Synaptic Vesicle Docking Research Articles

New Insights Into Interactions of Presynaptic Calcium Channel Subtypes and SNARE Proteins in Neurotransmitter Release.

Action potential (AP) induces presynaptic membrane depolarization and subsequent opening of Ca2+ channels, and then triggers neurotransmitter release at the active zone of presynaptic terminal. Presynaptic Ca2+ channels and SNARE proteins (SNAREs) interactions form a large signal transfer complex, which are core components for exocytosis. Ca2+ channels serve to regulate the activity of Ca2+ channels through direct binding and indirect activation of active zone proteins and SNAREs. The activation of Ca2+ channels promotes synaptic vesicle recruitment, docking, priming, fusion and neurotransmission release. Intracellular calcium increase is a key step for the initiation of vesicle fusion. Various voltage-gated calcium channel (VGCC) subtypes exert different physiological functions. Until now, it has not been clear how different subtypes of calcium channels integrally regulate the release of neurotransmitters within 200 μs of the AP arriving at the active zone of synaptic terminal. In this mini review, we provide a brief overview of the structure and physiological function of Ca2+ channel subtypes, interactions of Ca2+ channels and SNAREs in neurotransmitter release, and dynamic fine-tune Ca2+ channel activities by G proteins (Gβγ), multiple protein kinases and Ca2+ sensor (CaS) proteins.

MIR137 schizophrenia-associated locus controls synaptic function by regulating synaptogenesis, synapse maturation and synaptic transmission.

The MIR137 locus is a replicated genetic risk factor for schizophrenia. The risk-associated allele is reported to increase miR-137 expression and miR-137 overexpression alters synaptic transmission in mouse hippocampus. We investigated the cellular mechanisms underlying these observed effects in mouse hippocampal neurons in culture. First, we correlated the risk allele to expression of the genes in the MIR137 locus in human postmortem brain. Some evidence for increased MIR137HG expression was observed, especially in hippocampus of the disease-associated genotype. Second, in mouse hippocampal neurons, we confirmed previously observed changes in synaptic transmission upon miR-137 overexpression. Evoked synaptic transmission and spontaneous release were 50% reduced. We identified defects in release probability as the underlying cause. In contrast to previous observations, no evidence was obtained for selective synaptic vesicle docking defects. Instead, ultrastructural morphometry revealed multiple effects of miR-137 overexpression on docking, active zone length and total vesicle number. Moreover, proteomic analyses of neuronal protein showed that expression of Syt1 and Cplx1, previously reported as downregulated upon miR-137 overexpression, was unaltered. Immunocytochemistry of synapses overexpressing miR-137 showed normal Synaptotagmin1 and Complexin1 protein levels. Instead, our proteomic analyses revealed altered expression of genes involved in synaptogenesis. Concomitantly, synaptogenesis assays revealed 31% reduction in synapse formation. Taken together, these data show that miR-137 regulates synaptic function by regulating synaptogenesis, synaptic ultrastructure and synapse function. These effects are plausible contributors to the increased schizophrenia risk associated with miR-137 overexpression.

Mechanisms of α-Synuclein Induced Synaptopathy in Parkinson's Disease.

Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition, α-Syn is able to interact with and reduce the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly contributes to the formation of toxic α-Syn oligomers. Together these data suggest a vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic dysfunction and impaired neuronal communication, ultimately causing synaptopathy and progressive neurodegeneration in Parkinson's disease.

