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.

Sporadic Creutzfeldt-Jakob disease subtype-specific alterations of the brain proteome: impact on Rab3a recycling.

Sporadic Creutzfeldt-Jakob disease (sCJD) is characterized by wide clinical and pathological variability, which is mainly influenced by the conformation of the misfolded prion protein, and by the methionine and valine polymorphism at codon 129 of the prion protein gene. This heterogeneity likely implies differences in the molecular cascade that leads to the development of certain disease phenotypes. In this study, we investigated the proteome of the frontal cortex of patients with the two most common sCJD subtypes (MM1 and VV2) using 2D-DIGE and MS. Analysis of 2D maps revealed that 46 proteins are differentially expressed in the sCJD. Common differential expression was detected for seven proteins, four showed opposite direction of differential expression, and the remaining ones displayed subtype-specific alteration. The highest number of differentially expressed proteins was associated with signal transduction and neuronal activity. Moreover, functional groups of proteins involved in cell cycle and death, as well as in structure and motility included subtype-specific expressed proteins exclusively. The expression of Rab GDP dissociation inhibitor alpha, which regulates Rab3a-mediated neurotransmitter release, was affected in both sCJD subtypes that were analyzed. Therefore, we also investigated as to whether Rab3a recycling is altered. Indeed, we found an accumulation of the membrane-associated form, thus the active one, which suggests that dysfunction of the Rab3a-mediated exocytosis might be implicated in sCJD pathology.

Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons.

Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.

Analysis on the emerging role of Rab3 GTPase-activating protein in Warburg Micro and Martsolf syndrome.

Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Recently mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GTPase-activating protein were found to cause Warburg Micro syndrome and Martsolf syndrome, respectively, both of which exhibit mental retardation. We have found that loss of p130 in mice results in inhibition of Ca2+-dependent glutamate release from cerebrocortical synaptosomes and alters short-term plasticity in the hippocampal CA1 region, probably through the accumulation of the GTP-bound form of Rab3A. Here, we describe the procedures for the measurement of the GTP-bound pool of Rab3A with pull-down assay using mouse brains and the biochemical method for the measurement of glutamate release from mouse synaptosomes.

Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post-mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, synaptosome-associated protein 25 (SNAP 25) in HD brains of grades I-IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre-synaptic transmitter release may underlie some of the symptoms of HD.

Rab3 GTPase-activating protein regulates synaptic transmission and plasticity through the inactivation of Rab3.

Rab3A small G protein is a member of the Rab family and is most abundant in the brain, where it is localized on synaptic vesicles. Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and this change in activity is associated with the trafficking cycle of synaptic vesicles at nerve terminals. Rab3 GTPase-activating protein (GAP) stimulates the GTPase activity of Rab3A and is expected to determine the timing of the dissociation of Rab3A from synaptic vesicles, which may be coupled with synaptic vesicle exocytosis. Rab3 GAP consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150. Recently, mutations in p130 were found to cause Warburg Micro syndrome with severe mental retardation. Here, we generated p130-deficient mice and found that the GTP-bound form of Rab3A accumulated in the brain. Loss of p130 in mice resulted in inhibition of Ca(2+)-dependent glutamate release from cerebrocortical synaptosomes and altered short-term plasticity in the hippocampal CA1 region. Thus, Rab3 GAP regulates synaptic transmission and plasticity by limiting the amount of the GTP-bound form of Rab3A.

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes.

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca2+ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca2+ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca2+ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca2+ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.

Alternative splicing in the first alpha-helical region of the Rab-binding domain of Rim regulates Rab3A binding activity: is Rim a Rab3 effector protein during evolution?

Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim1Delta56-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim1Delta56-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.

Transient increase in rab 3A and synaptobrevin immunoreactivity after mild hypoxia in neonatal rats.

