Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 µM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) was reduced with increasing [Ca2+]. Overall, we find that specific lipids differentially modulate Ca2+ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca2+, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

Delivering synaptic protein mRNAs via extracellular vesicles ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

BACKGROUND: Synaptic dysfunction with reduced synaptic protein levels is a core feature of Alzheimer's disease (AD). Synaptic proteins play a central role in memory processing, learning, and AD pathogenesis. Evidence suggests that synaptic proteins in plasma neuronal-derived extracellular vesicles (EVs) are reduced in patients with AD. However, it remains unclear whether levels of synaptic proteins in EVs are associated with hippocampal atrophy of AD and whether upregulating the expression of these synaptic proteins has a beneficial effect on AD. METHODS: In this study, we included 57 patients with AD and 56 healthy controls. We evaluated their brain atrophy through magnetic resonance imaging using the medial temporal lobe atrophy score. We measured the levels of four synaptic proteins, including synaptosome-associated protein 25 (SNAP25), growth-associated protein 43 (GAP43), neurogranin, and synaptotagmin 1 in both plasma neuronal-derived EVs and cerebrospinal fluid (CSF). We further examined the association of synaptic protein levels with brain atrophy. We also evaluated the levels of these synaptic proteins in the brains of 5×FAD mice. Then, we loaded rabies virus glycoprotein-engineered EVs with messenger RNAs (mRNAs) encoding GAP43 and SNAP25 and administered these EVs to 5×FAD mice. After treatment, synaptic proteins, dendritic density, and cognitive function were evaluated. RESULTS: The results showed that GAP43, SNAP25, neurogranin, and synaptotagmin 1 were decreased in neuronal-derived EVs but increased in CSF in patients with AD, and the changes corresponded to the severity of brain atrophy. GAP43 and SNAP25 were decreased in the brains of 5×FAD mice. The engineered EVs efficiently and stably delivered these synaptic proteins to the brain, where synaptic protein levels were markedly upregulated. Upregulation of synaptic protein expression could ameliorate cognitive impairment in AD by promoting dendritic density. This marks the first successful delivery of synaptic protein mRNAs via EVs in AD mice, yielding remarkable therapeutic effects. CONCLUSIONS: Synaptic proteins are closely related to AD processes. Delivery of synaptic protein mRNAs via EVs stands as a promising effective precision treatment strategy for AD, which significantly advances the current understanding of therapeutic approaches for the disease.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation.

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

miRNA-363-3p Hinders Proliferation, Migration, Invasion and Autophagy of Thyroid Cancer Cells by Controlling SYT1 Transcription to affect NF-κB.

BACKGROUND: Thyroid cancer (TC) is a frequent endocrine malignant tumor with various pathologic types. miRNA-363-3p plays a pivotal part in the occurrence, development, prognosis, and treatment of cancer. OBJECTIVE: To explore the mechanism of miRNA-363-3p in TC and provide a new idea for targeted therapy of TC. METHODS: Differential miRNAs and downstream target mRNAs in TC tissues were predicted with bioinformatics analysis. Expression levels of miRNA-363-3p and Synaptotagmin I (SYT1) in TC cells were ascertained by qRT-PCR. Cell migration, invasion, and proliferation were detected by wound healing assay, transwell assay, colony formation assay, CCK-8, and BrdU fluorescence experiment, respectively. Flow cytometry was utilized to detect the levels of apoptosis and necrosis. Immunofluorescence assay was used for detecting autophagosome formation in cells, and the expression levels of autophagy-related proteins, as well as NF-κB related proteins, were measured by western blot. Dual-luciferase reporter gene assay was applied for detecting the interaction between miRNA-363-3p and SYT1. RESULTS: miRNA-363-3p was prominently down-regulated in TC cells. miRNA-363-3p overexpression suppressed migration, invasion, and proliferation, promoting apoptosis and necrosis of TC cells. As the downstream target of miRNA-363-3p, SYT1 was up-regulated in TC cells. SYT1 overexpression reversed the inhibition of TC cell proliferation, invasion, migration, and autophagy mediated by miRNA-363-3p overexpression. In addition, miRNA-363-3p overexpression inhibited the activation of the NF-κB pathway in cells, while further overexpression of SYT1 weakened the inhibition of miRNA-363-3p overexpression on the NF-κB pathway. CONCLUSION: miRNA-363-3p affected the NF-κB signaling pathway by down-regulating SYT1 expression to inhibit the malignant progression of TC cells, providing theoretical support for the treatment of TC.

