Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons.
Ontology highlight
ABSTRACT:
SUBMITTER: Vevea JD
PROVIDER: S-EPMC8452306 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Myelination of axons by oligodendrocytes enables fast saltatory conduction. Oligodendrocytes are responsive to neuronal activity, which has been shown to induce changes to myelin sheaths, potentially to optimize conduction and neural circuit function. However, the cellular bases of activity-regulated myelination in vivo are unclear, partly due to the difficulty of analyzing individual myelinated axons over time. Activity-regulated myelination occurs in specific neuronal subtypes and can be mediated by synaptic vesicle fusion, but several questions remain: it is unclear whether vesicular fusion occurs stochastically along axons or in discrete hotspots during myelination and whether vesicular fusion regulates myelin targeting, formation, and/or growth. It is also unclear why some neurons, but not others, exhibit activity-regulated myelination. Here, we imaged synaptic vesicle fusion in individual neurons in living zebrafish and documented robust vesicular fusion along axons during myelination. Surprisingly, we found that axonal vesicular fusion increased upon and required myelination. We found that axonal vesicular fusion was enriched in hotspots, namely the heminodal non-myelinated domains into which sheaths grew. Blocking vesicular fusion reduced the stable formation and growth of myelin sheaths, and chemogenetically stimulating neuronal activity promoted sheath growth. Finally, we observed high levels of axonal vesicular fusion only in neuronal subtypes that exhibit activity-regulated myelination. Our results identify a novel "feedforward" mechanism whereby the process of myelination promotes the neuronal activity-regulated signal, vesicular fusion that, in turn, consolidates sheath growth along specific axons selected for myelination.
| S-EPMC8445327 | biostudies-literature
Project description:The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.
| S-EPMC7282819 | biostudies-literature
Project description:Early exposure to anesthetics may interfere with synaptic development and lead to cognitive deficits. We previously demonstrated a decrease in vesicles docked at and within 100 nm from the presynaptic membrane in hippocampal nerve terminals of neonatal rats after anesthesia. Hence, we designed this study to assess the effects of neonatal anesthesia on synapsin 1 (Syn1) and synaptotagmin 1 (Syt1), two key regulators of vesicle docking and fusion. To test the link between changes in Syn1 and Syt1 and behavioral deficits observed after neonatal anesthesia, we also assessed retention memory and fear conditioning in adolescent rats after neonatal anesthesia. Pups received a combination of clinical anesthetics, then Syn1 and Syt1 mRNA and protein expression were determined at the peak (postnatal day 8, P8), part-way through (P12) and end of synaptogenesis (P24) in the CA1-subiculum by qPCR and western blotting. Anesthesia decreased Syn1 and Syt1 mRNA expression at P8 (P<0.01 and <0.001) and P12 (P=0.001 and 0.017), but not P24 (P=0.538 and 0.671), and impaired Syn1, p-Syn1, and Syt1 protein levels at P8 (P=0.038, 0.041, and 0.004, respectively), P12 (P<0.001, P=0.001, and P<0.0001), and P24 (P=0.025, 0.031, and 0.001). Anesthetic-challenged rats displayed deficient long-term retention memory (P=0.019) and hippocampus-dependent fear conditioning (P<0.001). These results suggest that anesthetics alter Syn1 and Syt1 during synapse assembly and maturation, raising the possibility that anesthetic interference with Syn1 and Syt1 could initiate changes in synaptic function that contribute to the cognitive deficits observed after neonatal anesthesia.
| S-EPMC6510243 | biostudies-literature
Project description:The γ-secretase complex is composed of four membrane protein subunits, including presenilin as the catalytic component with aspartyl protease activity. The enzyme cleaves within the transmembrane domain of >70 different type I integral membrane proteins and has been dubbed "the proteasome of the membrane". The most studied substrates include the Notch family of receptors, involved in cell differentiation, and the amyloid precursor protein (APP), involved in the pathogenesis of Alzheimer's disease. A central mechanistic question is how γ-secretase recognizes helical transmembrane substrates and carries out processive proteolysis. Recent findings addressing substrate recognition and processing will be discussed, including the role of protease subunit nicastrin as a gatekeeper, the effects of Alzheimer-causing mutations in presenilin on processive proteolysis of APP, and evidence that three pockets in the active site (S1', S2', and S3') determine carboxypeptidase cleavage of substrate in intervals of three residues. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.
| S-EPMC6899174 | biostudies-literature
Project description:Many presynaptic transcripts have been observed in axons, yet their role in synapse development remains unknown. Using visually and pharmacologically isolated presynaptic terminals from dissociated rat hippocampal neurons, we found that ribosomes and β-catenin mRNA preferentially localize to recently formed boutons. Locally translated β-catenin accumulates at presynaptic terminals, where it regulates synaptic vesicle release dynamics. Thus, local translation of β-catenin is a newly described mechanism for axons to independently functionalize nerve terminals at great distances from cellular somata.
| S-EPMC3651888 | biostudies-literature
Project description:Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.
| S-EPMC2268828 | biostudies-other
Project description:Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.
| S-EPMC3084856 | biostudies-literature
Project description:Amyloid β-peptide, the principal component of characteristic cerebral plaques of Alzheimer's disease (AD), is produced through intramembrane proteolysis of the amyloid precursor protein (APP) by γ-secretase. Despite the importance in the pathogenesis of AD, the mechanisms of intramembrane proteolysis and substrate processing by γ-secretase remain poorly understood. Here, complementary all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method and biochemical experiments were combined to investigate substrate processing of wildtype and mutant APP by γ-secretase. The GaMD simulations captured spontaneous activation of γ-secretase, with hydrogen bonded catalytic aspartates and water poised for proteolysis of APP at the ε cleavage site. Furthermore, GaMD simulations revealed that familial AD mutations I45F and T48P enhanced the initial ε cleavage between residues Leu49-Val50, while M51F mutation shifted the ε cleavage site to the amide bond between Thr48-Leu49. Detailed analysis of the GaMD simulations allowed us to identify distinct low-energy conformational states of γ-secretase, different secondary structures of the wildtype and mutant APP substrate, and important active-site subpockets for catalytic function of the enzyme. The simulation findings were highly consistent with experimental analyses of APP proteolytic products using mass spectrometry and Western blotting. Taken together, the GaMD simulations and biochemical experiments have enabled us to elucidate the mechanisms of γ-secretase activation and substrate processing, which should facilitate rational computer-aided drug design targeting this functionally important enzyme.
| S-EPMC7318072 | biostudies-literature
Project description:Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development.
| S-EPMC4396464 | biostudies-literature
Project description:Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.
| S-EPMC6733930 | biostudies-literature