STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.

Lesions and mutations of the DISC1 (Disrupted-in-schizophrenia-1) gene have been linked to major depression, schizophrenia, bipolar disorder and autism, but the influence of DISC1 on synaptic transmission remains poorly understood. Using two independent genetic approaches-RNAi and a DISC1 KO mouse-we examined the impact of DISC1 on the synaptic vesicle (SV) cycle by population imaging of the synaptic tracer vGpH in hippocampal neurons. DISC1 loss-of-function resulted in a marked decrease in SV exocytic rates during neuronal stimulation and was associated with reduced Ca(2+) transients at nerve terminals. Impaired SV release was efficiently rescued by elevation of extracellular Ca(2+), hinting at a link between DISC1 and voltage-gated Ca(2+) channels. Accordingly, blockade of N-type Cav2.2 channels mimics and occludes the effect of DISC1 inactivation on SV exocytosis, and overexpression of DISC1 in a heterologous system increases Cav2.2 currents. Collectively, these results show that DISC1-dependent enhancement of SV exocytosis is mediated by Cav2.2 and point to aberrant glutamate release as a probable endophenotype of major psychiatric disorders.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is conventionally considered to be solely dependent on local protein synthesis. Given the impact of epigenetics on memory, the intriguing question is whether epigenetic regulation influences mGluR-LTD as well. G9a/GLP histone lysine methyltransferase complex is crucial for brain development and goal-directed learning as well as for drug-addiction. In this study, we analyzed whether the epigenetic regulation by G9a/GLP complex affects mGluR-LTD in CA1 hippocampal pyramidal neurons of 5-7 weeks old male Wistar rats. In hippocampal slices with intact CA1 dendritic regions, inhibition of G9a/GLP activity abolished mGluR-LTD. The inhibition of this complex upregulated the expression of plasticity proteins like PKMζ, which mediated the prevention of mGluR-LTD expression by regulating the NSF-GluA2-mediated trafficking of AMPA receptors towards the postsynaptic site. G9a/GLP inhibition during the induction of mGluR-LTD also downregulated the protein levels of phosphorylated-GluA2 and Arc. Interestingly, G9a/GLP inhibition could not impede the mGluR-LTD when the cell-body was severed. Our study highlights the role of G9a/GLP complex in intact neuronal network as a bidirectional switch; when turned on, it facilitates the expression of mGluR-LTD, and when turned off, it promotes the expression of long-term potentiation.

Altered epigenetic mechanisms are implicated in the cognitive decline associated with neurodegenerative diseases such as in Alzheimer's disease (AD). AD is the most prevalent form of dementia worldwide; amyloid plaques and neurofibrillary tangles are the histopathological hallmarks of AD. We have recently reported that the inhibition of G9a/GLP complex promotes long-term potentiation (LTP) and its associative mechanisms such as synaptic tagging and capture (STC). However, the role of this complex in plasticity impairments remains elusive. Here, we investigated the involvement of G9a/GLP complex in alleviating the effects of soluble Amyloid-β 1-42 oligomers (oAβ) on neuronal plasticity and associativity in the CA1 region of acute hippocampal slices from 5- to 7-week-old male Wistar rats. Our findings demonstrate that the regulation of G9a/GLP complex by inhibiting its catalytic activity reverses the amyloid-β oligomer-induced deficits in late-LTP and STC. This is achieved by releasing the transcription repression of the brain-derived neurotrophic factor (Bdnf) gene. The catalytic inhibition of G9a/GLP complex leads to the upregulation of Bdnf expression in the slices treated with oAβ. This further ensures the availability of BDNF that subsequently binds its receptor tyrosine kinase B (TrkB) and maintains the late-LTP. Furthermore, the capture of BDNF by weakly activated synapses re-establishes STC. Our findings regarding the reinstatement of functional plasticity and associativity in AD-like conditions provide the first evidence for the role of G9a/GLP complex in AD. We propose G9a/GLP complex as the possible target for preventing oAβ-induced plasticity deficits in hippocampal neurons.