A conserved electrostatic membrane-binding surface in synaptotagmin-like proteins revealed using molecular phylogenetic analysis and homology modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

Lethal variant in the C2A domain may cause severe SYT1-associated neurodevelopmental disorder in the newborns.

Synaptotagmin-1 (SYT1) plays a pivotal role in regulating presynaptic processes, including neurotransmitter release. SYT1 variants perturb synaptic vesicle endocytosis and exocytosis, resulting in a series of neurodevelopmental disorders defined as Baker-Gordon syndrome. Herein, we report the case of a newborn with dysmorphic facial appearance, severe hypotonia, poor feeding, gastroesophageal reflux, and an inability to eat and breathe, diagnosed with Baker-Gordon syndrome. A retrospective search was performed on a newborn with Baker-Gordon syndrome. Medical charts were reviewed, with focus on the clinical presentation, diagnostic process, and treatment outcomes. Whole-genome high-throughput DNA sequencing was performed to identify genetic variants. Whole-exome sequencing identified the likely pathogenic variant as SYT1 C.551 T > C(p.V184A). Sanger sequencing results indicated that this variant was a de novo mutation in a conservative site located in the C2A domain of the protein. The patient died at 57 days old because of severe feeding and breathing problems. Our findings of a novel lethal variant in the C2A domain of SYT1 in the youngest patient diagnosed infantile Baker-Gordon syndrome who presented with the most severe hypotonia reported to date expands the spectrum of SYT1- associated neurodevelopmental disorders.

Membrane lipids couple synaptotagmin to SNARE-mediated granule fusion in insulin-secreting cells.

Insulin secretion depends on the Ca2+-regulated fusion of granules with the plasma membrane. A recent model of Ca2+-triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to the membrane fusion machinery (Kiessling et al., 2018). Specifically, Ca2+-mediated binding of Syt1's C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different acyl chain compositions. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and that lipid disordering correlates with increased fusion. Addition of Syt1's C2AB domains significantly decreased lipid order in target membranes and increased SNARE-mediated fusion probability. Strikingly, Syt's action on both fusion and lipid order could be partially bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca2+/synaptotagmin-sensing to the SNARE fusion machinery in cells.

Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration.

Synaptotagmin-1 and synaptotagmin-7 are two prominent calcium sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding calcium, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examine the biophysical properties of these two synaptotagmin isoforms and compare their interactions with phospholipid membranes. We discover that synaptotagmin-1-membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a super-penetrator. We exploit these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to calcium.

MicroRNA-19a regulates milk fat metabolism by targeting SYT1 in bovine mammary epithelial cells.

MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3'-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows.

Synaptotagmin 1-triggered lipid signaling facilitates coupling of exo- and endocytosis.

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.

Cytosolic LPS-induced caspase-11 oligomerization and activation is regulated by extended synaptotagmin 1.

Caspase-11 (Casp-11) is known to induce pyroptosis and defends against cytosol-invading bacterial pathogens, but its regulation remains poorly defined. Here, we identified extended synaptotagmin 1 (E-Syt1), an endoplasmic reticulum protein, as a key regulator of Casp-11 oligomerization and activation. Macrophages lacking E-Syt1 exhibited reduced production of interleukin-1ß (IL-1ß) and impaired pyroptosis upon cytosolic lipopolysaccharide (LPS) delivery and cytosol-invasive bacterial infection. Moreover, cleavage of Casp-11 and its downstream substrate gasdermin D were significantly diminished in ESyt1-/- macrophages. Upon LPS stimulation, E-Syt1 underwent oligomerization and bound to the p30 domain of Casp-11 via its synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. E-Syt1 oligomerization and its interaction with Casp-11 facilitated Casp-11 oligomerization and activation. Notably, ESyt1-/- mice were susceptible to infection by cytosol-invading bacteria Burkholderia thailandensis while being resistant to LPS-induced endotoxemia. These findings collectively suggest that E-Syt1 may serve as a platform for Casp-11 oligomerization and activation upon cytosolic LPS sensing.

Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice.

In Parkinson's disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in Striatal Neurons by Regulating Substance P Secretion.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+ binding to the C2A domain of SYT9 triggered membrane fusion in vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENT Synaptotagmin 9 (SYT9) has been described as a Ca2+ sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+ sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

PERK recruits E-Syt1 at ER-mitochondria contacts for mitochondrial lipid transport and respiration.

The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.

Mitochondrial membrane biogenesis: A new pathway for lipid transport mediated by PERK/E-Syt1 complex.

Despite decades of extensive research, mitochondrial lipid transport is a process far from fully understood. In this issue, Sassano et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206008) identified a new complex, composed of E-Syt1 and PERK, which mediates lipid transport at ER-mitochondria contact sites and regulates mitochondrial functions in human cells.

SNARE protein VAMP-2, but not syntaxin-1, SNAP-25 and synaptotagmin 1, expressed in perisynaptic astrocytic processes in the CA1 area of the rat hippocampus.

OBJECTIVE: Perisynaptic astrocytic processes have been suggested as sites for the regulated release of neuroactive substances. However, very little is known about the molecular properties of regulated exocytosis in these processes. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins mediate synaptic vesicle exocytosis from neuronal cells and might be candidates for regulated exocytosis also from astrocytic processes. The expression of SNARE proteins in astrocytes, however, is not clarified. Thus, we aimed to investigate the localization and relative concentrations of neuronal SNARE proteins syntaxin-1, synaptosomal nerve-associated protein 25 (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2) (synaptobrevin-2) and calcium sensor synaptotagmin 1 in perisynaptic astrocytic processes compared to nerve terminals and dendrites. METHODS: We used quantitative immunogold electron microscopy of the rat hippocampus to investigate the localization and concentration of neuronal SNARE proteins. RESULTS: As expected, analysis of the immunogold data revealed a lower labeling density of SNARE proteins in the perisynaptic astrocytic processes than in presynaptic terminals. The same was also true when compared to dendrites. Contrary to VAMP-2, labeling intensities for syntaxin-1, SNAP-25 and synaptotagmin 1 were not distinguishable from background labeling in the processes. The relative concentration of VAMP-2 stands out, as the mean perisynaptic astrocytic process concentration of the protein was only 68 % lower than in presynaptic terminals and still 32 % higher than in dendrites. VAMP-2 was associated with small vesicles in the processes. Some gold particles were located over the astrocytic plasma membrane. CONCLUSION: VAMP-2 is expressed in perisynaptic astrocytic processes, with a concentration higher than in the dendrites. Our results are compatible with the role of VAMP-2 in exocytosis from perisynaptic astrocytic processes.

Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1.

Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10-100 µM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.

Axonal Domain Structure as a Putative Identifier of Neuron-Specific Vulnerability to Oxidative Stress in Cultured Neurons.

Several populations of neurons are purported to degenerate in Parkinson's disease (PD). One current hypothesis suggests that vulnerable neurons in PD share common characteristics including projecting to voluminous territories and having extremely long and branched axonal domains with large numbers of neurotransmitter release sites. In this study, we used a mouse in vitro culture system to compare the axonal domain of neuronal populations suspected to be vulnerable in PD to that of neuronal populations considered at a lesser risk. In the first category, we included dopamine (DA) neurons of the substantia nigra, noradrenergic neurons of the locus coeruleus (LC), serotonin neurons of the raphe nuclei (R), and cholinergic neurons of the dorsal motor nucleus of the vagus (DMV). In the second category, we included DA neurons of the ventral tegmental area, cholinergic neurons of the hypoglossal nucleus, and cholinergic interneurons of the dorsal striatum. Validating their differential vulnerability, we find that, when compared with neurons presumed to be resilient in PD, a larger proportion of neurons presumed to be vulnerable in PD degenerate in response to cell stress induced by hydrogen peroxide. We also find that they are endowed with larger axonal domains, that are more complex, have more axonal varicosities with a higher proportion of varicosities that are positive for synaptotagmin 1 (Syt-1). Notwithstanding the obvious limitations related to the dissection of small brain nuclei and to the growth of these neurons in vitro, these findings support the hypothesis that axonal domain structure is a key characteristic of neuronal vulnerability to oxidative stress.