Liprin-α3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses

The presynaptic active zone provides sites for vesicle docking and release at central nervous synapses and is essential for speed and accuracy of synaptic transmission. Liprin-α binds to several active zone proteins, and loss-of-function studies in invertebrates established important roles for Liprin-α in neurodevelopment and active zone assembly. However, Liprin-α localization and functions in vertebrates have remained unclear. We used stimulated emission depletion superresolution microscopy to systematically determine the localization of Liprin-α2 and Liprin-α3, the two predominant Liprin-α proteins in the vertebrate brain, relative to other active-zone proteins. Both proteins were widely distributed in hippocampal nerve terminals, and Liprin-α3, but not Liprin-α2, had a prominent component that colocalized with the active-zone proteins Bassoon, RIM, Munc13, RIM-BP, and ELKS. To assess Liprin-α3 functions, we generated Liprin-α3-KO mice by using CRISPR/Cas9 gene editing. We found reduced synaptic vesicle tethering and docking in hippocampal neurons of Liprin-α3-KO mice, and synaptic vesicle exocytosis was impaired. Liprin-α3 KO also led to mild alterations in active zone structure, accompanied by translocation of Liprin-α2 to active zones. These findings establish important roles for Liprin-α3 in active-zone assembly and function, and suggest that interplay between various Liprin-α proteins controls their active-zone localization.

Interaction between A-Synuclein and VAMP2 Promotes SNARE-Ependent Vesicle Docking and Fusion

Despites its central role in the pathogenesis of Parkinson disease, the normal biological function of α-synuclein remains largely unknown. Previous studies have shown both in-vitro and in-vivo that α-synuclein specifically binds to anionic membrane and could serve as a chaperone for the SNARE complex that mediates the docking and fusion of synaptic vesicles. Using a combination of solution NMR spectroscopy and single-molecule assays, we show here that α-synuclein promotes SNARE-dependent docking and fusion through its interaction with v-SNARE synaptobrevin 2 (VAMP2). We show that while the amphipathic N-terminal domain of α-synuclein binds to the surface of negatively charged vesicles, its C-terminal tail directly interacts with the C-terminal region of VAMP2. Our study highlights the critical role VAMP2 in recruiting α-synuclein in order to catalyze the SNARE-dependent vesicle fusion process and suggests that in non-pathological conditions α-synuclein is an important mediator of synaptic vesicle trafficking.

Hydrodynamics govern the pre-fusion docking time of synaptic vesicles.

Synaptic vesicle fusion is a crucial step in the neurotransmission process. Neurotransmitter-filled vesicles are pre-docked at the synapse by the mediation of ribbon structures and SNARE proteins at the ribbon synapses. An electrical impulse triggers the fusion process of pre-docked vesicles, leading to the formation of a fusion pore and subsequently resulting in the release of neurotransmitter into the synaptic cleft. In this study, a continuum model of lipid membrane along with lubrication theory is used to determine the traverse time of the synaptic vesicle under the influence of hydrodynamic forces. We find that the traverse time is strongly dependent on how fast the driving force decays or grows with closure of the gap between the vesicle and the plasma membrane. If the correct behaviour is chosen, the traverse time obtained is of the order of a few hundred milliseconds and lies within the experimentally obtained value of approximately 250 ms (Zenisek D, Steyer JA, Almers W. 2000 Nature406, 849-854 (doi:10.1038/35022500)). We hypothesize that there are two different force behaviours, which complies with the experimental findings of pre-fusion docking of synaptic vesicles at the ribbon synapses. The common theme in the proposed force models is that the driving force has to very rapidly increase or decrease with the amount of clamping.

A phenomenological model of the synapse between the inner hair cell and auditory nerve: Implications of limited neurotransmitter release sites

Peterson and Heil [Hear. Res., In Press] have argued that the statistics of spontaneous spiking in auditory nerve fibers (ANFs) can be best explained by a model with a limited number of synaptic vesicle docking (release) sites (∼4) and a relatively-long average redocking time (∼16–17 ms) for each of the sites. In this paper we demonstrate how their model can be: i) generalized to also describe sound-driven ANF responses and ii) incorporated into a well-established and widely-used model of the entire auditory periphery [Zilany et al., J. Acoust. Soc. Am. 135, 283–286, 2014]. The responses of the new model exhibit substantial improvement in several measures of ANF spiking statistics, and predicted physiological forward-masking and rate-level functions from the new model structure are shown to also better match published physiological data.

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.