L-type CaV1.3 calcium channels are expressed on the dendrites and soma of neurons, and there is a paucity of information about its role in hippocampal plasticity. Here, by genetic targeting to ablate CaV1.3 RNA editing, we demonstrate that unedited CaV1.3ΔECS mice exhibited improved learning and enhanced long-term memory, supporting a functional role of RNA editing in behavior. Significantly, the editing paradox that functional recoding of CaV1.3 RNA editing sites slows Ca2+-dependent inactivation to increase Ca2+ influx but reduces channel open probability to decrease Ca2+ influx was resolved. Mechanistically, using hippocampal slice recordings, we provide evidence that unedited CaV1.3 channels permitted larger Ca2+ influx into the hippocampal pyramidal neurons to bolster neuronal excitability, synaptic transmission, late long-term potentiation, and increased dendritic arborization. Of note, RNA editing of the CaV1.3 IQ-domain was found to be evolutionarily conserved in mammals, which lends support to the importance of the functional recoding of the CaV1.3 channel in brain function.

Synaptic cooperation and competition are important components of synaptic plasticity that tune synapses for the formation of associative long-term plasticity, a cellular correlate of associative long-term memory. We have recently reported that coincidental activation of weak synapses within the vicinity of potentiated synapses will alter the cooperative state of synapses to a competitive state thus leading to the slow decay of long-term plasticity, but the molecular mechanism underlying this is still unknown. Here, using acute hippocampal slices of rats, we have examined how increasing extracellular dopamine concentrations interact and/or affect electrically induced long-term potentiation (LTP) in the neighboring synapses. We demonstrate that D1/D5-receptor-mediated potentiation at the CA1 Schaffer collateral synapses differentially regulates synaptic co-operation and competition. Further investigating the molecular players involved, we reveal an important role for extracellular signal-regulated kinases-1 and 2 (ERK1/2) as signal integrators and dose-sensors. Interestingly, a sustained activation of ERK1/2 pathway seems to be involved in the differential regulation of synaptic associativity. The concentration-dependent effects of the modulatory transmitter, as demonstrated for dopaminergic signaling in the present study, might offer additional computational power by fine tuning synaptic associativity processes for establishing long-term associative memory in neural networks.

Aging is associated with impaired plasticity and memory. Altered epigenetic mechanisms are implicated in the impairment of memory with advanced aging. Histone deacetylase 3 (HDAC3) is an important negative regulator of memory. However, the role of HDAC3 in aged neural networks is not well established. Late long-term potentiation (late-LTP), a cellular correlate of memory and its associative mechanisms such as synaptic tagging and capture (STC) were studied in the CA1 area of hippocampal slices from 82-84 week old rats. Our findings demonstrate that aging is associated with deficits in the magnitude of LTP and impaired STC. Inhibition of HDAC3 augments the late-LTP and re-establishes STC. The augmentation of late-LTP and restoration of STC is mediated by the activation of nuclear factor kappa B (NFκB) pathway. We provide evidence for the promotion of associative plasticity in aged neural networks by HDAC3 inhibition and hence propose HDAC3 and NFκB as the possible therapeutic targets for treating age -related cognitive decline.

The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor-mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.

Aging is associated with decline in cognitive functions, prominently in the memory consolidation and association capabilities. Hippocampus plays a crucial role in the formation and maintenance of long-term associative memories, and a significant body of evidence shows that impairments in hippocampal function correlate with aging-related memory loss. A number of studies have implicated alterations in hippocampal synaptic plasticity, such as long-term potentiation (LTP), in age-related cognitive decline although exact mechanisms underlying are not completely clear. Zinc deficiency and the resultant adverse effects on cognition have been well studied. However, the role of excess of zinc in synaptic plasticity, especially in aging, is not addressed well. Here, we have investigated the hippocampal zinc levels and the impairments in synaptic plasticity, such as LTP and synaptic tagging and capture (STC), in the CA1 region of acute hippocampal slices from 82- to 84-week-old male Wistar rats. We report increased zinc levels in the hippocampus of aged rats and also deficits in the tetani-induced and dopaminergic agonist-induced late-LTP and STC. The observed deficits in synaptic plasticity were restored upon chelation of zinc using a cell-permeable chelator. These data suggest that functional plasticity and associativity can be successfully established in aged neural networks by chelating zinc with cell-permeable chelating agents.

