Effects of bound nucleotides on the secondary structure, thermal stability, and phosphorylation of Rab3A.

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.

Molecular and functional interactions of alpha-synuclein with Rab3a.

Alpha-synuclein (a-Syn) is a presynaptic protein, the misfolding of which is associated with Parkinson's disease. Rab GTPases are small guanine nucleotide binding proteins that play key roles in vesicle trafficking and have been associated with a-Syn function and dysfunction. a-Syn is enriched on synaptic vesicles, where it has been reported to interact with GTP-bound Rab3a, a master regulator of synaptic vesicle trafficking. a-Syn is known to bind weakly to Rab8a in solution via a positively charged patch, but the physiological implications of such interactions have not been explored. Here, we investigate direct interactions between a-Syn and Rab3a in solution and on lipid membranes using NMR spectroscopy. We find that the C terminus of a-Syn interacts with Rab3a in a manner similar to its previously reported interaction with Rab8a. While weak in solution, we demonstrate that this interaction becomes stronger when the proteins are bound to a membrane surface. The Rab3a binding site for a-Syn is similar to the surface that contacts the Rab3a effector rabphilin-3A, which modulates the enzymatic activity of Rab3a. Accordingly, we show that a-Syn inhibits GTP hydrolysis by Rab3a and that inhibition is more potent on the membrane surface, suggesting that their interaction may be functionally relevant. Finally, we show that phosphorylation of a-Syn residue Ser 129, a modification associated with Parkinson's disease pathology, enhances its interactions with Rab3a and increases its ability to inhibit Rab3a GTP hydrolysis. These results represent the first observation of a functional role for synuclein-Rab interactions and for a-Syn Ser 129 phosphorylation.

Small Rab GTPases in Intracellular Vesicle Trafficking: The Case of Rab3A/Raphillin-3A Complex in the Kidney.

Small Rab GTPases, the largest group of small monomeric GTPases, regulate vesicle trafficking in cells, which are integral to many cellular processes. Their role in neurological diseases, such as cancer and inflammation have been extensively studied, but their implication in kidney disease has not been researched in depth. Rab3a and its effector Rabphillin-3A (Rph3A) expression have been demonstrated to be present in the podocytes of normal kidneys of mice rats and humans, around vesicles contained in the foot processes, and they are overexpressed in diseases with proteinuria. In addition, the Rab3A knockout mice model induced profound cytoskeletal changes in podocytes of high glucose fed animals. Likewise, RphA interference in the Drosophila model produced structural and functional damage in nephrocytes with reduction in filtration capacities and nephrocyte number. Changes in the structure of cardiac fiber in the same RphA-interference model, open the question if Rab3A dysfunction would produce simultaneous damage in the heart and kidney cells, an attractive field that will require attention in the future.

Soluble α-synuclein facilitates priming and fusion by releasing Ca2+ from the thapsigargin-sensitive Ca2+ pool in PC12 cells.

α-Synuclein is associated with Parkinson's disease, and is mainly localized in presynaptic terminals and regulates exocytosis, but its physiological roles remain controversial. Here, we studied the effects of soluble and aggregated α-synuclein on exocytosis, and explored the molecular mechanism by which α-synuclein interacts with regulatory proteins, including Rab3A, Munc13-1 (also known as Unc13a) and Munc18-1 (also known as STXBP1), in order to regulate exocytosis. Through fluorescence recovery after photobleaching experiments, overexpressed α-synuclein in PC12 cells was found to be in a monomeric form, which promotes exocytosis. In contrast, aggregated α-synuclein induced by lactacystin treatment inhibits exocytosis. Our results show that α-synuclein is involved in vesicle priming and fusion. α-Synuclein and phorbol 12-myristate 13-acetate (PMA), which is known to enhance vesicle priming mediated by Rab3A, Munc13-1 and Munc18-1, act on the same population of vesicles, but regulate priming independently. Furthermore, the results show a novel effects of α-synuclein on mobilizing Ca2+ release from thapsigargin-sensitive Ca2+ pools to enhance the ATP-induced [Ca2+]i increase, which enhances vesicle fusion. Our results provide a detailed understanding of the action of α-synuclein during the final steps of exocytosis.

Synaptic loss in schizophrenia: a meta-analysis and systematic review of synaptic protein and mRNA measures.

