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GFP-avictoria antibody

RRID:AB_303395

Antibody ID

AB_303395

Target Antigen

GFP aequorea victoria

Proper Citation

(Abcam Cat# ab290, RRID:AB_303395)

Clonality

polyclonal antibody

Comments

ENCODE PROJECT External validation for lot# GR158277-1 is available under ENCODE ID: ENCAB976SGG. This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP and EGFP.

Host Organism

rabbit

Vendor

Abcam Go To Vendor

Cat Num

ab290 also ENCAB976SGG

Publications that use this research resource

Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors.

  • Pavlyukov MS
  • Cancer Cell
  • 2018 Jul 9

Literature context:


Abstract:

Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, we demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, we identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Funding information:
  • Cancer Research UK - (United Kingdom)
  • NCI NIH HHS - R01 CA183991()
  • NCI NIH HHS - R01 CA201402()
  • NINDS NIH HHS - R01 NS083767()

The basal interstitial nucleus (BIN) of the cerebellum provides diffuse ascending inhibitory input to the floccular granule cell layer.

  • Jaarsma D
  • J. Comp. Neurol.
  • 2018 Jun 26

Literature context:


Abstract:

The basal interstitial nucleus (BIN) in the white matter of the vestibulocerebellum has been defined more than three decades ago, but has since been largely ignored. It is still unclear which neurotransmitters are being used by BIN neurons, how these neurons are connected to the rest of the brain and what their activity patterns look like. Here, we studied BIN neurons in a range of mammals, including macaque, human, rat, mouse, rabbit and ferret, using tracing, immunohistological and electrophysiological approaches. We show that BIN neurons are GABAergic and glycinergic, that in primates they also express the marker for cholinergic neurons choline acetyl transferase (ChAT), that they project with beaded fibers to the glomeruli in the granular layer of the ipsilateral floccular complex, and that they are driven by excitation from the ipsilateral and contralateral medio-dorsal medullary gigantocellular reticular formation. Systematic analysis of co-distribution of the inhibitory synapse marker VIAAT, labeled BIN axons and Golgi cell marker mGluR2 indicate that BIN axon terminals complement Golgi cell axon terminals in glomeruli, accounting for a considerable proportion (> 20%) of the inhibitory terminals in the granule cell layer of the floccular complex. Together, these data show that BIN neurons represent a novel and relevant inhibitory input to the part of the vestibulocerebellum that controls compensatory and smooth pursuit eye movements. This article is protected by copyright. All rights reserved.

Funding information:
  • NCI NIH HHS - 2R15CA113747-02(United States)

Hippocampal NF-κB accounts for stress-induced anxiety behaviors via enhancing nNOS-CAPON-Dexras1 coupling.

  • Zhu LJ
  • J. Neurochem.
  • 2018 Jun 2

Literature context:


Abstract:

Anxiety disorders are associated with a high social burden worldwide. Recently, increasing evidence suggests that nuclear factor kappa B (NF-κB) has significant implications for psychiatric diseases, including anxiety and depressive disorders. However, the molecular mechanisms underlying the role of NF-κB in stress-induced anxiety behaviors are poorly understood. In this study, we show that chronic mild stress (CMS) and glucocorticoids dramatically increased the expression of NF-κB subunits p50 and p65, phosphorylation and acetylation of p65, and the level of nuclear p65 in vivo and in vitro, implicating activation of NF-κB signaling in chronic stress-induced pathological processes. Using the novelty-suppressed feeding (NSF) and elevated-plus maze (EPM) tests, we found that treatment with pyrrolidine dithiocarbamate (PDTC; intrahippocampal infusion), an inhibitor of NF-κB, rescued the CMS- or glucocorticoid-induced anxiogenic behaviors in mice. Microinjection of PDTC into the hippocampus reversed CMS-induced upregulation of neuronal nitric oxide synthase (nNOS), carboxy-terminal PDZ ligand of nNOS (CAPON) and dexamethasone-induced ras protein 1 (Dexras1) and dendritic spine loss of DG granule cells. Moreover, overexpression of CAPON by infusing LV-CAPON-L-GFP into the hippocampus induced nNOS-Dexras1 interaction and anxiety-like behaviors, and inhibition of NF-κB by PDTC reduced the LV-CAPON-L-GFP-induced increases in nNOS-Dexras1 complex and anxiogenic-like effects in mice. These findings indicate that hippocampal NF-κB mediates anxiogenic behaviors, probably via regulating the association of nNOS-CAPON-Dexras1, and uncover a novel approach to the treatment of anxiety disorders. This article is protected by copyright. All rights reserved.

Funding information:
  • NIGMS NIH HHS - GM GM69373(United States)

Planar cell polarity signalling coordinates heart tube remodelling through tissue-scale polarisation of actomyosin activity.

  • Merks AM
  • Nat Commun
  • 2018 Jun 4

Literature context:


Abstract:

Development of a multiple-chambered heart from the linear heart tube is inherently linked to cardiac looping. Although many molecular factors regulating the process of cardiac chamber ballooning have been identified, the cellular mechanisms underlying the chamber formation remain unclear. Here, we demonstrate that cardiac chambers remodel by cell neighbour exchange of cardiomyocytes guided by the planar cell polarity (PCP) pathway triggered by two non-canonical Wnt ligands, Wnt5b and Wnt11. We find that PCP signalling coordinates the localisation of actomyosin activity, and thus the efficiency of cell neighbour exchange. On a tissue-scale, PCP signalling planar-polarises tissue tension by restricting the actomyosin contractility to the apical membranes of outflow tract cells. The tissue-scale polarisation of actomyosin contractility is required for cardiac looping that occurs concurrently with chamber ballooning. Taken together, our data reveal that instructive PCP signals couple cardiac chamber expansion with cardiac looping through the organ-scale polarisation of actomyosin-based tissue tension.

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

ZL006 promotes migration and differentiation of transplanted neural stem cells in male rats after stroke.