The CD137-CD137 ligand (CD137L) costimulatory system is a critical immune checkpoint with pathophysiological implications in autoimmunity. In this study, we investigated the role of CD137L-mediated costimulation on renal, cutaneous and cerebral manifestations in lupus and the underlying immunological mechanism. Lupus-prone C57BL/6lpr-/- (B6.lpr) mice were crossed to C57BL/6.CD137L-/- mice to obtain CD137L-deficient B6.lpr [double knock out (DKO)] mice. We investigated the extent of survival, glomerulonephritis, skin lesions, cerebral demyelination, immune deviation and long-term synaptic plasticity among the two mouse groups. Cytokine levels, frequency of splenic leukocyte subsets and phenotypes were compared between DKO, B6.lpr and B6.WT mice. A 22 month observation of 226 DKO and 137 B6.lpr mice demonstrated significantly more frequent proliferative glomerulonephritis, larger skin lesions and shorter survival in DKO than in B6.lpr mice. Conversely, microglial activation and cerebral demyelination were less pronounced while long-term synaptic plasticity, was superior in DKO mice. Splenic Th17 cells were significantly higher in DKO than in B6.lpr and B6.WT mice while Th1 and Th2 cell frequencies were comparable between DKO and B6.lpr mice. IL-10 and IL-17 expression by T cells was not affected but there were fewer IL-10-producing myeloid (CD11b+) cells, and also lower serum IL-10 levels in DKO than in B6.lpr mice. The absence of CD137L causes an immune deviation toward Th17, fewer IL-10-producing CD11b+ cells and reduced serum IL-10 levels which potentially explain the more severe lupus in DKO mice while leading to reduced microglia activation, lesser cerebral damage and less severe neurological deficits.

Progressive memory loss is one of the most common characteristics of Alzheimer's disease (AD), which has been shown to be caused by several factors including accumulation of amyloid β peptide (Aβ) plaques and neurofibrillary tangles. Synaptic plasticity and associative plasticity, the cellular basis of memory, are impaired in AD. Recent studies suggest a functional relevance of microRNAs (miRNAs) in regulating plasticity changes in AD, as their differential expressions were reported in many AD brain regions. However, the specific role of these miRNAs in AD has not been elucidated. We have reported earlier that late long-term potentiation (late LTP) and its associative mechanisms such as synaptic tagging and capture (STC) were impaired in Aβ (1-42)-induced AD condition. This study demonstrates that expression of miR-134-5p, a brain-specific miRNA is upregulated in Aβ (1-42)-treated AD hippocampus. Interestingly, the loss of function of miR-134-5p restored late LTP and STC in AD. In AD brains, inhibition of miR-134-5p elevated the expression of plasticity-related proteins (PRPs), cAMP-response-element binding protein (CREB-1) and brain-derived neurotrophic factor (BDNF), which are otherwise downregulated in AD condition. The results provide the first evidence that the miR-134-mediated post-transcriptional regulation of CREB-1 and BDNF is an important molecular mechanism underlying the plasticity deficit in AD; thus demonstrating the critical role of miR-134-5p as a potential therapeutic target for restoring plasticity in AD condition.

Metabotropic glutamate receptors (mGluRs) play an important role in synaptic plasticity and memory and are largely classified based on amino acid sequence homology and pharmacological properties. Among group III metabotropic glutamate receptors, mGluR7 and mGluR4 show high relative expression in the rat hippocampal area CA2. Group III metabotropic glutamate receptors are known to down-regulate cAMP-dependent signaling pathways via the activation of Gi/o proteins. Here, we provide evidence that inhibition of group III mGluRs by specific antagonists permits an NMDA receptor- and protein synthesis-dependent long-lasting synaptic potentiation in the apparently long-term potentiation (LTP)-resistant Schaffer collateral (SC)-CA2 synapses. Moreover, long-lasting potentiation of these synapses transforms a transient synaptic potentiation of the entorhinal cortical (EC)-CA2 synapses into a stable long-lasting LTP, in accordance with the synaptic tagging/capture hypothesis (STC). Furthermore, this study also sheds light on the role of ERK/MAPK protein signaling and the downregulation of STEP protein in the group III mGluR inhibition-mediated plasticity in the hippocampal CA2 region, identifying them as critical molecular players. Thus, the regulation of group III mGluRs provides a conducive environment for the SC-CA2 synapses to respond to events that could lead to activity-dependent synaptic plasticity.

