The Synaptic Vesicle Glycoprotein 2: Structure, Function, and Disease

Subscriber access provided by RUTGERS UNIVERSITY

Review

The synaptic vesicle glycoprotein 2: structure, function, and disease relevance Kristen Stout, Amy Dunn, Carlie Hoffman, and Gary W Miller ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00351 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1 The synaptic vesicle glycoprotein 2: structure, function, and disease relevance

Kristen Stout1*, Amy Dunn2, Carlie Hoffman3, Gary W Miller4*

1. Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL 2. The Jackson Laboratory, Bar Harbor, ME 3. Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 4. Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York City, NY

*Correspondence to [email protected] or [email protected]

Acknowledgements: We kindly acknowledge Dr. Lauren P Shapiro for her editorial role in the preparation of this manuscript. This work was supported by the following NIH grants: ES012870, ES023839, NS089242, DA037652, AG058396.

Author Contributions: KAS– primary writer and figure creator. ARD – major writer and editor. CAH – minor writer and editor. GWM – advisor and editor.

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

2 Abstract The synaptic vesicle glycoprotein 2 (SV2) family is comprised of three paralogs: SV2A, SV2B, and SV2C. In vertebrates, SV2s are 12-transmembrane proteins present on every secretory vesicle, including synaptic vesicles, and are critical to neurotransmission. Structural and functional studies suggest that SV2 proteins may play several roles to promote proper vesicular function. Among these roles are their potential to stabilize the transmitter content of vesicles, to maintain and orient the releasable pool of vesicles, and to regulate vesicular calcium sensitivity to ensure efficient, coordinated release of transmitter. The SV2 family is highly relevant to human health in a number of ways. First, SV2A plays a role in neuronal excitability, and as such is the specific target for the antiepileptic drug levetiracetam. SV2 proteins also act as the target by which potent neurotoxins, particularly botulinum, gain access to neurons and exert their toxicity. Both SV2B and SV2C are increasingly implicated in diseases such as Alzheimer’s disease and Parkinson’s disease. Interestingly, despite decades of intensive research, their exact function remains elusive. Thus, SV2 proteins are intriguing in their potentially diverse roles within the presynaptic terminal, and several recent developments have enhanced our understanding and appreciation of the protein family. Here we review the structure and function of SV2 proteins as well as their relevance to disease and therapeutic development.

Keywords: vesicle, SV2, neurotransmission


Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3 Chemical neurotransmission is essential for neuronal communication. In this process Ca2+ floods into the presynaptic terminal, triggering release of neurotransmitters into the synapse, where they can act on postsynaptic receptors. The speed at which this communication occurs is evolutionarily important. As such, neurons evolved highly efficient mechanisms for neurotransmission, allowing a neuron to release transmitter in less than a millisecond following a Ca2+ trigger1. To hasten neurotransmission, neurotransmitters (GABA, glutamate, monoamines, acetylcholine, etc.) are sequestered into synaptic vesicles by vesicular transporters. Vesicular transporters are powered by a proton electrochemical gradient, established by a vesicular ATPase2. Filled vesicles are trafficked to the active zone, a specialized portion of the synaptic membrane where neurotransmission occurs. Through elegant protein interactions, vesicles dock to the active zone and are primed for Ca2+ influx1,3. Following fusion, vesicles are recycled and the process begins anew. Neurotransmission is complicated, involving dozens of proteins that each work in a unique way to accomplish the goal of neurotransmitter release. One of the protein families involved in neurotransmission is the synaptic vesicle glycoprotein 2 (SV2) family. SV2 proteins are found in every neurosecretory vesicle in the human body. Such a ubiquitous presence in the vesicular proteome suggests that SV2 proteins are essential to vesicular function. However, no definitive function for any family member has been identified. Here, we discuss structural, functional, and pathological properties of SV2 proteins as described in the literature. Structure SV2 proteins are members of the major facilitator superfamily, a large family of membrane transporters expressed widely throughout bacteria, archaea, and eukarya4. Like other major facilitator superfamily proteins, SV2s have 12 transmembrane domains, with cytosolic N- and C-termini. Additionally, SV2s have a large Nglycosylated intraluminal loop between transmembrane domains 7 and 8 (Figure 15). Three paralogs of SV2 exist in vertebrates: SV2A (chromosome 16), SV2B (chromosome 157), and SV2C (chromosome 58). These paralogs share 61-64% sequence homology and 80% structural homology9. Each protein also has splice variants. In addition to the full sequence, SV2A has a structural variant that lacks amino acids 683-742 as well as 5 known ACS Paragon Plus Environment