  • Wang DL
  • J. Neurosci. Res.
  • 2018 Jun 8

Literature context:


Abstract:

New strategies must be developed to resolve the problems of stroke treatment. In recent years, stem cell-based therapy after stroke has come into the public and academic lens. Previously we have shown that uncoupling neuronal nitric oxide synthase (nNOS) from the postsynaptic density protein-95 (PSD-95) by ZL006, a small molecular compound, can ameliorate ischemic damage and promote neuronal differentiation of endogenous neural stem cells (NSCs) in focal cerebral ischemic male rats. In this study, we transplanted exogenous NSCs into the ipsilateral hemisphere of male rats in combination with ZL006 treatment after ischemic stroke. We show that ZL006 treatment facilitates the migration of transplanted NSCs into the ischemia-injured area and promotes neuronal differentiation of these cells, which is not due to a direct effect of ZL006 on exogenous NSCs but is associated with increased phosphorylation of cAMP response element-binding protein (CREB) in neurons and favorable microenvironment. Moreover, improved functional outcome in the ZL006-treated group was also found. Taken together, our data indicate that ZL006, uncoupling nNOS-PSD-95 in neurons, positively regulates the fate of transplanted NSCs and benefits the functional outcome after stroke in male rats.

Excitatory and Inhibitory Neurons Utilize Different Ca2+ Sensors and Sources to Regulate Spontaneous Release.

  • Courtney NA
  • Neuron
  • 2018 Jun 6

Literature context:


Abstract:

Spontaneous neurotransmitter release (mini) is an important form of Ca2+-dependent synaptic transmission that occurs in the absence of action potentials. A molecular understanding of this process requires an identification of the underlying Ca2+ sensors. Here, we address the roles of the relatively low- and high-affinity Ca2+ sensors, synapotagmin-1 (syt1) and Doc2α/β, respectively. We found that both syt1 and Doc2 regulate minis, but, surprisingly, their relative contributions depend on whether release was from excitatory or inhibitory neurons. Doc2α promoted glutamatergic minis, while Doc2β and syt1 both regulated GABAergic minis. We identified Ca2+ ligand mutations in Doc2 that either disrupted or constitutively activated the regulation of minis. Finally, Ca2+ entry via voltage-gated Ca2+ channels triggered miniature GABA release by activating syt1, but had no effect on Doc2-driven minis. This work reveals an unexpected divergence in the regulation of spontaneous excitatory and inhibitory transmission in terms of both Ca2+ sensors and sources.

Funding information:
  • Intramural NIH HHS - (United States)
  • NIGMS NIH HHS - T32 GM008692()

A transcriptomics resource reveals a transcriptional transition during ordered sarcomere morphogenesis in flight muscle.

  • Spletter ML
  • Elife
  • 2018 May 30

Literature context:


Abstract:

Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study sarcomerogenesis, we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, most sarcomeric components group in two clusters, which are strongly induced after all myofibrils have been assembled, indicating a transcriptional transition during myofibrillogenesis. Following myofibril assembly, many short sarcomeres are added to each myofibril. Subsequently, all sarcomeres mature, reaching 1.5 µm diameter and 3.2 µm length and acquiring stretch-sensitivity. The efficient induction of the transcriptional transition during myofibrillogenesis, including the transcriptional boost of sarcomeric components, requires in part the transcriptional regulator Spalt major. As a consequence of Spalt knock-down, sarcomere maturation is defective and fibers fail to gain stretch-sensitivity. Together, this defines an ordered sarcomere morphogenesis process under precise transcriptional control - a concept that may also apply to vertebrate muscle or heart development.

Funding information:
  • Agence Nationale de la Recherche - ANR ACHN()
  • Agence Nationale de la Recherche - ANR-10-INBS-04- 01()
  • Agence Nationale de la Recherche - ANR-11- LABX-0054()
  • Aix-Marseille Université - ANR-11-IDEX-0001-02()
  • Center for Integrated Protein Science München - the Boehringer Ingelheim Fonds()
  • European Molecular Biology Organization - EMBO-LTR 688-2011()
  • European Molecular Biology Organization - EMBO-YIP()
  • H2020 European Research Council - ERC Grant 310939()
  • National Institute for Health Research - 5F32AR062477()
  • NEI NIH HHS - EY16707(United States)
  • NIAMS NIH HHS - F32 AR062477()

Targeted cortical reorganization using optogenetics in non-human primates.

  • Yazdan-Shahmorad A
  • Elife
  • 2018 May 29

Literature context:


Abstract:

Brain stimulation modulates the excitability of neural circuits and drives neuroplasticity. While the local effects of stimulation have been an active area of investigation, the effects on large-scale networks remain largely unexplored. We studied stimulation-induced changes in network dynamics in two macaques. A large-scale optogenetic interface enabled simultaneous stimulation of excitatory neurons and electrocorticographic recording across primary somatosensory (S1) and motor (M1) cortex (Yazdan-Shahmorad et al., 2016). We tracked two measures of network connectivity, the network response to focal stimulation and the baseline coherence between pairs of electrodes; these were strongly correlated before stimulation. Within minutes, stimulation in S1 or M1 significantly strengthened the gross functional connectivity between these areas. At a finer scale, stimulation led to heterogeneous connectivity changes across the network. These changes reflected the correlations introduced by stimulation-evoked activity, consistent with Hebbian plasticity models. This work extends Hebbian plasticity models to large-scale circuits, with significant implications for stimulation-based neurorehabilitation.

Funding information:
  • American Heart Association - Post-doctoral fellowship()
  • Defense Advanced Research Projects Agency - W911NF-14-2-0043()
  • Department of Health - NIHR-RP-011-053(United Kingdom)
  • National Science Foundation - Graduate student fellowship()

MUTE Directly Orchestrates Cell-State Switch and the Single Symmetric Division to Create Stomata.

  • Han SK
  • Dev. Cell
  • 2018 May 7

Literature context:


Abstract:

Precise cell division control is critical for developmental patterning. For the differentiation of a functional stoma, a cellular valve for efficient gas exchange, the single symmetric division of an immediate precursor is absolutely essential. Yet, the mechanism governing this event remains unclear. Here we report comprehensive inventories of gene expression by the Arabidopsis bHLH protein MUTE, a potent inducer of stomatal differentiation. MUTE switches the gene expression program initiated by SPEECHLESS. MUTE directly induces a suite of cell-cycle genes, including CYCD5;1, in which introduced expression triggers the symmetric divisions of arrested precursor cells in mute, and their transcriptional repressors, FAMA and FOUR LIPS. The regulatory network initiated by MUTE represents an incoherent type 1 feed-forward loop. Our mathematical modeling and experimental perturbations support a notion that MUTE orchestrates a transcriptional cascade leading to a tightly restricted pulse of cell-cycle gene expression, thereby ensuring the single cell division to create functional stomata.

Funding information:
  • NIDCR NIH HHS - T32-DE007288(United States)

Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity.

  • Roh HC
  • Cell Metab.
  • 2018 May 1

Literature context:


Abstract:

Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological "whitening," but it is unknown whether or to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.