Although synaptic loss is thought to be core to the pathophysiology of schizophrenia, the nature, consistency and magnitude of synaptic protein and mRNA changes has not been systematically appraised. Our objective was thus to systematically review and meta-analyse findings. The entire PubMed database was searched for studies from inception date to the 1st of July 2017. We selected case-control postmortem studies in schizophrenia quantifying synaptic protein or mRNA levels in brain tissue. The difference in protein and mRNA levels between cases and controls was extracted and meta-analysis conducted. Among the results, we found a significant reduction in synaptophysin in schizophrenia in the hippocampus (effect size: -0.65, p < 0.01), frontal (effect size: -0.36, p = 0.04), and cingulate cortices (effect size: -0.54, p = 0.02), but no significant changes for synaptophysin in occipital and temporal cortices, and no changes for SNAP-25, PSD-95, VAMP, and syntaxin in frontal cortex. There were insufficient studies for meta-analysis of complexins, synapsins, rab3A and synaptotagmin and mRNA measures. Findings are summarised for these, which generally show reductions in SNAP-25, PSD-95, synapsin and rab3A protein levels in the hippocampus but inconsistency in other regions. Our findings of moderate-large reductions in synaptophysin in hippocampus and frontal cortical regions, and a tendency for reductions in other pre- and postsynaptic proteins in the hippocampus are consistent with models that implicate synaptic loss in schizophrenia. However, they also identify potential differences between regions and proteins, suggesting synaptic loss is not uniform in nature or extent.

Cocaine Self-Administration and Abstinence Modulate NMDA Receptor Subunits and Active Zone Proteins in the Rat Nucleus Accumbens.

Cocaine-induced plasticity in the glutamatergic transmission and its N-methyl-d-aspartate (NMDA) receptors are critically involved in the development of substance use disorder. The presynaptic active zone proteins control structural synaptic plasticity; however, we are still far from understanding the molecular determinants important for cocaine seeking behavior. The aim of this study was to investigate the effect of cocaine self-administration and different conditions of cocaine forced abstinence on the composition of the NMDA receptor subunits and on the levels of active zone proteins, i.e., Ras-related protein 3A (Rab3A), Rab3 interacting molecules 1 (RIM1) and mammalian uncoordinated protein 13 (Munc13) in the rat nucleus accumbens. We found an up-regulation of the accumbal levels of GluN1 and GluN2A following cocaine self-administration that was paralleled by an increase of Munc13 and RIM1 levels. At the same time, we also demonstrated that different conditions of cocaine abstinence abolished changes in NMDA receptor subunits (except for higher GluN1 levels after cocaine abstinence with extinction training), while an increase in the Munc13 concentration was shown in rats housed in an enriched environment. In conclusion, cocaine self-administration is associated with the specific up-regulation of the NMDA receptor subunit composition and is related with new presynaptic targets controlling neurotransmitter release. Moreover, changes observed in cocaine abstinence with extinction training and in an enriched environment in the levels of NMDA receptor subunit and in the active zone protein, respectively, may represent a potential regulatory step in cocaine-seeking behavior.

A Phosphoproteomic Screen Identifies a Guanine Nucleotide Exchange Factor for Rab3A Protein as a Mitogen-activated Protein (MAP) Kinase Phosphatase-5-regulated MAP Kinase Target in Interleukin 6 (IL-6) Secretion and Myogenesis.

The mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-regulated MAPK targets in the control of skeletal muscle function and regenerative myogenesis have not been established. To identify MKP-5-regulated MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice. We discovered a novel MKP-5-regulated MAPK substrate called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyperphosphorylated on a phospho-MAPK motif in skeletal muscle of MKP-5-deficient mice. GRAB was found to be phosphorylated by JNK on serine 169. Myoblasts overexpressing a phosphorylation-defective mutant of GRAB containing a mutation at Ser-169 to Ala-169 (GRAB-S169A) inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser-169 was required for the secretion of the promyogenic cytokine interleukin 6 (IL-6). Consistent with this observation, MKP-5-deficient mice exhibited increased circulating IL-6 expression as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP-5-mediated regulation of JNK negatively regulates phosphorylation of GRAB, which subsequently controls secretion of IL-6. These data support the notion that MKP-5 serves as a negative regulator of MAPK-dependent signaling of critical skeletal muscle signaling pathways.