1. In the present work we describe the short term effects of mild neonatal hypoxia on the synapse as assessed by the immunoreactivity (IR) of two synaptic proteins: rab 3A and synaptobrevin (VAMP). 2. Using the sensitive methodology of immunoblotting, we measured rab 3A and VAMP-IR in homogenates from the cerebral cortex, hippocampus, and corpus striatum of control (breathing room air) and hypoxiated (breathing 95.5% N2-6.5% O2 for 70 min) 4-day-old rats at 1, 2, and 6 h after the end of the hypoxia. Immunostaining with examination by light microscopy was performed using the synaptic protein-specific antibodies on fixed brain sections from animals belonging to the same litter and submitted to hypoxia. 3. A transient increase of VAMP-IR was observed in the hippocampus and corpus striatum, and for rab 3A in the striatum, 1 h after initiating reoxygenation. At the following time points the values returned to control levels. This effect was less clearly observed in the immunostained sections. 4. Mild hypoxia has an effect on sensitive brain regions, eliciting an increase in the IR of at least two proteins involved in the synaptic vesicle cycle. The transient nature of this effect possibly indicates the activation of endogenous neuroprotective mechanisms.

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique.

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca(2+) channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca(2+) channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca(2+) channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca(2+) channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.

Reduction of the synaptic protein rab3a in the thalamus and connecting brain regions in post-mortem schizophrenic brains.

Although the psychotic symptoms in schizophrenia can be alleviated by treatment with dopaminergic receptor antagonists, the etiology and underlying neurochemical pathology remains obscure. Both neuropathological and magnetic resonance imaging studies have found evidence for neuronal loss and atrophy in the thalamus in schizophrenia, implicating this key structure for gating information to cortical areas in the pathophysiology. Recent studies have also found evidence of synaptic loss in the thalamus in schizophrenia. To further examine possible synaptic disturbances, we studied the synaptic related protein rab3a as a marker for synaptic density, using both quantitative Western blotting and immunohistochemistry. The material consisted of brains from 22 schizophrenic patients (mean age 79.3 years), and 24 control subjects (74.8 years). Reduced rab3a protein levels were found in the left thalamus in schizophrenia (0.47 +/- 0.17 vs. 1.00 +/- 0.18; p < 0.0001), while a less marked decrease was found also in the right thalamus (0.75 +/- 0.13 vs. 1.00 +/- 0.09; p < 0.0001). Immunohistochemistry, performed on two schizophrenic and two control brains, revealed that rab3a immunoreactivity was most reduced in the left anterior and mediodorsal thalamic nuclei. Therefore, we extended the study to brain regions connected these thalamic nuclei. Reduced rab3a protein levels were found schizophrenia also in the frontal cortex, hippocampus, gyrus cinguli, and parietal cortex, while no significant differences were found in the temporal cortex, or in cerebellum. The reduction in rab3a was not found to be secondary to confounding factors such as age-differences, post-mortem delay time, generalized brain atrophy, or antipsychotic medication. Therefore, the reduction of rab3a probably reflects synaptic disturbances, possibly synaptic loss, in the limbic system and neocortical areas, in schizophrenia. This part of the brain is known to be involved in behavioral and emotional control, and thus to be crucial for higher mental functions, suggesting that synaptic disturbances in the limbic system may be of importance in the development of psychotic symptoms in schizophrenia.

Cyclosporine A-induced hypertension involves synapsin in renal sensory nerve endings.

The calcineurin inhibitor cyclosporine A (CsA) has emerged as a major cause of secondary hypertension in humans, but the underlying pathogenetic mechanisms have remained enigmatic. Synapsins are a family of synaptic vesicle phosphoproteins that are essential for normal regulation of neurotransmitter release at synapses. In addition to synaptic vesicles, synapsins and other vesicle proteins are found on microvesicles in sensory nerve endings in peripheral tissues. However, the functions of the sensory microvesicles in general, and of synapsins in particular, are unknown. We now demonstrate in a mouse model that CsA raises blood pressure by stimulating renal sensory nerve endings that contain synapsin-positive microvesicles. In knockout mice lacking synapsin I and II, sensory nerve endings are normally developed but not stimulated by CsA whereas a control stimulus, capsaicin, is fully active. The reflex activation of efferent sympathetic nerve activity and the increase in blood pressure by CsA seen in control are greatly attenuated in synapsin-deficient mice. These results provide a mechanistic explanation for CsA-induced acute hypertension and suggest that synapsins could serve as a drug target in this refractory condition. Furthermore, these data establish evidence that synapsin-containing sensory microvesicles perform an essential role in sensory transduction and suggest a role for synapsin phosphorylation in this process.