Funding information:
  • NIDDK NIH HHS - P30DK52574(United States)
  • NIDDK NIH HHS - R01 DK085171()
  • NIDDK NIH HHS - R01 DK102170()
  • NIDDK NIH HHS - R01 DK102173()
  • NIDDK NIH HHS - R01 DK113669()

Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin.

  • Lee JJ
  • J. Cell Biol.
  • 2018 May 7

Literature context:


Abstract:

The Parkinson's disease factors PINK1 and parkin are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila melanogaster expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues, including Parkinson's disease-relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies, we did not observe any substantial impact on basal mitophagy. Because these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.

Funding information:
  • Medical Research Council - MC_UP_1501/1()
  • NCI NIH HHS - CA111294(United States)

Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability.

  • Kwasna D
  • Mol. Cell
  • 2018 Apr 5

Literature context:


Abstract:

Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.

Funding information:
  • NIA NIH HHS - R21 AG040683(United States)

Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ.

  • Pollard DJ
  • MBio
  • 2018 Apr 10

Literature context:


Abstract:

Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.IMPORTANCE Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. Citrobacter rodentium is a mouse pathogen used to model EPEC and EHEC pathogenesis in vivo Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors in vivo are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the in vivo processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type C. rodentium or an espJ catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response in vivo.

Funding information:
  • NIGMS NIH HHS - GM07739(United States)

Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

  • Hou W
  • Dev. Cell
  • 2018 Apr 23

Literature context:


Abstract:

The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain. Consistently, PRMT2 phenocopied Cobl functions in both gain- and loss-of-function studies. Both PRMT2- and Cobl-promoted dendritogenesis relied on methylation. PRMT2 effects require both its catalytic domain and SH3 domain. Cobl-mediated dendritic arborization required PRMT2, complex formation with PRMT2, and PRMT2's catalytic activity. Mechanistic studies reveal that Cobl methylation is key for Cobl actin binding. Therefore, arginine methylation is a regulatory mechanism reaching beyond controlling nuclear processes. It also controls a major, cytosolic, cytoskeletal component shaping neuronal cells.

Funding information:
  • NHLBI NIH HHS - HL24415(United States)

Hippo Signaling Plays an Essential Role in Cell State Transitions during Cardiac Fibroblast Development.

  • Xiao Y
  • Dev. Cell
  • 2018 Apr 23

Literature context:


Abstract:

During development, progenitors progress through transition states. The cardiac epicardium contains progenitors of essential non-cardiomyocytes. The Hippo pathway, a kinase cascade that inhibits the Yap transcriptional co-factor, controls organ size in developing hearts. Here, we investigated Hippo kinases Lats1 and Lats2 in epicardial diversification. Epicardial-specific deletion of Lats1/2 was embryonic lethal, and mutant embryos had defective coronary vasculature remodeling. Single-cell RNA sequencing revealed that Lats1/2 mutant cells failed to activate fibroblast differentiation but remained in an intermediate cell state with both epicardial and fibroblast characteristics. Lats1/2 mutant cells displayed an arrested developmental trajectory with persistence of epicardial markers and expanded expression of Yap targets Dhrs3, an inhibitor of retinoic acid synthesis, and Dpp4, a protease that modulates extracellular matrix (ECM) composition. Genetic and pharmacologic manipulation revealed that Yap inhibits fibroblast differentiation, prolonging a subepicardial-like cell state, and promotes expression of matricellular factors, such as Dpp4, that define ECM characteristics.

Funding information:
  • NIAAA NIH HHS - R01 AA020401(United States)

Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.

  • Sorkina T
  • Elife
  • 2018 Apr 9

Literature context:


Abstract:

Clathrin-independent endocytosis (CIE) mediates internalization of many transmembrane proteins but the mechanisms of cargo recruitment during CIE are poorly understood. We found that the cell-permeable furopyrimidine AIM-100 promotes dramatic oligomerization, clustering and CIE of human and mouse dopamine transporters (DAT), but not of their close homologues, norepinephrine and serotonin transporters. All effects of AIM-100 on DAT and the occupancy of substrate binding sites in the transporter were mutually exclusive, suggesting that AIM-100 may act by binding to DAT. Surprisingly, AIM-100-induced DAT endocytosis was independent of dynamin, cholesterol-rich microdomains and actin cytoskeleton, implying that a novel endocytic mechanism is involved. AIM-100 stimulated trafficking of internalized DAT was also unusual: DAT accumulated in early endosomes without significant recycling or degradation. We propose that AIM-100 augments DAT oligomerization through an allosteric mechanism associated with the DAT conformational state, and that oligomerization-triggered clustering leads to a coat-independent endocytosis and subsequent endosomal retention of DAT.

Funding information:
  • National Institutes of Health - DA014204()
  • NIAID NIH HHS - U01 AI101981(United States)

Structural Basis of Formation of the Microtubule Minus-End-Regulating CAMSAP-Katanin Complex.

  • Jiang K
  • Structure
  • 2018 Mar 6

Literature context:


Abstract:

CAMSAP/Patronin family members regulate the organization and stability of microtubule minus ends in various systems ranging from mitotic spindles to differentiated epithelial cells and neurons. Mammalian CAMSAP2 and CAMSAP3 bind to growing microtubule minus ends, where they form stretches of stabilized microtubule lattice. The microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. Here, by using biochemical, biophysical, and structural approaches, we reveal that a short helical motif conserved in CAMSAP2 and CAMSAP3 binds to the heterodimer formed by the N- and C-terminal domains of katanin subunits p60 and p80, respectively. The identified CAMSAP-katanin binding mode is supported by mutational analysis and genome-editing experiments. It is strikingly similar to the one seen in the ASPM-katanin complex, which is responsible for microtubule minus-end regulation in mitotic spindles. Our work provides a general molecular mechanism for the cooperation of katanin with major microtubule minus-end regulators.

Funding information:
  • NHLBI NIH HHS - R01 HL70748(United States)

Color Processing in the Early Visual System of Drosophila.

  • Schnaitmann C
  • Cell
  • 2018 Jan 11

Literature context:


Abstract:

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that color-opponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently.

Widespread optogenetic expression in macaque cortex obtained with MR-guided, convection enhanced delivery (CED) of AAV vector to the thalamus.

