voltage-gated Na(+) channels Na(v)1.8 and Na(v)1.9 are expressed by a subset of primary sensory neurons and have been implicated in various pain states. Although recent studies suggest involvement of TTX-R Na(+) channels in sensory synaptic transmission and spinal pain processing, it remains unknown whether TTX-R Na(+) channels are expressed and function presynaptically. We examined expression of TTX-R channels at sensory synapses formed between rat dorsal root ganglion (DRG) and spinal cord (SC) neurons in a DRG/SC co-culture system. Immunostaining showed extensive labeling of presynaptic axonal boutons with Na(v)1.8- and Na(v)1.9-specific antibodies. Measurements using the fluorescent Na(+) indicator SBFI demonstrated action potential-induced presynaptic Na(+) entry that was resistant to tetrodotoxin (TTX) but was blocked by lidocaine. Furthermore, presynaptic [Ca(2+)](i) elevation in response to a single action potential was not affected by TTX in TTX-resistant DRG neurons. Finally, glutamatergic synaptic transmission was not inhibited by TTX in more than 50% of synaptic pairs examined; subsequent treatment with lidocaine completely blocked these TTX-resistant excitatory postsynaptic currents. Taken together, these results provide evidence for presynaptic expression of functional TTX-R Na(+) channels that may be important for shaping presynaptic action potentials and regulating transmitter release at the first sensory synapse.

Mislocalization of h channel subunits underlies h channelopathy in temporal lobe epilepsy.

  • Shin M
  • Neurobiol. Dis.
  • 2008 Oct 16

Literature context:


Abstract:

Many animal models of temporal lobe epilepsy (TLE) begin with status epilepticus (SE) followed by a latency period. Increased hippocampal pyramidal neuron excitability may contribute to seizures in TLE. I(h), mediated by h channels, regulates intrinsic membrane excitability by modulating synaptic integration and dampening dendritic calcium signaling. In a rat model of TLE, we found bidirectional changes in h channel function in CA1 pyramidal neurons. 1-2 d after SE, before onset of spontaneous seizures, physiological parameters dependent upon h channels were augmented and h channel subunit surface expression was increased. 28-30 d following SE, after onset of spontaneous seizures, h channel function in dendrites was reduced, coupled with diminished h channel subunit surface expression and relocalization of subunits from distal dendrites to soma. These results implicate h channel localization as a molecular mechanism influencing CA1 excitability in TLE.

Molecular dynamics of photoreceptor synapse formation in the developing chick retina.

  • Wahlin KJ
  • J. Comp. Neurol.
  • 2008 Feb 10

Literature context:


Abstract:

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT-PCR), dissociated retinal cells, laser-capture microdissection (LCM), immunocytochemistry and confocal microscopy. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5-8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin-2, syntaxin-3, synaptotagmin, glutamate receptors -1, -4, and -5, homer-1 and -2, and tenascin-R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated.

Funding information:
  • NIMH NIH HHS - MH48866(United States)

Capabilities of neurexins in the chick ciliary ganglion.

  • Ross BS
  • Dev Neurobiol
  • 2008 Feb 15

Literature context:


Abstract:

Transcellular interactions between neuroligins (NL) and beta-neurexin have been widely documented to promote maturation and function of both glutamatergic and GABAergic synapses. Recently it has been shown that neuroligin-1 plays a similar role at nicotinic synapses on chick ciliary ganglion neurons in culture, acting from the postsynaptic side to enhance transmitter release from adjacent cholinergic terminals and boost nicotinic input to the cells. We show here that the ciliary ganglion expresses three forms of neuroligin as well as two beta-neurexins and an alpha-neurexin. Overexpression of the beta-neurexins, but not the alpha-neurexin, can induce clustering of endogenous PSD-95 in adjacent neurons, presumably engaging neuroligin in the postsynaptic cell. The trans effects of beta-neurexins are selective; though both alpha3- and alpha7-containing nicotinic receptors are available on opposing cells, beta-neurexins induce coclustering of alpha3- but not alpha7-containing nicotinic receptors. Overexpression of other putative synaptogenic molecules, including SynCAM and L1, are ineffective at trans-clustering of PSD-95 on adjacent neurons. The beta-neurexins also exert a cis effect, coclustering presynaptic markers along with beta-neurexin in neurites juxtaposed to postsynaptic proteins, consistent with organizing presynaptic components as well. Striated muscle, the synaptic target of ciliary neurons in vivo, also expresses neuroligin. The results demonstrate that NL and neurexins are present at multiple sites in nicotinic cholinergic pathways and suggest the possibility of both cis- and trans-interactions to influence nicotinic signaling.