Dynamic Partitioning of Synaptic Vesicle Pools by the SNARE-Binding Protein Tomosyn.

Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools. SIGNIFICANCE STATEMENT: Although information transfer at central synapses via sustained high-frequency neural activity requires coordinated synaptic vesicle (SV) recycling, the mechanism(s) by which synapses sense and dynamically modify SV pools to match network demands remains poorly defined. To advance understanding, we quantified SV pool sizes and their sensitivity to neural activity while altering Tomo1 expression, a putative regulator of the presynaptic Readily Releasable Pool. Remarkably, we find Tomo1 actions to extend beyond the Readily Releasable Pool to mediate the Total Recycling Pool and SV Resting Pool distribution, and this action is sensitive to neural activity through Cdk5 phosphorylation of Tomo1. Moreover, Tomo1 appears to exert these actions through interaction with Rab3A-GTP and synapsin proteins. Together, our results argue that Tomo1 is a central mediator of SV availability for neurotransmission.

Reducing Endogenous α-Synuclein Mitigates the Degeneration of Selective Neuronal Populations in an Alzheimer's Disease Transgenic Mouse Model.

UNLABELLED: Alzheimer's disease (AD) is characterized by the progressive accumulation of amyloid ß (Aß) and microtubule associate protein tau, leading to the selective degeneration of neurons in the neocortex, limbic system, and nucleus basalis, among others. Recent studies have shown that α-synuclein (α-syn) also accumulates in the brains of patients with AD and interacts with Aß and tau, forming toxic hetero-oligomers. Although the involvement of α-syn has been investigated extensively in Lewy body disease, less is known about the role of this synaptic protein in AD. Here, we found that reducing endogenous α-syn in an APP transgenic mouse model of AD prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. Together, these results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of Aß. SIGNIFICANCE STATEMENT: Reducing endogenous α-synuclein (α-syn) in an APP transgenic mouse model of Alzheimer's disease (AD) prevented the degeneration of cholinergic neurons, ameliorated corresponding deficits, and recovered the levels of Rab3a and Rab5 proteins involved in intracellular transport and sorting of nerve growth factor and brain-derived neurotrophic factor. These results suggest that α-syn might participate in mechanisms of vulnerability of selected neuronal populations in AD and that reducing α-syn might be a potential approach to protecting these populations from the toxic effects of amyloid ß.

Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP.

UNLABELLED: Brain-derived neurotrophic factor (BDNF) is essential for neuronal differentiation and survival. We know that BDNF levels decline in the brains of patients with Huntington's disease (HD), a neurodegenerative disease caused by the expression of mutant huntingtin protein (mHtt), and furthermore that administration of BDNF in HD mice is protective against HD neuropathology. BDNF is produced in neurons, but astrocytes are also an important source of BDNF in the brain. Nonetheless, whether mHtt affects astrocytic BDNF in the HD brain remains unknown. Here we investigated astrocytes from HD140Q knock-in mice and uncovered evidence that mHtt decreases BDNF secretion from astrocytes, which is mediated by exocytosis in astrocytes. Our results demonstrate that mHtt associates with Rab3a, a small GTPase localized on membranes of dense-core vesicles, and prevents GTP-Rab3a from binding to Rab3-GAP1, disrupting the conversion of GTP-Rab3a into GDP-Rab3a and thus impairing the docking of BDNF vesicles on plasma membranes of astrocytes. Importantly, overexpression of Rab3a rescues impaired BDNF vesicle docking and secretion from HD astrocytes. Moreover, ATP release and the number of ATP-containing dense-core vesicles docking are decreased in HD astrocytes, suggesting that the exocytosis of dense-core vesicles is impaired by mHtt in HD astrocytes. Further, Rab3a overexpression reduces reactive astrocytes in the striatum of HD140Q knock-in mice. Our results indicate that compromised exocytosis of BDNF in HD astrocytes contributes to the decreased BDNF levels in HD brains and underscores the importance of improving glial function in the treatment of HD. SIGNIFICANCE STATEMENT: Huntington's disease (HD) is an inherited neurodegenerative disorder that affects one in every 10,000 Americans. To date, there is no effective treatment for HD, in part because the pathogenic mechanism driving the disease is not fully understood. The dysfunction of astrocytes is known to contribute to the pathogenesis of HD. One important role of astrocytes is to synthesize and release brain-derived neurotrophic factor (BDNF), which is vital for neuronal survival, development, and function. We found that mutant huntingtin protein (mHtt) at the endogenous level decreases BDNF secretion from astrocytes by disrupting the conversion of GTP-Rab3a into GDP-Rab3a and that overexpressing Rab3a can rescue this deficient BDNF release and early neuropathology in HD knock-in mouse brain. Our study suggests that astrocytic Rab3a is a potential therapeutic target for HD treatment.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores.