  • Yazdan-Shahmorad A
  • J. Neurosci. Methods
  • 2018 Jan 1

Literature context:


Abstract:

BACKGROUND: In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Previous work has shown that parenchymal delivery of adeno-associated virus (AAV) in the thalamus by convection-enhanced delivery (CED) can lead to large-scale transduction via axonal transport in distal areas including cortex. We used this approach to obtain widespread cortical expression of light-sensitive ion channels. NEW METHOD: AAV vectors co-expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein (YFP) genes were infused into thalamus of three rhesus macaques under MR-guided CED. After six to twelve weeks recovery, in vivo optical stimulation and single cell recording in the cortex was carried out using an optrode in anesthetized animals. Post-mortem immunostaining against YFP was used to estimate the distribution and level of expression of ChR2 in thalamus and cortex. RESULTS: Histological analysis revealed high levels of transduction in cortical layers. The patterns of expression were consistent with known thalamo-cortico-thalamic circuits. Dense expression was seen in thalamocortiocal axonal fibers in layers III, IV and VI and in pyramidal neurons in layers V and VI, presumably corticothalamic neurons. In addition we obtained reliable in vivo light-evoked responses in cortical areas with high levels of expression. COMPARISON WITH EXISTING METHODS: Thalamic CED is very efficient in achieving large expressing areas in comparison to convectional techniques both in minimizing infusion time and in minimizing damage to the brain. CONCLUSION: MR-guided CED infusion into thalamus provides a simplified approach to transduce large cortical areas by thalamo-cortico-thalamic projections in primate brain.

Funding information:
  • NIGMS NIH HHS - T32 GM007739(United States)

α-synuclein Induces Mitochondrial Dysfunction through Spectrin and the Actin Cytoskeleton.

  • Ordonez DG
  • Neuron
  • 2018 Jan 3

Literature context:


Abstract:

Genetics and neuropathology strongly link α-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related α-synucleinopathies. Here we describe a new Drosophila model of α-synucleinopathy based on widespread expression of wild-type human α-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant α-synuclein aggregation. We use results of forward genetic screening and genetic analysis in our new model to demonstrate that α-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse α-synucleinopathy model and in postmortem brain tissue from patients with α-synucleinopathy. Importantly, we provide evidence that the interaction of α-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for α-synuclein induced neurodegeneration.

Funding information:
  • NCI NIH HHS - U01 CA111275(United States)
  • NIA NIH HHS - R01 AG044113()
  • NICHD NIH HHS - U54 HD090255()
  • NIGMS NIH HHS - R01 GM084947()
  • NIH HHS - P40 OD018537()
  • NINDS NIH HHS - R01 NS083391()
  • NINDS NIH HHS - R01 NS086074()
  • NINDS NIH HHS - R01 NS092093()
  • NINDS NIH HHS - R01 NS098821()

Natural Parasite Exposure Induces Protective Human Anti-Malarial Antibodies.

  • Triller G
  • Immunity
  • 2017 Dec 19

Literature context:


Abstract:

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.

Funding information:
  • NIAID NIH HHS - F32 AI114113()
  • NIEHS NIH HHS - 5R01 ES010807(United States)

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

  • Sigg MA
  • Dev. Cell
  • 2017 Dec 18

Literature context:


Abstract:

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Funding information:
  • NIA NIH HHS - R01 AG027849(United States)
  • NIAMS NIH HHS - R01 AR054396()
  • NIGMS NIH HHS - R01 GM095941()

11β-HSD2 SUMOylation Modulates Cortisol-Induced Mineralocorticoid Receptor Nuclear Translocation Independently of Effects on Transactivation.

  • Jiménez-Canino R
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) has an essential role in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids to inactive 11-ketosteroids. Congenital deficiency of 11β-HSD2 causes a form of salt-sensitive hypertension known as the syndrome of apparent mineralocorticoid excess. The disease phenotype, which ranges from mild to severe, correlates well with reduction in enzyme activity. Furthermore, polymorphisms in the 11β-HSD2 coding gene (HSD11B2) have been linked to high blood pressure and salt sensitivity, major cardiovascular risk factors. 11β-HSD2 expression is controlled by different factors such as cytokines, sex steroids, or vasopressin, but posttranslational modulation of its activity has not been explored. Analysis of 11β-HSD2 sequence revealed a consensus site for conjugation of small ubiquitin-related modifier (SUMO) peptide, a major posttranslational regulatory event in several cellular processes. Our results demonstrate that 11β-HSD2 is SUMOylated at lysine 266. Non-SUMOylatable mutant K266R showed slightly higher substrate affinity and decreased Vmax, but no effects on protein stability or subcellular localization. Despite mild changes in enzyme activity, mutant K266R was unable to prevent cortisol-dependent MR nuclear translocation. The same effect was achieved by coexpression of wild-type 11β-HSD2 with sentrin-specific protease 1, a protease that catalyzes SUMO deconjugation. In the presence of 11β-HSD2-K266R, increased nuclear MR localization did not correlate with increased response to cortisol or increased recruitment of transcriptional coregulators. Taken together, our data suggests that SUMOylation of 11β-HSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation.

A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis.

  • Altemose N
  • Elife
  • 2017 Oct 26

Literature context:


Abstract:

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers.

Funding information:
  • NCRR NIH HHS - S10 RR029731-01(United States)

SKIP controls flowering time via the alternative splicing of SEF pre-mRNA in Arabidopsis.

  • Cui Z
  • BMC Biol.
  • 2017 Sep 11

Literature context:


Abstract:

BACKGROUND: Similar to other eukaryotes, splicing is emerging as an important process affecting development and stress tolerance in plants. Ski-interacting protein (SKIP), a splicing factor, is essential for circadian clock function and abiotic stress tolerance; however, the mechanisms whereby it regulates flowering time are unknown. RESULTS: In this study, we found that SKIP is required for the splicing of serrated leaves and early flowering (SEF) pre-messenger RNA (mRNA), which encodes a component of the ATP-dependent SWR1 chromatin remodeling complex (SWR1-C). Defects in the splicing of SEF pre-mRNA reduced H2A.Z enrichment at FLC, MAF4, and MAF5, suppressed the expression of these genes, and produced an early flowering phenotype in skip-1 plants. CONCLUSIONS: Our findings indicate that SKIP regulates SWR1-C function via alternative splicing to control the floral transition in Arabidopsis thaliana.

Myelinogenic Plasticity of Oligodendrocyte Precursor Cells following Spinal Cord Contusion Injury.