Page 4 of 33

4 single nucleotide polymorphisms (SNPs)10,11. SV2B has a structural variant lacking amino acids 1-151 and 5 known SNPs11,12. SV2C also has a structural variant with an altered and truncated N-terminus and also has 5 SNPs11. The function of these variants has not been established. Two other paralogous proteins, SV2-related protein (SVOP) and SVOP-like protein (SVOPL), share 20-22% sequence homology with SV2s, though they are not glycosylated. Orthologues of these proteins (SVOP/SVOPL) are present in invertebrates, including Drosophila melangoster and Caenorhabditis elegans. Expression While SV2s are found in every neurosecretory vesicle, individual paralogs vary in their expression profiles. SV2A is the most ubiquitous, with expression in every structure in the brain to varying degrees9. SV2B expression is more restricted, with strongest expression in the trigeminal and motor nuclei and very little expression in the globus pallidus, dentate gyrus of the hippocampus, cerebellum, and substantia nigra pars reticulata9. SV2C expression is the most limited, localizing to evolutionarily old brain regions, with strong expression throughout the striatum, midbrain, and ventral pallidum and very little expression in the neocortex13,14. At the vesicular level, SV2 is sorted with high precision; each synaptic vesicle typically contains five copies of the SV2 protein, with very little intravesicular variation15. Genetic ablation of SV2A results in compensatory upregulation of SV2B/C, presumably maintaining total levels of SV2 in the animal16,17. SV2A and SV2B are co-expressed in a large number of neurons and immunoprecipitation of intact vesicles reveals colabeling of individual vesicles9. SV2C may also colocalize with other SV2 proteins, though this has not been directly assessed. Co- expression of SV2 paralogues has been suggested to indicate functional redundancy9, however, expression patterns also suggest paralog-dependent function. While SV2A is found in both excitatory and inhibitory neurons, SV2B is limited to excitatory synapses9. SV2C is expressed in 70% of dopaminergic neurons but is also expressed strongly in GABAergic medium spiny neurons of the striatum13,18,19. This expression correlates with lower levels of SV2A and SV2B in the striatum9. Additionally, while SV2A and SV2B are not expressed in cell bodies9, SV2C is present in TH-positive midbrain dopamine neuron cell bodies18,19, ACS Paragon Plus Environment

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 suggesting SV2C may play a role in somatodendritic dopamine release. SV2C is also localized to cell bodies of striatal GABAergic medium spiny neurons18,19. SV2s are also expressed peripherally, both in the peripheral nervous system and in neuroendocrine cells9,20. Paralogue specific expression has not been well described in the periphery, but we report here what limited information is available. SV2A is expressed in peripheral sympathetic synapses, where it controls transmitter release21. SV2B is the primary paralogue expressed in the retina22-24. SV2s are expressed in the motor axons that innervate muscle fibers; while SV2B and SV2C are expressed in all motoneurons, SV2A is selectively expressed in slow motoneurons25. Neuroendocrine cells also express SV220. A pan-SV2 monoclonal antibody showed robust staining in the human gastrointestinal tract, adrenal medulla, pancreas, thyroid/parathyroid and pancreas20. Though paralogue specific investigation hasn’t been thoroughly reported, SV2B is transiently expressed in embryonic kidney9. All three paralogues are expressed in pancreas, with SV2A and SV2C expressed on insulin-containing granules, and participate in glucose-evoked insulin release26. SV2A is also expressed in platelets27. Function The precise function of SV2 remains elusive despite excellent and extensive experimental analysis. Most of the literature centers on the function of SV2A, which is the molecular target of the anti-epileptic drug levetiracetam28-33. Over the course of the last twenty years SV2 has been investigated for many potential functions, including: vesicular transport, stabilizing vesicular loading of neurotransmitter, anchoring vesicular proteins, assisting in vesicle trafficking, regulating calcium sensitivity, and interacting with the extracellular matrix.