Microglia are the main form of immune defense in the central nervous system. Microglia express phosphatidylinositol 3-kinase (PI3K), which has been shown to play a significant role in synaptic plasticity in neurons and inflammation via microglia. This study shows that microglial PI3K is regulated epigenetically through histone modifications and posttranslationally through sumoylation and is involved in long-term potentiation (LTP) by modulating the expression of brain-derived neurotrophic factor (BDNF), which has been shown to be involved in neuronal synaptic plasticity. Sodium butyrate, a histone deacetylase inhibitor, upregulates PI3K expression, the phosphorylation of its downstream effectors, AKT and cAMP response element-binding protein (CREB), and the expression of BDNF in microglia, suggesting that BDNF secretion is regulated in microglia via epigenetic regulation of PI3K. Further, knockdown of SUMO1 in BV2 microglia results in a decrease in the expression of PI3K, the phosphorylation of AKT and CREB, as well as the expression of BDNF. These results suggest that microglial PI3K is epigenetically regulated by histone modifications and posttranslationally modified by sumoylation, leading to altered expression of BDNF. Whole-cell voltage-clamp showed the involvement of microglia in neuronal LTP, as selective ablation or disruption of microglia with clodronate in rat hippocampal slices abolished LTP. However, LTP was rescued when the same hippocampal slices were treated with active PI3K or BDNF, indicating that microglial PI3K/AKT signaling contributes to LTP and synaptic plasticity. Understanding the mechanisms by which microglial PI3K influences synapses provides insights into the ways it can modulate synaptic transmission and plasticity in learning and memory.

STIM2 is an integral membrane protein of the endoplasmic reticulum (ER) that regulates the activity of plasma membrane (PM) channels at ER-PM contact sites. Recent studies show that STIM2 promotes spine maturation and surface expression of the AMPA receptor (AMPAR) subunit GluA1, hinting at a probable role in synaptic plasticity. Here, we used a Stim2 cKO mouse to explore the function of STIM2 in Long-Term Potentiation (LTP) and Depression (LTD), two widely-studied models of synaptic plasticity implicated in information storage. We found that STIM2 is required for the stable expression of both LTP and LTD at CA3-CA1 hippocampal synapses. Altered plasticity in Stim2 cKO mice is associated with subtle alterations in the shape and density of dendritic spines in CA1 neurons. Further, surface delivery of GluA1 in response to LTP-inducing chemical manipulations was markedly reduced in excitatory neurons derived from Stim2 cKO mice. GluA1 endocytosis following chemically-induced LTD was also impaired in Stim2 cKO neurons. We conclude that STIM2 facilitates synaptic delivery and removal of AMPARs and regulates activity-dependent changes in synaptic strength through a unique mode of communication between the ER and the synapse.

Presynaptic assembly involves the specialization of a patch of axonal membrane into a complex structure that supports synaptic vesicle exocytosis and neurotransmitter release. In mammalian neurons, presynaptic assembly is widely studied in a co-culture assay, where a synaptogenic cue expressed at the surface of a heterologous cell induces presynaptic differentiation in a contacting axon. This assay has led to the discovery of numerous synaptogenic proteins, but has not been used to probe neuronal mechanisms regulating presynaptic induction. The identification of regulatory pathways that fine-tune presynaptic assembly is hindered by the lack of adequate tools to quantitatively image this process. Here, we introduce an image-processing algorithm that identifies presynaptic clusters in mammalian co-cultures and extracts a range of synapse-specific parameters. Using this software, we assessed the intrinsic variability of this synaptic induction assay and probed the effect of eight neuronal microRNAs on presynaptic assembly. Our analysis revealed a novel role for miR-27b in augmenting the density of presynaptic clusters. Our software is applicable to a wide range of synaptic induction protocols (including spontaneous synaptogenesis observed in neuron cultures) and is a valuable tool to determine the subtle impact of disease-associated genes on presynaptic assembly.