Funding information:
  • NICHD NIH HHS - P01 HD075750(United States)

Transient expression of LIM-domain transcription factors is coincident with delayed maturation of photoreceptors in the chicken retina.

  • Fischer AJ
  • J. Comp. Neurol.
  • 2008 Feb 1

Literature context:


Abstract:

In the retina of warm-blooded vertebrates, photoreceptors are specified many days before the onset of synaptogenesis and the expression of photopigments. The factors that regulate the maturation of photoreceptors in the developing retina remain unknown. We report here that photoreceptors transiently express LIM-domain transcription factors during the development of the chicken retina. We examined the differentiation of photoreceptors through the normal course of embryonic development and at the far periphery of the postnatal retina, where the differentiation of photoreceptors is slowed and persists across a spatial gradient. In the embryonic retina, we find visinin-positive photoreceptors that transiently express Islet2 and Lim3 starting at E8 and ending around E15, but persisting in far peripheral regions of the retina through the first 2 weeks of postnatal development. During early stages of photoreceptor maturation, there is coincident and transient expression of the LIM-domain factors with axonin1, a cell surface glycoprotein that is a member of the immunoglobulin superfamily. Coincident with the downregulation of Islet2 and Lim3, we find the upregulation of calbindin, red/green opsin, rhodopsin, and a synaptic marker in the outer plexiform layer (OPL; dystrophin). In the periphery of the postnatal retina, photoreceptors that express Islet2, Lim3, and axonin1 do not overlap with photoreceptors that express calbindin, red/green opsin, rhodopsin, and dystrophin. We propose that Islet2 and Lim3 may promote the expression of genes that are involved in the early stages of differentiation but may suppress the expression of genes that are required in the mature photoreceptors.

Funding information:
  • NIMH NIH HHS - P50MH103222(United States)

Developmental regulation of muscleblind-like (MBNL) gene expression in the chicken embryo retina.

  • Huang H
  • Dev. Dyn.
  • 2008 Jan 27

Literature context:


Abstract:

Muscleblind-like (MBNL) is a CCCH zinc finger-containing RNA-binding protein required for the development of both muscle and photoreceptors in Drosophila; it is conserved evolutionarily, and it is associated in humans with the muscular disease myotonic dystrophy. Its role in the development of vertebrate retinal cells, however, remains unknown. As an initial approach to its investigation, we have cloned three chick muscleblind genes, characterized their isoforms, and examined their expression patterns in the chick embryo retina. The relative levels of expression of the MBNL genes increased during embryonic development. In situ hybridization (ISH) showed that the three MBNL mRNAs had widespread patterns of expression at all the developmental stages examined. Of interest, the temporal and spatial patterns of protein expression, detected by immunocytochemistry with antibodies against MBNL1 and MBNL2, were much more restricted than those seen by ISH. At early stages (ED5-7), for example, MBNL1 and MBNL2 mRNAs were present throughout the retina, but immunoreactivity for the corresponding proteins was largely restricted to the periphery of the optic cup (presumptive iris/ciliary epithelium/ciliary margin zone). MBNL1 and MBNL2 immunoreactivity became detectable at the fundus at later stages, but was limited to a very small subset of the cells that had ISH signals for the cognate mRNAs (particularly ganglion cells and photoreceptors). Within photoreceptors, MBNL1 and MBNL2 immunoreactivity first appeared in their inner segments; MBNL2 remained there, but MBNL1 became subsequently localized to their synaptic terminals. These expression patterns are consistent with the possibility that MBNLs may regulate photoreceptor development in the chick retina, much as MBL does in Drosophila, and suggest that the expression of MBNL1 and MBNL2 may be regulated posttranscriptionally.