Vesicular transport. The major facilitator superfamily is home to all vesicular neurotransmitter transporters. Given their family heritage and structural similarities, it was initially assumed that SV2 functioned as a novel neurotransmitter transporter14,34-36. However, despite efforts to this end, no direct evidence of SV2-mediated ACS Paragon Plus Environment


Page 6 of 33

6 neurotransmitter transport exists. Interestingly, the protein does act as a physical transporter for botulinum and tetanus neurotoxins37,38. Additionally, evidence suggests that SV2 may act as a sugar transporter when localized to the plasmalemmal membrane during vesicle fusion39. One of the most interesting aspects of transmembrane vesicular proteins is their altered interactome through the phases of neurotransmission. During exocytosis luminal proteins become extracellular proteins, completely changing potential binding partners (Figure 2). Botulinum toxins, the most potent neurotoxins known, make use of this aspect of SV2s, binding to N-glycosylated luminal domain 4 and hijacking an endocytotic ride into the presynaptic neuron37,40-46. Botulinum toxin serotype alters the affinity of the toxins for specific SV2 paralogs (reviewed by Davies et al.47). Likewise, tetanus toxin also gains entry to central neurons via SV2mediated endocytosis38. In addition to neurotoxins, it has been proposed that SV2s may transport sugars39. Recombinant human SV2A expressed in a hexose-transporter deficient Saccharomyces cerevisiae strain survived exclusively on galactose-containing medium. Further, radioactive galactose transport was levetiracetam- and proton gradientsensitive, thus suggesting SV2A acts as a galactose symporter in yeast. Sugar transport function has yet to be demonstrated in higher order models, as the assay is inherently more difficult. Transport activity for SV2 in vertebrates is only apparent during fusion, when SV2 is appropriately localized to transport a substrate from the synaptic cleft into the neuron. Traditional vesicular uptake assays would not capture such a phenomenon, as the protein is not exposed to the synaptic cleft in intact vesicles. From a bioenergetic standpoint, sugar transport coupled to vesicular fusion is a practical idea, potentially allowing increased energy substrate transport during times of high synaptic activity. The idea is reminiscent of acetylcholine transmission, where plasmalemmal choline acetyltransferase localizes to cholinergic synaptic vesicles, allowing increased recycling of transmitter during stimulation48.

Neurotransmitter loading. Loading neurotransmitter into a vesicle requires substantial energy requirements. A v-type ATPase establishes a proton gradient, acidifying the lumen of the vesicle. This gradient drives transport, ACS Paragon Plus Environment

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7 exchanging protons for neurotransmitter. The resultant accumulation of neurotransmitter in the vesicle also produces a gradient. Data suggest that SV2 assists in this process. An orthologous SV2 protein in the electric organ of Diplobatis ommata fish (60-62% homology with SV2A/B and 80% homology with SV2C) creates a proteoglycan matrix within the lumen of synaptic vesicles49-51. Though SV2 proteins have predicted molecular weights ranging from 77-83 kDa based on their amino acid structure, glycosylation of luminal asparagine residues increases their size to ~95 kDa for lightly glycosylated and ~250 kDa for heavily glycosylated SV2 proteins51. Proteoglycans are negatively charged52, which allows them to adsorb positively charged or zwitterionic neurotransmitters, such as monoamines, glycine, and GABA. This proteoglycan matrix binds 95% of neurotransmitter and ATP within the vesicle, requiring ionic exchange for release and thereby regulating neurotransmission50. The importance of the intravesicular proteoglycan matrix to vesicular uptake, storage, and release of neurotransmitter has not been addressed, either in D. ommata or higher order organisms, though changes in vesicular size have been demonstrated in mice in response to neurotransmitter loading53,54; this phenomenon requires SV2 expression53.

Anchor of vesicle structure and cycling. Over 80 integral membrane proteins reside on the synaptic vesicle membrane. The transmembrane domains of these proteins are thought to occupy 25% of the overall vesicle membrane surface55. Additionally, reconstructed images of the frog neuromuscular junction obtained via electron tomography indicate that luminal macromolecules occupy 10% of the vesicle’s volume, contacting the luminal membrane at 25 sites56,57. This luminal assembly has a bilateral shape consisting of four arms radiating out from a central focal point and is found, nearly identically, in all synaptic vesicles56. The points at which the density contacts the luminal membrane are associated with the macromolecules that regulate fusion to the active zone, and as the vesicle traffics to the active zone its orientation is brought into precise and consistent alignment56. Possible transmembrane vesicular protein candidates that contribute to the luminal density include SV2, synaptobrevin, and synaptotagmin56. Of these proteins, SV2 is the proposed mainstay of luminal protein assembly due to the length of its luminal domain, which is greater than the diameter of the vesicle lumen. Given that five ACS Paragon Plus Environment