Prion protein deposition and abnormal synaptic protein expression in the cerebellum in Creutzfeldt-Jakob disease.

Prion protein (PrP(C)) is a cell membrane-anchored glycoprotein, which is replaced by a pathogenic protease-resistant, beta-sheet-containing isoform (PrP(CJD) or PrP(SC)) in human and animal prion encephalopathies, including sporadic Creutzfeldt-Jakob disease. Cell fractionation methods show that PrP(C) localizes in presynaptic membrane-enriched fractions. Following infection, abnormal PrP accumulates in nerve cell processes and synaptic regions. The present study examines the possible correlation between abnormal PrP deposition and the expression of synaptic proteins controlling neurotransmission in the cerebellum of six 129 Met/Met sporadic cases of Creutzfeldt-Jakob disease. Aggregates of protease-resistant PrP-positive granules, reminiscent of cerebellar glomeruli, were found in the granular cell layer, whereas fine punctate PrP-immunoreactive deposits occurred in the molecular layer. Small numbers of diffuse, irregular plaque-like PrP deposits in the molecular and granular cell layers were present in every case. The somas of Purkinje cells, and stellate, basket and Golgi neurons, were not immunostained. PrP-immunoreactive fibres were found in the album of the cerebellum and hilus of the dentate nucleus. Punctate PrP deposition decorated the neuropil of the dentate nucleus and the surface of dentate neurons. Synaptic protein expression was examined with synaptophysin, synapsin-1, synaptosomal-associated protein of 25,000 mol. wt, syntaxin-1 and Rab3a immunohistochemistry. Reduced synaptophysin, synapsin-1, synaptosomal-associated protein of 25,000 mol. wt, syntaxin-1 and Rab3a immunoreactivity was noted in the granular cell layer in every case, but reduced expression was inconstant in the molecular layer. Synaptophysin accumulated in axon torpedoes, thus indicating abnormal axon transport. Expression of synaptic proteins was relatively preserved in the dentate nucleus, although synaptophysin immunohistochemistry disclosed large coarse pericellular terminals in Creutzfeldt-Jakob disease, instead of the fine granular terminals in control cases, around the soma of dentate neurons. Finally, Rab3a accumulated in the cytoplasm of Purkinje cells, thus suggesting major anomalies in Rab3a transport. These observations demonstrate, for the first time, abnormal expression of crucial synaptic proteins in the cerebellum of cases with Creutzfeldt-Jakob disease. However, abnormal PrP deposition is not proportional to the degree of reduction of synaptic protein expression in the different layers of the cerebellar cortex and in the dentate nucleus. Therefore, it remains to be elucidated how abnormal PrP impacts on the metabolism of proteins linked to exocytosis and neurotransmission, and how abnormal PrP deposition results in eventual synaptic loss.

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells.

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immunoelectron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.

The synaptic-vesicle-specific proteins rab3a and synaptophysin are reduced in thalamus and related cortical brain regions in schizophrenic brains.

Two synaptic-vesicle proteins, rab3a and synaptophysin, have been studied on post-mortem brain tissues of schizophrenics and healthy controls. We found significantly reduced levels of rab3a in thalamus (p<0.001); for both proteins in gyrus cinguli and hippocampus (p<0.0001); for rab3a in frontal and parietal cortex (p<0.05); and no differences in temporal cortex or cerebellum in schizophrenics compared with controls. Reduced synaptic density may be a prominent feature of the molecular neuropathology of schizophrenia.