  • Assinck P
  • J. Neurosci.
  • 2017 Sep 6

Literature context:


Abstract:

Spontaneous remyelination occurs after spinal cord injury (SCI), but the extent of myelin repair and identity of the cells responsible remain incompletely understood and contentious. We assessed the cellular origin of new myelin by fate mapping platelet-derived growth factor receptor α (PDGFRα), Olig2+, and P0+ cells following contusion SCI in mice. Oligodendrocyte precursor cells (OPCs; PDGFRα+) produced oligodendrocytes responsible for de novo ensheathment of ∼30% of myelinated spinal axons at injury epicenter 3 months after SCI, demonstrating that these resident cells are a major contributor to oligodendrocyte regeneration. OPCs also produced the majority of myelinating Schwann cells in the injured spinal cord; invasion of peripheral myelinating (P0+) Schwann cells made only a limited contribution. These findings reveal that PDGFRα+ cells perform diverse roles in CNS repair, as multipotential progenitors that generate both classes of myelinating cells. This endogenous repair might be exploited as a therapeutic target for CNS trauma and disease.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to profound functional deficits, though substantial numbers of axons often survive. One possible explanation for these deficits is loss of myelin, creating conduction block at the site of injury. SCI leads to oligodendrocyte death and demyelination, and clinical trials have tested glial transplants to promote myelin repair. However, the degree and duration of myelin loss, and the extent and mechanisms of endogenous repair, have been contentious issues. Here, we use genetic fate mapping to demonstrate that spontaneous myelin repair by endogenous oligodendrocyte precursors is much more robust than previously recognized. These findings are relevant to many types of CNS pathology, raising the possibility that CNS precursors could be manipulated to repair myelin in lieu of glial transplantation.

Funding information:
  • NIDDK NIH HHS - DK072473(United States)

The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation.

  • Zhao YG
  • Mol. Cell
  • 2017 Sep 21

Literature context:


Abstract:

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

Parallel Inhibitory and Excitatory Trigemino-Facial Feedback Circuitry for Reflexive Vibrissa Movement.

  • Bellavance MA
  • Neuron
  • 2017 Aug 2

Literature context:


Abstract:

Animals employ active touch to optimize the acuity of their tactile sensors. Prior experimental results and models lead to the hypothesis that sensory inputs are used in a recurrent manner to tune the position of the sensors. A combination of electrophysiology, intersectional genetic viral labeling and manipulation, and classical tracing allowed us to identify second-order sensorimotor loops that control vibrissa movements by rodents. Facial motoneurons that drive intrinsic muscles to protract the vibrissae receive a short latency inhibitory input, followed by synaptic excitation, from neurons located in the oralis division of the trigeminal sensory complex. In contrast, motoneurons that retract the mystacial pad and indirectly retract the vibrissae receive only excitatory input from interpolaris cells that further project to the thalamus. Silencing this feedback alters retraction. The observed pull-push circuit at the lowest-level sensorimotor loop provides a mechanism for the rapid modulation of vibrissa touch during exploration of peri-personal space.

Drosophila CRY Entrains Clocks in Body Tissues to Light and Maintains Passive Membrane Properties in a Non-clock Body Tissue Independent of Light.

  • Agrawal P
  • Curr. Biol.
  • 2017 Aug 21

Literature context:


Abstract:

Circadian (∼24 hr) clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor CRYPTOCHROME (CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading TIMELESS (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, we generated a GFP-tagged-cry transgene that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.

Molecular and Circuit-Dynamical Identification of Top-Down Neural Mechanisms for Restraint of Reward Seeking.

  • Kim CK
  • Cell
  • 2017 Aug 24

Literature context:


Abstract:

Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well, even for rewards of high value. In humans and rodents, the medial prefrontal cortex (mPFC) has been implicated in suppressing reward seeking; however, despite vital significance in health and disease, the neural circuitry through which mPFC regulates reward seeking remains incompletely understood. Here, we show that a specific subset of superficial mPFC projections to a subfield of nucleus accumbens (NAc) neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. A highly resolved subpopulation of these top-down projecting neurons, identified by 2-photon Ca2+ imaging and activity-dependent labeling to recruit the relevant neurons, was found capable of suppressing reward seeking. This natural activity-resolved mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.

The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions.

  • Merklinger E
  • Elife
  • 2017 Jul 19

Literature context:


Abstract:

Molecule clustering is an important mechanism underlying cellular self-organization. In the cell membrane, a variety of fundamentally different mechanisms drive membrane protein clustering into nanometre-sized assemblies. To date, it is unknown whether this clustering process can be dissected into steps differentially regulated by independent mechanisms. Using clustered syntaxin molecules as an example, we study the influence of a cytoplasmic protein domain on the clustering behaviour. Analysing protein mobility, cluster size and accessibility to myc-epitopes we show that forces acting on the transmembrane segment produce loose clusters, while cytoplasmic protein interactions mediate a tightly packed state. We conclude that the data identify a hierarchy in membrane protein clustering likely being a paradigm for many cellular self-organization processes.

IGF1-Dependent Synaptic Plasticity of Mitral Cells in Olfactory Memory during Social Learning.

  • Liu Z
  • Neuron
  • 2017 Jul 5

Literature context:


Abstract:

During social transmission of food preference (STFP), mice form long-term memory of food odors presented by a social partner. How does the brain associate a social context with odor signals to promote memory encoding? Here we show that odor exposure during STFP, but not unconditioned odor exposure, induces glomerulus-specific long-term potentiation (LTP) of synaptic strength selectively at the GABAergic component of dendrodendritic synapses of granule and mitral cells in the olfactory bulb. Conditional deletion of synaptotagmin-10, the Ca2+ sensor for IGF1 secretion from mitral cells, or deletion of IGF1 receptor in the olfactory bulb prevented the socially relevant GABAergic LTP and impaired memory formation after STFP. Conversely, the addition of IGF1 to acute olfactory bulb slices elicited the GABAergic LTP in mitral cells by enhancing postsynaptic GABA receptor responses. Thus, our data reveal a synaptic substrate for a socially conditioned long-term memory that operates at the level of the initial processing of sensory information.

Funding information:
  • NIMH NIH HHS - P50 MH086403()

SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs.

  • Nozawa RS
  • Cell
  • 2017 Jun 15

Literature context:


Abstract:

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

Red Blood Cell Invasion by the Malaria Parasite Is Coordinated by the PfAP2-I Transcription Factor.

  • Santos JM
  • Cell Host Microbe
  • 2017 Jun 14

Literature context:


Abstract:

Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor PfAP2-I, belonging to the Apicomplexan AP2 (ApiAP2) family, that is responsible for regulating the expression of genes involved in RBC invasion. Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of target genes. Although PfAP2-I contains three AP2 DNA-binding domains, only one is required for binding of the target genes during blood stage development. Furthermore, we find that PfAP2-I associates with several chromatin-associated proteins, including the Plasmodium bromodomain protein PfBDP1 and that complex formation is associated with transcriptional regulation. As a key regulator of red blood cell invasion, PfAP2-I represents a potential new antimalarial therapeutic target.