Page 8 of 33

8 copies of SV2 localize to all synaptic vesicles15, it is likely that these large intravesicular loops interact to form a backbone that anchors all other transmembrane vesicular proteins (Figure 2). Molecular evidence exists to support this hypothesis: large vesicular protein complexes between SV2 and other vesicular proteins have been identified58-60. Additionally, the negatively charged proteoglycan matrix may contribute to this phenomenon. When the pH drops below the isoelectric point of proteins, such as in the acidified lumen of a synaptic vesicle, the backbone becomes positively charged, promoting interaction of the luminal domains of integral vesicular proteins with the matrix and thereby stabilizing protein orientation. Thus, SV2 may act as the master regulator of vesicle structure, anchoring all other transvesicular proteins into their appropriate orientation to enable efficient association with vesicular fusion machinery.

Vesicle trafficking. Vesicle trafficking is vital to neurotransmission, ensuring that the vesicle is brought to the active zone and appropriately anchored for fusion, then recycled once neurotransmission occurs. The involvement of SV2 in the synaptic vesicle cycle has been controversial. Deletion of SV2A in neurosecretory adrenal chromaffin cells reduces the size of the readily releasable pool by 50%17. This reduction is correlated with decreased high molecular weight SNARE complex formation, suggesting that SV2 acts prior to fusion17. Interestingly, the kinetics of vesicle fusion are unaltered, suggesting that the trafficking deficit, and not calcium affinity, dictates the SV2 deletion phenotype. This initial decrease in synaptic release has been demonstrated in retinal neurons61, cortical neurons62, hippocampal neurons63, striatal slice19, and peripheral sympathetic neurons21. Additionally, neurons lacking SV2 show delayed recovery of the readily releasable pool following stimulusinduced depletion21,61. It has been postulated that deficits in the readily releasable pool are due to neuronal adaptation to chronically elevated calcium levels in SV2 ablated neurons61.

Regulation of calcium sensitivity. Mice lacking SV2A have decreased calcium-induced neurotransmission in both excitatory and inhibitory neurons16,62,63. Overall, cultured hippocampal neurons from SV2A/B double knockout mice have reduced synaptic release but, interestingly, display synaptic facilitation during stimulus trains ACS Paragon Plus Environment

Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9 and increased paired-pulse response compared with SV2B knockout neurons, which are not significantly different than wildtype neurons62,64. These increases are calcium-dependent, as treatment with chelator EGTA-AM abolishes the enhanced synaptic activity without affecting the total reduction in stimulated release in SV2A/B double knockouts62. Given its structure, it was originally hypothesized that SV2 functioned as a calcium transporter64. However, this hypothesis has been challenged26,62. Though calcium is required for the observed synaptic facilitation, deletion of SV2A/B does not change synaptic calcium affinity: while total release is reduced there is no change in relative inhibitory postsynaptic current (IPSC) amplitudes at escalating calcium dose, treatment with ionophore ionomycin does not produce differential results in double knockouts, and EGTA-AM treatment produces identical relative decreases in train release regardless of SV2 presence62. These complexities led to the hypothesis that SV2 doesn’t directly transport calcium but rather renders primed vesicles more calcium responsive62. As loss of SV2A is lethal around 3 weeks of age, all experiments in SV2A knockout mice were conducted in early postnatal mice in the absence of available temporally-inducible SV2A knockout models. This is problematic, as SV2 expression varies through development, indicating the functional consequence of the protein may change over the lifespan of the animal9,23,24. As such, the consequence of SV2B deletion was interrogated in adult retinal ribbon synapses, where SV2B is the major paralog23,24. Calcium concentration, both resting and stimulus-induced, was elevated two-fold in SV2B-null mature rod bipolar neurons compared to wildtype mice61. This effect was concurrent with decreased readily releasable pool size, decreased calcium sensitivity during exocytosis, early facilitation during initial pulses of stimulation trains, and increased time to recovery of membrane capacitance61. Normalizing elevated calcium concentration in neurons from SV2B-null mice rescued aberrant neurotransmission. Conversely, elevating calcium concentration in wildtype neurons induced similar secretory deficits, suggesting that the phenotype induced by SV2B deletion is due to altered calcium homeostasis61. These data again suggest that SV2 may function as a calcium transporter, sequestering calcium into synaptic vesicles61, though no direct evidence of calcium transport has been established. Vesicular uptake assays with radiolabeled calcium are necessary to directly address the capacity of SV2 to transport calcium. ACS Paragon Plus Environment


