Mechanisms that ensure robust long-term performance of synaptic transmission over a wide range of activity are crucial for the integrity of neuronal networks, for processing sensory information and for the ability to learn and store memories. Recent experiments have revealed that such robust performance requires a tight coupling between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. Distinct presynaptic scaffolding proteins are essential for fulfilling this requirement, providing either ultrastructural coordination or acting as signalling hubs.

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Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.

https://www.embopress.org/doi/pdf/10.1002/embj.201386357

Synaptic vesicle recycling: steps and principles

Homeostatic mechanisms are required to control formation and maintenance of synaptic connections to maintain the general level of neural impulse activity within normal limits. How genes controlling these processes are co-coordinately regulated during homeostatic synaptic plasticity is unknown. MicroRNAs (miRNAs) exert regulatory control over mRNA stability and translation and may contribute to local and activity-dependent posttranscriptional control of synapse-associated mRNAs. However, identifying miRNAs that function through posttranscriptional gene silencing at synapses has remained elusive. Using a bioinformatics screen to identify sequence motifs enriched in the 3'UTR of rapidly destabilized mRNAs, we identified a developmentally and activity-regulated miRNA (miR-485) that controls dendritic spine number and synapse formation in an activity-dependent homeostatic manner. We find that many plasticity-associated genes contain predicted miR-485 binding sites and further identify the presynaptic protein SV2A as a target of miR-485. miR-485 negatively regulated dendritic spine density, postsynaptic density 95 (PSD-95) clustering, and surface expression of GluR2. Furthermore, miR-485 overexpression reduced spontaneous synaptic responses and transmitter release, as measured by miniature excitatory postsynaptic current (EPSC) analysis and FM 1-43 staining. SV2A knockdown mimicked the effects of miR-485, and these effects were reversed by SV2A overexpression. Moreover, 5 d of increased synaptic activity induced homeostatic changes in synaptic specializations that were blocked by a miR-485 inhibitor. Our findings reveal a role for this previously uncharacterized miRNA and the presynaptic protein SV2A in homeostatic plasticity and nervous system development, with possible implications in neurological disorders (e.g., Huntington and Alzheimer's disease), where miR-485 has been found to be dysregulated.

https://www.pnas.org/content/pnas/108/28/11650.full.pdf

MicroRNA regulation of homeostatic synaptic plasticity.

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

https://www.jneurosci.org/content/jneuro/31/4/1461.full.pdf

Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision.

Late-onset Alzheimer's disease (LOAD) risk is strongly influenced by genetic factors such as the presence of the apolipoprotein E e4 allele (referred to here as APOE4), as well as non-genetic determinants including ageing. To pursue mechanisms by which these affect human brain physiology and modify LOAD risk, we initially analysed whole-transcriptome cerebral cortex gene expression data in unaffected APOE4 carriers and LOAD patients. APOE4 carrier status was associated with a consistent transcriptomic shift that broadly resembled the LOAD profile. Differential co-expression correlation network analysis of the APOE4 and LOAD transcriptomic changes identified a set of candidate core regulatory mediators. Several of these--including APBA2, FYN, RNF219 and SV2A--encode known or novel modulators of LOAD associated amyloid beta A4 precursor protein (APP) endocytosis and metabolism. Furthermore, a genetic variant within RNF219 was found to affect amyloid deposition in human brain and LOAD age-of-onset. These data implicate an APOE4 associated molecular pathway that promotes LOAD.

Integrative genomics identifies APOE ε4 effectors in Alzheimer's disease

Synaptic vesicles are specialized cycling endosomes that contain a unique constellation of membrane proteins. Proteins are sorted to vesicles by short amino acid sequences that serve as binding sites for clathrin adaptor proteins. Here we show that a tyrosine-based endocytosis motif in the vesicle protein SV2 is required for trafficking to synaptic vesicles of both SV2 and the calcium sensor protein synaptotagmin. Aberrant neurotransmission in cultured hippocampal neurons lacking SV2 was rescued by expression of wild-type SV2A, but not by SV2A-Y46A, a mutant containing a disrupted endocytosis motif in SV2A's cytoplasmic N terminus. Neurons expressing SV2A-Y46A had significantly more SV2 on the plasma membrane, indicating reduced internalization. A screen for proteins that preferentially bound wild-type SV2A identified multiple endocytosis-related proteins, and in vitro binding studies confirmed binding to the clathrin adaptors AP2, EPS15, and amphiphysin 2/Bin1. Neurons lacking SV2 contained less synaptotagmin and had a higher proportion of synaptotagmin on the plasma membrane. Expression of either wild-type SV2A or SV2A-Y46A restored synaptotagmin expression levels; however, only wild-type SV2A restored a normal proportion of synaptotagmin on the plasma membrane. These findings indicate that SV2 influences the expression and trafficking of synaptotagmin via separate mechanisms. Synaptic vesicles immunoisolated from SV2A/B double knock-out mice had significantly less synaptotagmin than vesicles isolated from wild-type mice. Our results indicate that SV2 plays a major role in regulating the amount of synaptotagmin in synaptic vesicles and provide an explanation for the observation that synapses lacking SV2 have fewer vesicles competent for calcium-induced fusion.

