Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed \"homeostatic synaptic plasticity.\" We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A was required for the increase in synaptic AMPARs following prolonged activity blockade, but the increase in mEPSC amplitudes was not always accompanied by an increase in postsynaptic AMPAR levels, suggesting other factors may contribute. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that neuronal Rab3A is required for homeostatic synaptic plasticity and that it does so partially through regulation of the surface expression of AMPA receptors.

Cognitive dysfunction is associated with methamphetamine use disorder (MUD). Here, we used genetic and pharmacological approaches to examine the involvement of either Group 2 metabotropic glutamate (mGlu2) or mGlu3 receptors in memory deficit induced by methamphetamine in mice. Methamphetamine treatment (1 mg/kg, i.p., once a day for 5 d followed by 7 d of withdrawal) caused an impaired performance in the novel object recognition test in wild-type mice, but not in mGlu2-/- or mGlu3-/- mice. Memory deficit in wild-type mice challenged with methamphetamine was corrected by systemic treatment with selectively negative allosteric modulators of mGlu2 or mGlu3 receptors (compounds VU6001966 and VU0650786, respectively). Methamphetamine treatment in wild-type mice caused large increases in levels of mGlu2/3 receptors, the Type 3 activator of G-protein signaling (AGS3), Rab3A, and the vesicular glutamate transporter, vGlut1, in the prefrontal cortex (PFC). Methamphetamine did not alter mGlu2/3-mediated inhibition of cAMP formation but abolished the ability of postsynaptic mGlu3 receptors to boost mGlu5 receptor-mediated inositol phospholipid hydrolysis in PFC slices. Remarkably, activation of presynaptic mGlu2/3 receptors did not inhibit but rather amplified depolarization-induced [3H]-D-aspartate release in synaptosomes prepared from the PFC of methamphetamine-treated mice. These findings demonstrate that exposure to methamphetamine causes changes in the expression and function of mGlu2 and mGlu3 receptors, which might alter excitatory synaptic transmission in the PFC and raise the attractive possibility that selective inhibitors of mGlu2 or mGlu3 receptors (or both) may be used to improve cognitive dysfunction in individuals affected by MUD.

The goal of this study was to demonstrate the functions and specific mechanism of long non-coding RNA (lncRNA) GNAS-AS1 in lung adenocarcinoma. Levels of lncRNA GNAS-AS1, microRNA (miR)-433-3p, and Rab3A were assessed by quantitative real-time PCR (qRT-PCR). The target-binding sites of lncRNA GNAS-AS1, miR-433-3p, and Rab3A were predicted and confirmed by bioinformatics tool (StarBase) and a dual-luciferase reporter system. Cell proliferation and apoptosis were checked using MTT and flow cytometry, respectively. Additionally, the levels of apoptosis-related and epithelial-mesenchymal transition (EMT)-associated genes in A549 cells were analyzed by qRT-PCR and western blot. We found that lncRNA GNAS-AS1 was upregulated, miR-433-3p was low-expressed, and Rab3A was overexpressed in lung adenocarcinoma tissues and cell lines. LncRNA GNAS-AS1 interacted with miR-433-3p and negatively regulated miR-433-3p levels. Rab3A was a direct target of miR-433-3p. Downregulation of lncRNA GNAS-AS1 remarkably suppressed cell proliferation, promoted cell apoptosis, decreased B-cell lymphoma-2 (Bcl-2) expression, enhanced the Bcl-2-Associated X (Bax) level, promoted E-cadherin expression, and reduced N-cadherin and Rab3A levels. However, the miR-433-3p inhibitor reversed all these findings. Similarly, the inhibitory effects of miR-433-3p mimic on A549 cells were reversed by the Rab3A-plasmid. In conclusion, lncRNA GNAS-AS1 downregulation suppressed lung adenocarcinoma cell proliferation and EMT through the miR-433-3p/Rab3A axis.

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.

We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein.

Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of Ca2+-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) as well as Rab3a, which we have previously described to be a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.

Synaptic impairment may be the main cause of cognitive dysfunction in brain aging that is probably due to a reduction in synaptic contact between the axonal buttons and dendritic spines. Rho proteins including the small GTPase Rac1 have become key regulators of neuronal morphogenesis that supports synaptic plasticity. Small Rho- and Ras-GTPases are post-translationally modified by the isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively. For all GTPases, anchoring in the plasma membrane is essential for their activation by guanine nucleotide exchange factors (GEFs). Rac1-specific GEFs include the protein T lymphoma invasion and metastasis 1 (Tiam1). Tiam1 interacts with the TrkB receptor to mediate the brain-derived neurotrophic factor (BDNF)-induced activation of Rac1, resulting in cytoskeletal rearrangement and changes in cellular morphology. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) acts as a highly affine-selective TrkB receptor agonist and causes the dimerization and autophosphorylation of the TrkB receptor and thus the activation of downstream signaling pathways. In the current study, we investigated the effects of 7,8-DHF on cerebral lipid isoprenoid and Rho protein levels in male C57BL/6 mice aged 3 and 23 months. Aged mice were daily treated with 100 mg/kg b.w. 7,8-DHF by oral gavage for 21 days. FPP, GGPP, and cholesterol levels were determined in brain tissue. In the same tissue, the protein content of Tiam1 and TrkB in was measured. The cellular localization of the small Rho-GTPase Rac1 and small Rab-GTPase Rab3A was studied in total brain homogenates and membrane preparations. We report the novel finding that 7,8-DHF restored levels of the Rho proteins Rac1 and Rab3A in membrane preparations isolated from brains of treated aged mice. The selective TrkB agonist 7,8-DHF did not affect BDNF and TrkB levels, but restored Tiam1 levels that were found to be reduced in brains of aged mice. FPP, GGPP, and cholesterol levels were significantly elevated in brains of aged mice but not changed by 7,8-DHF treatment. Hence, 7,8-DHF may be useful as pharmacological tool to treat age-related cognitive dysfunction although the underlying mechanisms need to be elucidated in detail.

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.

Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases.

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.