Funding information:
  • NIAID NIH HHS - R01 AI076276()
  • NIAID NIH HHS - R01 AI125565()
  • NIGMS NIH HHS - P50 GM071508()
  • NIGMS NIH HHS - R01 GM114141()

RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis.

  • Di Marco S
  • Mol. Cell
  • 2017 Jun 1

Literature context:


Abstract:

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

Learning shapes the aversion and reward responses of lateral habenula neurons.

  • Wang D
  • Elife
  • 2017 May 31

Literature context:


Abstract:

The lateral habenula (LHb) is believed to encode negative motivational values. It remains unknown how LHb neurons respond to various stressors and how learning shapes their responses. Here, we used fiber-photometry and electrophysiology to track LHb neuronal activity in freely-behaving mice. Bitterness, pain, and social attack by aggressors intensively excite LHb neurons. Aversive Pavlovian conditioning induced activation by the aversion-predicting cue in a few trials. The experience of social defeat also conditioned excitatory responses to previously neutral social stimuli. In contrast, fiber photometry and single-unit recordings revealed that sucrose reward inhibited LHb neurons and often produced excitatory rebound. It required prolonged conditioning and high reward probability to induce inhibition by reward-predicting cues. Therefore, LHb neurons can bidirectionally process a diverse array of aversive and reward signals. Importantly, their responses are dynamically shaped by learning, suggesting that the LHb participates in experience-dependent selection of behavioral responses to stressors and rewards.

Repression of Interstitial Identity in Nephron Progenitor Cells by Pax2 Establishes the Nephron-Interstitium Boundary during Kidney Development.

  • Naiman N
  • Dev. Cell
  • 2017 May 22

Literature context:


Abstract:

The kidney contains the functional units, the nephrons, surrounded by the renal interstitium. Previously we discovered that, once Six2-expressing nephron progenitor cells and Foxd1-expressing renal interstitial progenitor cells form at the onset of kidney development, descendant cells from these populations contribute exclusively to the main body of nephrons and renal interstitial tissues, respectively, indicating a lineage boundary between the nephron and renal interstitial compartments. Currently it is unclear how lineages are regulated during kidney organogenesis. We demonstrate that nephron progenitor cells lacking Pax2 fail to differentiate into nephron cells but can switch fates into renal interstitium-like cell types. These data suggest that Pax2 function maintains nephron progenitor cells by repressing a renal interstitial cell program. Thus, the lineage boundary between the nephron and renal interstitial compartments is maintained by the Pax2 activity in nephron progenitor cells during kidney organogenesis.

Funding information:
  • NIDDK NIH HHS - R01 DK094933()
  • NIDDK NIH HHS - R37 DK054364()
  • NIH HHS - R21 OD021437()

Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization.

  • Tortosa E
  • Neuron
  • 2017 May 17

Literature context:


Abstract:

Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/β Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.

Translation of CircRNAs.

  • Pamudurti NR
  • Mol. Cell
  • 2017 Apr 6

Literature context:


Abstract:

Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.

SNF2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance and Proper Replication.

  • Taneja N
  • Mol. Cell
  • 2017 Apr 6

Literature context:


Abstract:

Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells. Moreover, Fft3 also precludes nucleosome turnover at several euchromatic loci to prevent R-loop formation, ensuring proper replication progression. Our analyses show that overexpression of Clr4/Suv39h, which is also required for efficient replication through these loci, suppresses phenotypes associated with the loss of Fft3. This work uncovers a conserved factor critical for epigenetic inheritance of heterochromatin and describes a mechanism in which suppression of nucleosome turnover prevents formation of structural barriers that impede replication at fragile regions in the genome.

Funding information:
  • Intramural NIH HHS - Z01 BC010523-04()
  • Intramural NIH HHS - Z01 BC010523-05()
  • Intramural NIH HHS - Z99 CA999999()

An Interaction Landscape of Ubiquitin Signaling.

  • Zhang X
  • Mol. Cell
  • 2017 Mar 2

Literature context:


Abstract:

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.

Cell- and region-specific expression of depression-related protein p11 (S100a10) in the brain.

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

Literature context:


Abstract:

P11 (S100a10), a member of the S100 family of proteins, has widespread distribution in the vertebrate body, including in the brain, where it has a key role in membrane trafficking, vesicle secretion, and endocytosis. Recently, our laboratory has shown that a constitutive knockout of p11 (p11-KO) in mice results in a depressive-like phenotype. Furthermore, p11 has been implicated in major depressive disorder (MDD) and in the actions of antidepressants. Since depression affects multiple brain regions, and the role of p11 has only been determined in a few of these areas, a detailed analysis of p11 expression in the brain is warranted. Here we demonstrate that, although widespread in the brain, p11 expression is restricted to distinct regions, and specific neuronal and nonneuronal cell types. Furthermore, we provide comprehensive mapping of p11 expression using in situ hybridization, immunocytochemistry, and whole-tissue volume imaging. Overall, expression spans multiple brain regions, structures, and cell types, suggesting a complex role of p11 in depression. J. Comp. Neurol. 525:955-975, 2017. © 2016 Wiley Periodicals, Inc.

Funding information:
  • NINDS NIH HHS - R01 NS085232(United States)

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

  • Fu SJ
  • J. Neurosci.
  • 2017 Mar 1

Literature context:


Abstract:

Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and β subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin.

  • Ben-Yaacov A
  • Neuron
  • 2017 Mar 8

Literature context:


Abstract:

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain and critically contribute to synaptic plasticity and pathology. AMPAR trafficking and gating are tightly controlled by auxiliary transmembrane AMPAR regulatory proteins (TARPs). Here, using systematic domain swaps with the TARP-insensitive kainate receptor GluK2, we show that AMPAR interaction with the prototypical TARP stargazin/γ2 primarily involves the AMPAR membrane domains M1 and M4 of neighboring subunits, initiated or stabilized by the AMPAR C-tail, and that these interactions are sufficient to enable full receptor modulation. Moreover, employing TARP chimeras disclosed a key role in this process also for the TARP transmembrane domains TM3 and TM4 and extracellular loop 2. Mechanistically, our data support a two-step action in which binding of TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions.

Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane.

  • Weber P
  • Elife
  • 2017 Feb 27

Literature context:


Abstract:

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.