https://www.jneurosci.org/content/jneuro/30/16/5569.full.pdf

Cotrafficking of SV2 and synaptotagmin at the synapse

Among the proteins that mediate calcium-stimulated transmitter release, the synaptic vesicle protein 2 (SV2) stands out as a unique modulator specific to the neurons and endocrine cells of vertebrates. In synapses, SV2 regulates the expression and trafficking of the calcium sensor protein synaptotagmin, an action consistent with the reduced calcium-mediated exocytosis observed in neurons lacking SV2. Yet SV2 contains amino acid motifs consistent with it performing other actions that could regulate presynaptic functioning and that might underlie the mechanism of drug action. To test the role of these functional motifs, we performed a mutagenic analysis of SV2A and assessed the ability of mutant SV2A proteins to restore normal synaptic transmission in neurons from SV2A/B knockout mice. We report that SV2A-R231Q, harboring a mutation in a canonical transporter motif, restored normal synaptic depression (a measure of release probability and signature deficit of neurons lacking SV2). In contrast, normal synaptic depression was not restored by SV2A-W300A and SV2A-W666A, harboring mutations of conserved tryptophans in the 5th and 10th transmembrane domains. Although they did not rescue normal neurotransmission, SV2A-W300A and SV2A-W666A did restore normal levels of synaptotagmin expression and internalization. This indicates that tryptophans 300 and 666 support an essential action of SV2 that is unrelated to its role in synaptotagmin expression or trafficking. These results indicate that SV2 performs at least two actions at the synapse that contribute to neurotransmitter release.

SV2 regulates neurotransmitter release via multiple mechanisms

Hypothesis A functional vestibular prosthesis can be implanted in human such that electrical stimulation of each semicircular canal produces canal-specific eye movements while preserving vestibular and auditory function. Background A number of vestibular disorders could be treated with prosthetic stimulation of the vestibular end organs. We have previously demonstrated in rhesus monkeys that a vestibular neurostimulator, based on the Nucleus Freedom cochlear implant, can produce canal-specific electrically evoked eye movements while preserving auditory and vestibular function. An investigational device exemption has been obtained from the FDA to study the feasibility of treating uncontrolled Meniere's disease with the device. Methods The UW/Nucleus vestibular implant was implanted in the perilymphatic space adjacent to the three semicircular canal ampullae of a human subject with uncontrolled Meniere's disease. Preoperative and postoperative vestibular and auditory function was assessed. Electrically evoked eye movements were measured at 2 time points postoperatively. Results Implantation of all semicircular canals was technically feasible. Horizontal canal and auditory function were largely, but not totally, lost. Electrode stimulation in 2 of 3 canals resulted in canal-appropriate eye movements. Over time, stimulation thresholds increased. Conclusion Prosthetic implantation of the semicircular canals in humans is technically feasible. Electrical stimulation resulted in canal-specific eye movements, although thresholds increased over time. Preservation of native auditory and vestibular function, previously observed in animals, was not demonstrated in a single subject with advanced Meniere's disease.

Prosthetic implantation of the human vestibular system

Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a ∼1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions.

/pdf/levetiracetam-reverses-synaptic-deficits-produced-by-4o4ohsq533.pdf

Levetiracetam Reverses Synaptic Deficits Produced by Overexpression of SV2A

Animal experiments and limited data in humans suggest that electrical stimulation of the vestibular end organs could be used to treat loss of vestibular function. In this paper we demonstrate that ...

https://www.physiology.org/doi/pdf/10.1152/jn.00171.2013

Amy Nowack

Papers

Cotrafficking of SV2 and synaptotagmin at the synapse

SV2 regulates neurotransmitter release via multiple mechanisms

Prosthetic implantation of the human vestibular system

Levetiracetam Reverses Synaptic Deficits Produced by Overexpression of SV2A

Vestibular implantation and longitudinal electrical stimulation of the semicircular canal afferents in human subjects