Page 10 of 33

10 Additionally, it has been proposed that, in addition to vesicular expression, SV2s are expressed in the mitochondrial compartment65,66. As mitochondria function as a neuronal calcium sink67, it is possible that the observed calcium effects may be due to mitochondrial, rather than vesicular, calcium transport. The mechanism of calcium sensitivity may also be attributable to the interaction of SV2 with the calcium sensor, synaptotagmin60,68. This interaction is direct, phosphorylation-dependent, and sensitive to calcium, with an IC50 of approximately 10 µM69-72. While all three SV2 paralogs bind synaptotagmin-1, the N-terminus of SV2A and SV2C contain a unique site that is functionally important for neurotransmission: injection of N-terminal SV2A and C peptides into the presynaptic neuron inhibits evoked postsynaptic potential amplitude by 20-30% in a calcium-dependent manner71. Additionally, evidence suggests that SV2A and synaptotagmin are co-trafficked to vesicles73. Deletion of SV2A or mutation of its endocytic motif reduces vesicular localization of synaptotagmin73. The functional relevance of the SV2-synaptotagmin interaction has been debated as lentiviral expression of N-terminal ablated SV2 in cortical neurons of SV2A/B null mice rescues reduced train release and synaptic facilitation. These effects are indistinguishable from rescue with wildtype SV2, suggesting that the synaptotagmin-1 interaction is dispensable to SV2 function62. However, the lentiviral constructs used to rescue SV2 deletion were tagged with EGFP62, which contains a similar endocytotic motif to SV273. Thus, the observed rescue may be EGFP-dependent and could artificially mask the importance of SV2 in regulating synaptotagmin localization. Association of SV2s with the other 14 synaptotagmins has not been systematically assessed.

Potential interactions with the extracellular matrix. Upon vesicular fusion, the luminal domain of SV2 becomes the extracellular domain and is free to interact with extracellular matrix proteins. SV2 acts as a laminin-1 receptor74 (Figure 4). This is an activity-dependent interaction, as increased vesicular fusion results in increased SV2 in the plasmalemmal membrane. While the functional consequences of the interaction are unknown, it is possible that the SV2-laminin interaction slows vesicular recycling, perhaps dictating full versus transient vesicle fusion. This could be particularly important for neurotransmitters that are adsorbed into the proteoglycan matrix (monoamines, acetylcholine, GABA), which may not be fully released during transient fusion events49,75. ACS Paragon Plus Environment

Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11 Interestingly, pharmacological or genetic inhibition of laminin produces similar synaptic dysfunction to SV2 knockouts, including: lack of calcium sensitivity76, defects in plasticity77, and altered synaptic release78.

Paralog-dependent function. Genetic deletion of SV2 results in synaptic dysfunction. Interestingly, in several systems the loss of SV2A or SV2C results in compensatory upregulation of the other paralog, presumably to maintain total SV2 level17,26. Compensatory upregulation is insufficient to rescue aberrant neurotransmission, suggesting that the functions of SV2A and SV2C are not redundant17,26. Genetic deletion of SV2B does not appear to have the same regulatory effect on SV2 expression, as loss of SV2B in the retina of mice does not alter SV2A expression and reduces total SV2 expression in the eye by 50%22. These compensatory phenotypes could likely be avoided by excision of these proteins in adult animals using viral Cre-recombinase injection into animals expressing flanking loxP sites around the paralog of interest. Bypassing the neurodevelopmental window may uncover new and important roles for these proteins. SV2s in neurological disease SV2s have been implicated in several neurological diseases, providing some additional hints to their function. SV2A, as the molecular target for the antiepileptic drug (AED) levetiracetam (LEV) and its derivatives (e.g. seletracetam and brivaracetam), has been the subject of extensive research for its role in epilepsy. More recently, LEV has shown efficacy in treating symptoms and neurological features of mild cognitive impairment (MCI) and Alzheimer’s disease (AD). This, combined with evidence of altered SV2A and SV2B expression in AD models, suggests a function for SV2A in cognitive function and cell health. SV2C, as might be expected by its enriched expression in the basal ganglia, has been increasingly implicated in dopamine-centric diseases such as Parkinson’s disease (PD) and psychiatric conditions. The data tying SV2s to these various neurological diseases point to the importance of further development of SV2-targeting compounds for the treatment of neurological disease. Particularly considering their specialized anatomical distributions, targeting SV2s may be a strategy to focus treatment to relevant nuclei while minimizing off-target or undesirable effects. ACS Paragon Plus Environment