Funding information:
  • NINDS NIH HHS - R21 NS072588(United States)

The classical complement cascade mediates CNS synapse elimination.

  • Stevens B
  • Cell
  • 2007 Dec 14

Literature context:


Abstract:

During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

Funding information:
  • NINDS NIH HHS - NS37853(United States)

Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus.

  • Wenzel HJ
  • Epilepsia
  • 2007 Nov 16

Literature context:


Abstract:

PURPOSE: Mice lacking the Kv1.1 potassium channel alpha subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1(-/-) (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. METHODS: Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1(-/-) animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1(-/-) brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E. coli lacZ reporter gene. Vector-injected mice were examined to determine the extent of neuronal infection. RESULTS: Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1(-/-) mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed "ectopic" Kv1.1 alpha channel subunit expression. CONCLUSIONS: Kcna1 deletion in mice results in a seizure disorder that resembles--electrographically and neuropathologically--the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection.

Funding information:
  • NIMH NIH HHS - R01 MH084812(United States)

NMDA di-heteromeric receptor populations and associated proteins in rat hippocampus.

  • Al-Hallaq RA
  • J. Neurosci.
  • 2007 Aug 1

Literature context:


Abstract:

Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A- or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2B or NR2A, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day 7 (P7) hippocampus and P42 and 6-month-old CA1/CA2. Quantitative Western blot analysis revealed that 60-70% of NR2A and 70-85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A- or NR2B-specific interactions with synapse-associated proteins. Our results indicate that NR2A- or NR2B-containing NMDARs associate similarly with postsynaptic density-95 (PSD-95), synapse-associated protein 102, and PSD-93 at P42. However, NR2A-containing receptors coimmunoprecipitated a greater proportion of the synaptic proteins neuronal nitric oxide synthase, Homer, and beta-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, collapsin response mediator protein 2, which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist primarily in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes.

Funding information:
  • Wellcome Trust - 087618/Z/08/Z(United Kingdom)

Regulation of Kv1 channel trafficking by the mamba snake neurotoxin dendrotoxin K.

  • Vacher H
  • FASEB J.
  • 2007 Mar 1

Literature context:


Abstract:

Modulation of voltage-gated potassium (Kv) channel surface expression can profoundly affect neuronal excitability. Some, but not all, mammalian Shaker or Kv1 alpha subunits contain a dominant endoplasmic reticulum (ER) retention signal in their pore region, preventing surface expression of Kv1.1 homotetrameric channels and of heteromeric Kv1 channels containing more than one Kv1.1 subunit. The critical amino acid residues within this ER pore-region retention signal are also critical for high-affinity binding of snake dendrotoxins (DTX). This suggests that ER retention may be mediated by an ER protein with a domain structurally similar to that of DTX. One facet of such a model is that expression of soluble DTX in the ER lumen should compete for binding to the retention protein and allow for surface expression of retained Kv1.1. Here, we show that luminal DTX expression dramatically increased both the level of cell surface Kv1.1 immunofluorescence staining and the proportion of Kv1.1 with processed N-linked oligosaccharides. Electrophysiological analyses showed that luminal DTX expression led to significant increases in Kv1.1 currents. Together, these data showed that luminal DTX expression increases surface expression of functional Kv1.1 homotetrameric channels and support a model whereby a DTX-like ER protein regulates abundance of cell surface Kv1 channels.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/E004431/1(United Kingdom)

[Hygiene phase of periodontal therapy].

  • Ramfjord SP
  • Phillip J
  • 1990 Aug 1

Literature context:


Abstract:

Funding information:
  • NIGMS NIH HHS - GM-069801(United States)