Self-amplification of phosphoinositide 3-kinase (PI3K) signaling is believed to regulate asymmetric membrane extension and cell migration, but the molecular organization of the underlying feedback circuit is elusive. Here we use an inducible approach to synthetically activate PI3K and interrogate the feedback circuitry governing self-enhancement of 3'-phosphoinositide (3-PI) signals in NIH3T3 fibroblasts. Synthetic activation of PI3K initially leads to uniform production of 3-PIs at the plasma membrane, followed by the appearance of asymmetric and highly amplified 3-PI signals. A detailed spatiotemporal analysis shows that local self-amplifying 3-PI signals drive rapid membrane extension with remarkable directional persistence and initiate a robust migratory response. This positive feedback loop is critically dependent on the small GTPase HRas. Silencing of HRas abrogates local amplification of 3-PI signals upon synthetic PI3K activation and results in short-lived protrusion events that do not support cell migration. Finally, our data indicate that this feedback circuit is likely to operate during platelet-derived growth factor-induced random cell migration. We conclude that positive feedback between PI3K and HRas is essential for fibroblasts to spontaneously self-organize and generate a productive migratory response in the absence of spatial cues.

Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.

Exposure to juvenile stress was found to have long-term effects on the plasticity and quality of associative memory in adulthood, but the underlying mechanisms are still poorly understood.

Studying the complex mechanisms underlying breast cancer metastasis and therapy response necessitates relevant in vivo models, particularly syngeneic models with an intact immune system. Two syngeneic spontaneously metastatic sublines, D2A1-m1 and D2A1-m2, were generated from the poorly metastasising BALB/c-derived D2A1 cell line by serial in vivo passaging. In vivo and in vitro analyses revealed distinct and shared characteristics of the metastatic D2A1-m1 and D2A1-m2 sublines. In particular, D2A1-m1 cells are more aggressive in experimental metastasis assays, while D2A1-m2 cells are more efficient at disseminating from the primary tumour in spontaneous metastasis assays. Surprisingly, classical metastasis-associated in vitro phenotypes, such as enhanced proliferation, migration and invasion, are reduced in the sublines compared to the parental cell line. Further, evasion of immune control cannot fully explain their enhanced metastatic properties. By contrast, both sublines show increased resistance to apoptosis when cultured in non-adherent conditions and, for the D2A1-m2 subline, increased 3D tumour spheroid growth. Moreover, the enhanced spontaneous metastatic phenotype of the D2A1-m2 subline is associated with an increased ability to recruit an activated tumour stroma. The metastatic D2A1-m1 and D2A1-m2 cell lines provide additional syngeneic models for investigating the different steps of the metastatic cascade and thereby represent valuable tools for breast cancer researchers. Finally, this study highlights that morphology and cell behaviour in 2D cell-based assays cannot be used as a reliable predictor of metastatic behaviour in vivo.

Attempts have been made to use glycogen synthase kinase-3 beta (GSK3β) inhibitors for prophylactic treatment of neurocognitive conditions. However the use of lithium, a non-specific inhibitor of GSK3β results in mild cognitive impairment in humans. The effects of global GSK3β inhibition or knockout on learning and memory in healthy adult mice are also inconclusive. Our study aims to better understand the role of GSK3β in learning and memory through a more regionally, targeted approach, specifically performing lentiviral-mediated knockdown of GSK3β within the dentate gyrus (DG). DG-GSK3β-silenced mice showed impaired contextual fear memory retrieval. However, cue fear memory, spatial memory, locomotor activity and anxiety levels were similar to control. These GSK3β-silenced mice also showed increased induction and maintenance of DG long-term potentiation (DG-LTP) compared to control animals. Thus, this region-specific, targeted knockdown of GSK3β in the DG provides better understanding on the role of GSK3β in learning and memory.