Page 12 of 33

12 SV2A and epilepsy. The efficacy of LEV as an AED was known before identification of its molecular target, with FDA approval granted in 1999 for the treatment of partial onset seizures in adults. Serendipitously, the severe seizure phenotype of SV2A-KO mice was published in the same year16, though the LEV-SV2A interaction wasn’t discovered until 200432. This discovery spurred research into the role of SV2A in epilepsy and led to the development of various SV2A-targeting LEV-derivatives that also show efficacy in epilepsy79-83. LEV and its derivatives comprise the only class of AED that targets the synaptic vesicle to reduce hyperexcitability and epileptogenesis. The exact role SV2A plays in epilepsy has not been fully described, though various lines of evidence implicate the protein in epilepsy pathology. Epilepsy patients commonly have reduced SV2A gene and protein expression and this effect is recapitulated in animal models84-87, suggesting that reduced SV2A increases vulnerability for epileptogenesis. Alternatively, SV2A expression may increase during seizure kindling and status epilepticus88,89. Consistent with this, low frequency stimulation in the hippocampus in pharmacoresistant spontaneously epileptic rats increased SV2A expression and subsequently decreased seizure frequency90. In one strain of chicken, an SV2A mRNA splice variant that results in reduced SV2A protein expression leads to a photosensitive epileptic phenotype that can be rescued with LEV91. In humans with glioma-associated epilepsy, tumor level of SV2A expression is predictive of LEV-responsiveness, as patients with lower SV2A levels are more likely to be LEV nonresponders92. Beyond altered protein expression, there is no apparent SV2A polymorphism associated with epilepsy risk or variation in LEV efficacy93,94. However, homozygosity for a recessive missense mutation in SV2A results in disruption of GABA release in the hippocampus and LEV-nonresponsive epilepsy, among a host of other neurological abnormalities95,96. These data further indicate that normal SV2A expression and function is required to maintain proper neurotransmission, and a lack of SV2A function leads to seizure vulnerability. However, work by Nowack, et al. (2011) suggests that overexpression of SV2A also leads to altered excitability and release probability, and that these abnormalities are also reversed by LEV33. Precisely how LEV interacts with SV2A to reduce abnormal excitability and rescue seizure phenotypes is unknown. Vogl, et al. (2012) demonstrated that LEV inhibits presynaptic calcium channels21. Additionally, ACS Paragon Plus Environment

Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13 LEV enhances synaptic depression via impairing replenishment of the readily releasable pool (RRP) of vesicles, particularly during periods of heightened stimulus intensity or activity (e.g., epileptogenesis)97,98. Similarly, brivaracetam slows vesicle recycling to the RRP, but with significantly higher affinity to SV2A than its parent drug82,99,100, and potentially acts at an alternative binding site82,101. This suggests that LEV and brivaracetam inhibit SV2A’s role in trafficking vesicles to the RRP. Other pieces of evidence point to a likelihood of LEV inhibiting additional functions of SV2A, such as its potential galactose transport activity39. However, since a reduction in SV2A results in heightened vulnerability to seizures in humans and animals, it is possible that inhibition of the protein would be deleterious; rather, LEV may stabilize the protein in an ideal functional conformation. Thus, the function of SV2A may have a U-shaped relationship with seizure vulnerability, in that both high and low function or expression of SV2A may alter neurotransmission and promote pathogenesis. In this way, LEV may rescue deficits resultant from both reduced and overexpressed SV2A. Additionally, prophylactic LEV (i.e., LEV administered to asymptomatic, high-risk spontaneously epileptic animals) may be efficacious in preventing vulnerability to seizures, and protective against hippocampal degeneration in epilepsy102. Finally, administration of LEV to non-epileptic animals and humans does not produce similar hyperexcitability as seen with reduced or overexpressed SV2A.