Medium Optimization for Synaptobrevin Production Using Statistical Methods

Background: Botulinum toxin, the most potent biological toxin, has become a powerful therapeutic tool for a growing number of clinical applications. Molecular studies have identified a family of synaptic vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins) which have been implicated in synaptic vesicle docking and fusion with plasma membrane proteins. Materials and Methods: Using the synaptobrevin as a substrate for in vitro assay is the method to detect BoNT activity. We have been working on optimizations of bacterial expression conditions and media for high-level production of synaptobrevin peptide. Statistics-based experimental design was used to investigate the effect of medium components (E. coli strain, peptone, IPTG, yeast extract, ampicillin, and temperature) on synaptobrevin production by E. coli. Results: A 24 fractional factorial design with center points revealed that IPTG and temperature were the most significant factors, whereas the other factors were not important within the levels tested. This purpose was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for synaptobrevin production was found to be: IPTG 29 mM, peptone 10 g/L, yeast extract 5 g/L, temperature 23°C and ampicillin 100 mg/L. This medium was projected to produce, theoretically, 115 mg/L synaptobrevin. Conclusion: The optimum medium composition synaptobrevin production was found to be: BL21 (E.coli strain), LB medium (peptone 10 g/L, Yeast 5 g/L), Ampicillin (100 mg/L), IPTG (0.29 mg/L) and temperature (23°C). Keywords: synaptobrevin; E. coli; experimental designs; central composite design; medium optimization

A Multilevel Functional Study of aSNAP25At-Risk Variant for Bipolar Disorder and Schizophrenia

The synaptosomal-associated protein SNAP25 is a key player in synaptic vesicle docking and fusion and has been associated with multiple psychiatric conditions, including schizophrenia, bipolar disorder, and attention-deficit/hyperactivity disorder. We recently identified a promoter variant in SNAP25, rs6039769, that is associated with early-onset bipolar disorder and a higher gene expression level in human prefrontal cortex. In the current study, we showed that this variant was associated both in males and females with schizophrenia in two independent cohorts. We then combined in vitro and in vivo approaches in humans to understand the functional impact of the at-risk allele. Thus, we showed in vitro that the rs6039769 C allele was sufficient to increase the SNAP25 transcription level. In a postmortem expression analysis of 33 individuals affected with schizophrenia and 30 unaffected control subjects, we showed that the SNAP25b/SNAP25a ratio was increased in schizophrenic patients carrying the rs6039769 at-risk allele. Last, using genetics imaging in a cohort of 71 subjects, we showed that male risk carriers had an increased amygdala-ventromedial prefrontal cortex functional connectivity and a larger amygdala than non-risk carriers. The latter association has been replicated in an independent cohort of 121 independent subjects. Altogether, results from these multilevel functional studies are bringing strong evidence for the functional consequences of this allelic variation of SNAP25 on modulating the development and plasticity of the prefrontal-limbic network, which therefore may increase the vulnerability to both early-onset bipolar disorder and schizophrenia.SIGNIFICANCE STATEMENT Functional characterization of disease-associated variants is a key challenge in understanding neuropsychiatric disorders and will open an avenue in the development of personalized treatments. Recent studies have accumulated evidence that the SNARE complex, and more specifically the SNAP25 protein, may be involved in psychiatric disorders. Here, our multilevel functional studies are bringing strong evidence for the functional consequences of an allelic variation of SNAP25 on modulating the development and plasticity of the prefrontal-limbic network. These results demonstrate a common genetically driven functional alteration of a synaptic mechanism both in schizophrenia and early-onset bipolar disorder and confirm the shared genetic vulnerability between these two disorders.

The primed SNARE-complexin-synaptotagmin complex for neuronal exocytosis.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone.

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses.

Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1β are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

Transcription regulation mechanism of the syntaxin 1A gene via protein kinase A.