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.

Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca2+ -independent and Ca2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc.

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.

ADP ribosylation factor 6 (ARF6) promotes acrosomal exocytosis by modulating lipid turnover and Rab3A activation.

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5'-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.

Spire-1 contributes to the invadosome and its associated invasive properties.

Cancer cells have an increased ability to squeeze through extracellular matrix gaps that they create by promoting proteolysis of its components. Major sites of degradation are specialized micro-domains in the plasma membrane collectively named invadosomes where the Arp2/3 complex and formin proteins cooperate to spatio-temporally control actin nucleation and the folding of a dynamic F-actin core. At invadosomes, proper coupling of exo-endocytosis allows polarized delivery of proteases that facilitate degradation of ECM and disruption of the cellular barrier. We investigated the contribution of the actin nucleator Spire-1 to invadosome structure and function, using Src-activated cells and cancer cells. We found that Spire-1 is specifically recruited at invadosomes and is part of a multi-molecular complex containing Src kinase, the formin mDia1 and actin. Spire-1 interacts with the Rab3A GTPase, a key player in the regulation of exocytosis that is present at invadosomes. Finally, over- and under-expression of Spire-1 resulted in cells with an increased or decreased potential for matrix degradation, respectively, therefore suggesting a functional interplay of Spire-1 with both actin nucleation and vesicular trafficking that might impact on cell invasive and metastatic behavior.

Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions.

Quantitative Proteomic Analysis Reveals Molecular Adaptations in the Hippocampal Synaptic Active Zone of Chronic Mild Stress-Unsusceptible Rats.

BACKGROUND: While stressful events are recognized as an important cause of major depressive disorder, some individuals exposed to life stressors maintain normal psychological functioning. The molecular mechanism(s) underlying this phenomenon remain unclear. Abnormal transmission and plasticity of hippocampal synapses have been implied to play a key role in the pathoetiology of major depressive disorder. METHODS: A chronic mild stress protocol was applied to separate susceptible and unsusceptible rat subpopulations. Proteomic analysis using an isobaric tag for relative and absolute quantitation coupled with tandem mass spectrometry was performed to identify differential proteins in enriched hippocampal synaptic junction preparations. RESULTS: A total of 4318 proteins were quantified, and 89 membrane proteins were present in differential amounts. Of these, SynaptomeDB identified 81 (91%) having a synapse-specific localization. The unbiased profiles identified several candidate proteins within the synaptic junction that may be associated with stress vulnerability or insusceptibility. Subsequent functional categorization revealed that protein systems particularly involved in membrane trafficking at the synaptic active zone exhibited a positive strain as potential molecular adaptations in the unsusceptible rats. Moreover, through STRING and immunoblotting analysis, membrane-associated GTP-bound Rab3a and Munc18-1 appear to coregulate syntaxin-1/SNAP25/VAMP2 assembly at the hippocampal presynaptic active zone of unsusceptible rats, facilitating SNARE-mediated membrane fusion and neurotransmitter release, and may be part of a stress-protection mechanism in actively maintaining an emotional homeostasis. CONCLUSIONS: The present results support the concept that there is a range of potential protein adaptations in the hippocampal synaptic active zone of unsusceptible rats, revealing new investigative targets that may contribute to a better understanding of stress insusceptibility.

Intermediates in the guanine nucleotide exchange reaction of Rab8 protein catalyzed by guanine nucleotide exchange factors Rabin8 and GRAB.

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.

α-Synuclein membrane association is regulated by the Rab3a recycling machinery and presynaptic activity.

α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity.

ß-Adrenergic receptors activate exchange protein directly activated by cAMP (Epac), translocate Munc13-1, and enhance the Rab3A-RIM1α interaction to potentiate glutamate release at cerebrocortical nerve terminals.

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the ß-adrenergic receptor (ßAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of ßARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that ßARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.