RNA Binding to CBP Stimulates Histone Acetylation and Transcription.

  • Bose DA
  • Cell
  • 2017 Jan 12

Literature context:


Abstract:

CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes.

Funding information:
  • NCI NIH HHS - R01 CA078831()
  • NIMH NIH HHS - DP2 MH107055()

A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets.

  • Moe Y
  • Endocrinology
  • 2016 Dec 11

Literature context:


Abstract:

We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.

Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating Aldh1a3 pre-mRNA 3'-end processing.

  • La Rosa P
  • Elife
  • 2016 Nov 15

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Abstract:

The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in Khdrbs1-/- NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex.

Funding information:
  • NCRR NIH HHS - C06 RR018928(United States)

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

  • Lee WH
  • Elife
  • 2016 Nov 15

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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.

Functional Metabolomics Describes the Yeast Biosynthetic Regulome.

  • Mülleder M
  • Cell
  • 2016 Oct 6

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Abstract:

Genome-metabolism interactions enable cell growth. To probe the extent of these interactions and delineate their functional contributions, we quantified the Saccharomyces amino acid metabolome and its response to systematic gene deletion. Over one-third of coding genes, in particular those important for chromatin dynamics, translation, and transport, contribute to biosynthetic metabolism. Specific amino acid signatures characterize genes of similar function. This enabled us to exploit functional metabolomics to connect metabolic regulators to their effectors, as exemplified by TORC1, whose inhibition in exponentially growing cells is shown to match an interruption in endomembrane transport. Providing orthogonal information compared to physical and genetic interaction networks, metabolomic signatures cluster more than half of the so far uncharacterized yeast genes and provide functional annotation for them. A major part of coding genes is therefore participating in gene-metabolism interactions that expose the metabolism regulatory network and enable access to an underexplored space in gene function.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

  • Zhong FL
  • Cell
  • 2016 Sep 22

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Abstract:

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

A Two-Immunoglobulin-Domain Transmembrane Protein Mediates an Epidermal-Neuronal Interaction to Maintain Synapse Density.

  • Cherra SJ
  • Neuron
  • 2016 Jan 20

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Abstract:

Synaptic maintenance is essential for neural circuit function. In the C. elegans locomotor circuit, motor neurons are in direct contact with the epidermis. Here, we reveal a novel epidermal-neuronal interaction mediated by a two-immunoglobulin domain transmembrane protein, ZIG-10, that is necessary for maintaining cholinergic synapse density. ZIG-10 is localized at the cell surface of epidermis and cholinergic motor neurons, with high levels at areas adjacent to synapses. Loss of zig-10 increases the number of cholinergic excitatory synapses and exacerbates convulsion behavior in a seizure model. Mis-expression of zig-10 in GABAergic inhibitory neurons reduces GABAergic synapse number, dependent on the presence of ZIG-10 in the epidermis. Furthermore, ZIG-10 interacts with the tyrosine kinase SRC-2 to regulate the phagocytic activity of the epidermis to restrict cholinergic synapse number. Our studies demonstrate the highly specific roles of non-neuronal cells in modulating neural circuit function, through neuron-type-specific maintenance of synapse density.

The Expression of Tubb2b Undergoes a Developmental Transition in Murine Cortical Neurons.

  • Breuss M
  • J. Comp. Neurol.
  • 2015 Oct 15

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Abstract:

The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the β-tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC-transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8-week-old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8-week-old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton.

Activity-dependent FMRP requirements in development of the neural circuitry of learning and memory.

  • Doll CA
  • Development
  • 2015 Apr 1

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Abstract:

The activity-dependent refinement of neural circuit connectivity during critical periods of brain development is essential for optimized behavioral performance. We hypothesize that this mechanism is defective in fragile X syndrome (FXS), the leading heritable cause of intellectual disability and autism spectrum disorders. Here, we use optogenetic tools in the Drosophila FXS disease model to test activity-dependent dendritogenesis in two extrinsic neurons of the mushroom body (MB) learning and memory brain center: (1) the input projection neuron (PN) innervating Kenyon cells (KCs) in the MB calyx microglomeruli and (2) the output MVP2 neuron innervated by KCs in the MB peduncle. Both input and output neuron classes exhibit distinctive activity-dependent critical period dendritic remodeling. MVP2 arbors expand in Drosophila mutants null for fragile X mental retardation 1 (dfmr1), as well as following channelrhodopsin-driven depolarization during critical period development, but are reduced by halorhodopsin-driven hyperpolarization. Optogenetic manipulation of PNs causes the opposite outcome--reduced dendritic arbors following channelrhodopsin depolarization and expanded arbors following halorhodopsin hyperpolarization during development. Importantly, activity-dependent dendritogenesis in both neuron classes absolutely requires dfmr1 during one developmental window. These results show that dfmr1 acts in a neuron type-specific activity-dependent manner for sculpting dendritic arbors during early-use, critical period development of learning and memory circuitry in the Drosophila brain.

Isolation and immortalization of MIP-GFP neurons from the hypothalamus.

  • Wang ZC
  • Endocrinology
  • 2014 Jun 19

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Abstract:

The mouse insulin I promoter (MIP) construct was developed to eliminate the promoter activity detected with the rat insulin II promoter in specific hypothalamic neurons that may have unintended effects on glucose and energy homeostasis in transgenic models. Thus, the specificity of this novel construct must be validated prior to the widespread availability of derived Cre models. Although limited validation efforts have indicated a lack of MIP activity within neuronal tissue, the global immunohistochemical methodology used may not be specific enough to rule out the possibility of specific populations of neurons with MIP activity. To investigate possible MIP activity within the hypothalamus, primary hypothalamic isolates from MIP-green fluorescent protein reporter mice were analyzed after fluorescent-activated cell sorting. Primary hypothalamic neurons isolated from the MIP-green fluorescent protein mice were immortalized. Characterization detected the presence of hypothalamic neuropeptide Y (NPY) and agouti-related peptide, involved in the control of energy homeostasis, as well as confirmed insulin responsiveness in the cell lines. Moreover, because insulin was demonstrated to differentially regulate NPY expression within these MIP neurons, the promoter construct may be active in multiple hypothalamic NPY/agouti-related peptide subpopulations with unique physiological functions. MIP transgenic animals may therefore face similar limitations seen previously with rat insulin II promoter-based models.

Funding information:
  • NEI NIH HHS - R01 EY014454(United States)

Onecut1 is essential for horizontal cell genesis and retinal integrity.