Syntaxin 1A (Stx1a) is primarily involved in the docking of synaptic vesicles at active zones in neurons. Its gene is a TATA-less gene, with several transcription initiation sites, which is activated by the binding of Sp1 and acetylated histone H3 (H3) in the core promoter region (CPR) through the derepression of class I histone deacetylase (HDAC). In the present study, to clarify the factor characterizing Stx1a gene expression via the protein kinase A (PKA) pathway inducing the Stx1a mRNA, we investigated whether the epigenetic process is involved in the Stx1a gene transcription induced by PKA signaling. We found that the PKA activator forskolin induced Stx1a expression in non-neuronal cells, FRSK and 3Y1, which do not endogenously express Stx1a, unlike PC12. HDAC8 inhibition by shRNA knockdown and specific inhibitors induced Stx1a expression in FRSK. The PKA inhibitor H89 suppressed HDAC8-Ser39 phosphorylation, H3 acetylation and Stx1a induction by forskolin in FRSK cells. Finally, we also found that forskolin led to the dissociation of HDAC8-CPR interaction and the association of Sp1 and Ac-H3 to CPR in FRSK. The results of the current study suggest that forskolin phosphorylates HDAC8-Ser39 via the PKA pathway and increases histone H3 acetylation in cells expressing HDAC8, resulting in the induction of the Stx1a gene.

Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming

The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex.

A stochastic model of active zone material mediated synaptic vesicle docking and priming at resting active zones

Synaptic vesicles (SVs) fuse with the presynaptic membrane (PM) at specialized regions called active zones for synaptic transmission. SVs are associated with dense aggregates of macromolecules called active zone material (AZM) that has been thought to be involved in SV release. However, its role has recently begun to be elucidated. Several morphological studies proposed distinctively different AZM mediated SV docking and priming models: sequential and concurrent SV docking/priming. To explore ways to reconcile the contradictory models we develop a stochastic AZM mediated SV docking and priming model. We assume that the position of each connection site of the AZM macromolecules on their SV, directly linking the SV with the PM, varies by random shortening and lengthening of the macromolecules at resting active zones. We also perform computer simulations of SVs near the PM at resting active zones, and the results show that the distribution of the AZM connection sites can significantly affect the SV’s docking efficiency and distribution of its contact area with the PM, thus priming and that the area correlates with the shape of the SVs providing a way to account for seemingly irreconcilable observations reported about the spatial relationship of SVs with the PM at active zones.

Calcium Sensitive Ring-Like Oligomers of Synaptotagmin 1: Implications for Neurotransmitter Release

The synaptic vesicle protein, Synaptotagmin-1 (Syt1) is required to couple Ca2+ influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Using negative stain electron microscopy, we find that the Syt1 assembles into ring-like oligomeric structures on lipid monolayers under physiological ionic strength and lipid composition in the absence of free Ca2+. These rings oligomers vary in diameter between 19 - 42 nm (with an average size of 30 ± 5 nm), corresponding to 12- 25 molecules of Syt1. The ring-like oligomers are sensitive to Ca2+ and are disrupted rapidly by the physiological concentrations of free Ca2+. Analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, the circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, we find that both Syt1 ring formation and its disruption by Ca2+ principally involve well-established functional surfaces of the C2B domain which are important for neurotransmission. This includes the Ca2+ -independent binding of the polybasic motif of C2B domain with phosphatidylinositol 4,5-bisphosphate (PIP2) as a prerequisite for rings to assemble. This suggests that ring formation may be triggered at a very early step in synaptic vesicle docking, as C2B-PIP2 interaction is required for docking in vivo. Ca2+ binding to the C2B domain and re-orientation/insertion into the membrane, both required for triggering synaptic transmission, disrupts the ring oligomers. We advance a simple and novel mechanism wherein Syt1 ring oligomer act as reversible washer/spacer to synchronize neurotransmitter release to Ca2+ influx. Supporting this hypothesis, we find mutations in Syt1 that specifically disrupt the ring oligomer formation results in dysregulation neuroexocytosis in PC12 cells.

PtdInsP2 and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.

Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth

ObjectiveThe present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. DesignImmunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. ResultsPGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. ConclusionsThe present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses.

Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis.

Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥3.5 and a p value <0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca2+ and H+ homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.