476 research outputs found
Titration of syntaxin1 in Mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.
Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms
Structure of the murine rab3A gene: correlation of genomic organization with antibody epitopes
Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission
Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the
possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures
that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders
of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and
Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the
CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found
between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable
analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic
transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism.
Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases
Neuronal Calcium Sensor Synaptotagmin-9 Is Not Involved in the Regulation of Glucose Homeostasis or Insulin Secretion
BACKGROUND:Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7. METHODOLOGY/PRINCIPAL FINDINGS:In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. CONCLUSIONS:Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic β-cells
Synchronous Symmetry Breaking in Neurons with Different Neurite Counts
As neurons develop, several immature processes (i.e., neurites) grow out of the cell body. Over time, each neuron breaks symmetry when only one of its neurites grows much longer than the rest, becoming an axon. This symmetry breaking is an important step in neurodevelopment, and aberrant symmetry breaking is associated with several neuropsychiatric diseases, including schizophrenia and autism. However, the effects of neurite count in neuronal symmetry breaking have never been studied. Existing models for neuronal polarization disagree: some predict that neurons with more neurites polarize up to several days later than neurons with fewer neurites, while others predict that neurons with different neurite counts polarize synchronously. We experimentally find that neurons with different neurite counts polarize synchronously. We also show that despite the significant differences among the previously proposed models, they all agree with our experimental findings when the expression levels of the proteins responsible for symmetry breaking increase with neurite count. Consistent with these results, we observe that the expression levels of two of these proteins, HRas and shootin1, significantly correlate with neurite count. This coordinated symmetry breaking we observed among neurons with different neurite counts may be important for synchronized polarization of neurons in developing organisms
Bi-allelic variants in TSPOAP1, encoding the active zone protein RIMBP1, cause autosomal recessive dystonia
Dystonia is a debilitating hyperkinetic movement disorder, which can be transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense and missense variants in TSPOAP1, encoding the active zone RIM-binding protein 1 (RIMBP1), as a novel genetic cause of autosomal recessive dystonia in seven subjects from three unrelated families. Subjects carrying loss-of-function variants presented with juvenile-onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between dysfunction of the presynaptic active zone and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis
Harness the power of endogenous neural stem cells by biomaterials to treat spinal cord injury
VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission
Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca2+-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle–associated SNAREs
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