  • Wu F
  • J. Neurosci.
  • 2013 Aug 7

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Abstract:

Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (∼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.

A 5'-flanking region of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) gene directs its cell-specific androgen-regulated gene expression in testicular germ cells.

  • Kavarthapu R
  • Endocrinology
  • 2013 Jun 20

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Abstract:

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is a posttranscriptional regulator of genes that are essential for spermatid elongation and completion of spermatogenesis. It also prevents Leydig cells (LCs) from gonadotropin overstimulation of androgen production. In transgenic (Tg) mice carrying deletions of the GRTH 5'-flanking regions, we previously demonstrated that the -1085 bp to ATG contains the elements for basal and androgen-induced LC-specific expression. No expression in germ cells (GCs) was found with sequences extended up to -3.6 kb. To define regulatory regions of GRTH required for expression in GC, Tg mice were generated with 5'-flanking sequence 6.4 kb (6.4 Kb-Tg) and/or deletion using green fluorescent protein (GFP) as reporter gene in the present study. GFP was expressed in all lines. Immunohistochemistry analysis showed that 6.4 Kb-Tg directed GFP expression in both GCs and LCs. Deletion of the sequence -205 bp to -3.6 kb (6.4 Kb/del-Tg) directs GFP expression only in meiotic and haploid GCs. This indicated that the distal region -6.4 kb/-3.6 kb is required for GRTH cell-specific expression in GC. Also, it inhibits the expression of GRTH in LC directed by the 205-bp promoter, an effect that is neutralized by the -3.6-kb/-205-bp sequence. Androgen receptor antagonist, flutamide treatment prevents GFP/GRTH expression in Tg lines, demonstrating in vivo direct and indirect effects of endogenous androgen on LCs and GCs, respectively. Our studies have generated and characterized Tg lines that can be used to define requirements for cell-specific expression of the GRTH gene and to further advance our knowledge on the regulation of GRTH by androgen in GCs.

Funding information:
  • NINDS NIH HHS - R01 NS064577(United States)

DNA methyltransferases1 (DNMT1) and 3a (DNMT3a) colocalize with GAD67-positive neurons in the GAD67-GFP mouse brain.

  • Kadriu B
  • J. Comp. Neurol.
  • 2012 Jun 15

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Abstract:

DNA methylation is an epigenetic regulatory mechanism commonly associated with transcriptional silencing. DNA methyltransferases (DNMTs) are a family of related proteins that both catalyze the de novo formation of 5-methylcytosine and maintain these methylation marks in cell-specific patterns in virtually all mitotic cells of the body. In the adult brain, methylation occurs in progenitor cells of the neurogenic zones and in postmitotic neurons. Of the DNMTs, DNMT1 and DNMT3a are most highly expressed in postmitotic neurons. While it has been commonly thought all postmitotic neurons and glia express DNMTs at comparable levels, the coexpression of selected DNMTs with markers of distinct neurotransmitter phenotypes has not been previously examined in detail in the mouse. To this end, we analyzed the expression of DNMT1 and DNMT3a along with GAD67 in the brains of the glutamic acid decarboxylase67-enhanced green fluorescent protein (GAD67-GFP) knockin mice. After first confirming that GFP-immunopositive neurons were also GAD67-positive, we showed that in the motor cortex, piriform cortex, striatum, CA1 region of the hippocampus, dentate gyrus, and basolateral amygdala (BLA), GFP immunofluorescence coincided with the signal corresponding to DNMT1 and DNMT3a. A detailed examination of cortical neurons, showed that ≈30% of NeuN-immunopositive neurons were also DNMT1-positive. These data do not exclude the expression of DNMT1 or DNMT3a in glutamatergic neurons and glia. However, they suggest that their expression is low compared with the levels present in GABAergic neurons.

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

Increasing heterogeneity in the organization of synaptic inputs of mature olfactory bulb neurons generated in newborn rats.

  • Kelsch W
  • J. Comp. Neurol.
  • 2012 Apr 15

Literature context:


Abstract:

New neurons are added into the mammalian olfactory bulb throughout life, but it remains unknown whether the properties of new neurons generated in newborn animals differ from those added during adulthood. We compared the densities of glutamatergic synapses of granule cells (GCs) generated in newborn and adult rats over extended periods of time. We observed that, whereas adult-born GCs maintained stable cell-to-cell variability of synaptic densities soon after they integrated into the circuit, cell-to-cell variability of synaptic densities of neonatal-born GCs increased months after their integration. We also investigated whether the synaptic reorganization induced by sensory deprivation occurred differently in mature neonatal- and adult-born GCs. Sensory deprivation after new GCs had differentiated induced more pronounced changes in the synaptic densities of neonatal-born GCs than in adult-born GCs. These observations suggest that the synapses of mature neonatal-born GCs retain a higher degree of malleability in response to changes in neuronal activity than adult-born GCs.

Funding information:
  • Doris Duke Charitable Foundation - T32 AI007387(United States)

Differential gene expression in migrating cortical interneurons during mouse forebrain development.

  • Faux C
  • J. Comp. Neurol.
  • 2010 Apr 15

Literature context:


Abstract:

Gamma-aminobutyric acid (GABA)ergic interneurons play a vital role in modulating the activity of the cerebral cortex, and disruptions to their function have been linked to neurological disorders such as schizophrenia and epilepsy. These cells originate in the ganglionic eminences (GE) of the ventral telencephalon and undergo tangential migration to enter the cortex. Currently, little is known about the signaling mechanisms that regulate interneuron migration. We therefore performed a microarray analysis comparing the changes in gene expression between the GABAergic interneurons that are actively migrating into the cortex with those in the GE. We were able to isolate pure populations of GABAergic cells by fluorescence-activated cell sorting of cortex and GE from embryonic brains of glutamate decarboxylase 67 (GAD67)-green fluorescent protein (GFP) transgenic mice. Our microarray analysis identified a number of novel genes that were upregulated in migrating cortical interneurons at both E13.5 and E15.5. Many of these genes have previously been shown to play a role in cell migration of both neuronal and non-neuronal cell types. In addition, several of the genes identified are involved in the regulation of migratory processes, such as neurite outgrowth, cell adhesion, and remodeling of the actin cytoskeleton and microtubule network. Moreover, quantitative polymerase chain reaction and in situ hybridization analyses confirmed that the expression of some of these genes is restricted to cortical interneurons. These data therefore provide a framework for future studies aimed at elucidating the complexities of interneuron migration and, in turn, may reveal important genes that are related to the development of specific neurological disorders.