SV2s and Alzheimer’s disease. The coincidence of seizures in AD has been known for decades103-105. Abnormal hyperactivity within the hippocampus occurs in AD, and an increase in seizures in later stage AD is associated with a significant decline in cognitive function106. Furthermore, various animal models of AD display altered cortical excitability and spontaneous seizure phenotypes107-111. Various AEDs show efficacy in reducing seizures that occur in AD, though LEV reduces seizures more effectively than other classes of AEDs106. Further, several studies have indicated that LEV, unlike other AEDs, is able to rescue cognitive deficits to some degree in AD, amnestic MCI112 and animal models of AD with comorbid epilepsy110. In particular, LEV reduces the characteristic hippocampal hyperactivity observed in AD and improves memory performance113. LEV and other SV2A-targeting compounds also improve cognition in non-demented epilepsy patients114-116, as well as in nonACS Paragon Plus Environment


Page 14 of 33

14 epileptic aged mice and some mouse models of AD without seizure activity117,118. These data indicate that modulating SV2A activity, independent of reducing seizure activity, is effective in reversing some degree of cognitive decline in AD and aging. The mechanism behind cognitive improvement with LEV is unknown, although the evidence points to a number of possibilities. Pathological hippocampal hyperactivity in early stages of the AD prodromal period may heighten neurodegenerative vulnerability in hippocampal cells. In particular, asymptomatic individuals who are at high genetic risk for AD (ApoE4, familial AD mutation carriers) have hyperactive cells within CA3 of the hippocampus; dampening the activity of these cells with LEV improves memory. CA3 hyperactivity may interfere with memory formation and retrieval and increase the likelihood of neurodegeneration. Dampening hyperactivity with LEV treatment improves memory function113. However, this is not likely the only explanation for the efficacy of LEV in AD, as it would be expected that any AED, not just LEV, would show similar results on cognition. LEV stimulated neuritogenesis and increased synaptic marker expression in an in vivo model of late-onset AD, possibly through direct interaction with newly discovered mitochondrial SV2A65. This may counter the regional loss of synapses observed in MCI/AD progression119,120, and may be an additional mechanism behind the cognitive improvement seen with LEV treatment. SV2B may also play a role in AD pathogenesis. Heese et al. (2001) revealed an upregulation of SV2B in vivo after treatment of cells with the AD-associated cytotoxic A1-42. This treatment stimulated the production of a variant SV2B mRNA transcript not seen in untreated cells. The protein product of this transcript variant is identical to SV2B, but the modification is thought to result in a more stable transcript and a protein with a differential posttranslational modification profile121. Furthermore, SV2B knockout appears to protect against toxicity and cognitive deficits in mice induced by injected A oligomers122. Finally, radioligands for SV2A have been proposed as a tool for identifying AD-related neurodegeneration and loss of synaptic density via PET imaging47,123,124. Though these SV2A-specific tracers were developed because SV2A is a universal synaptic marker, they may prove to be particularly useful in specifically assessing SV2A function and expression in AD and epilepsy in vivo. ACS Paragon Plus Environment

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15

SV2C and Parkinson’s disease. SV2C is the least abundant and most regionally-restricted of its family members. When it was discovered by Janz and Sudhof (1999), the investigators immediately postulated that it may be important in basal-ganglia functions and PD14. Indeed, subsequent studies have supported this idea. SV2C was recently identified as a genetic modifier of PD risk in smokers: the minor alleles at multiple loci in the promoter region of the SV2C gene confer a significantly increased risk for PD in smokers, reversing the normally protective effect of nicotine consumption in PD125. The expression patterns of SV2C were further described by Dardou, et al. (2011) and Dunn, et al. (2018), indicating that SV2C is in a majority of dopamine cells in the substantia nigra, as well as in other neuron types in the basal ganglia18,19,126. This group also provided the first experimental evidence that SV2C may be linked with PD by showing that SV2C mRNA expression increases after intoxication by the dopaminergic toxicant, MPTP13. Recent work by Yang, et al. (2018) showed that lead, a toxicant commonly linked with PD127, inhibits SV2C expression via the transcription repressor neuron-restrictive silencer factor128. More recently, our group and others have shown a more direct relationship between SV2C and PD. The expression of SV2C is significantly and specifically disrupted in PD. This disruption in PD may contribute to disease pathogenesis, as suggested by our studies into the effect of SV2C-knockout in vivo. Ablation of SV2C leads to reduced dopamine release and impaired motor function in mice19. SV2C, then, is positively associated with dopamine neuron function. These findings that SV2C promotes dopaminergic function are consistent with data indicating that vesicular function, particularly in dopamine neurons, is crucial for cell integrity129. These effects seem to have bearing in the human population as well. Patients treated with statins for blood pressure have reduced PD risk; interestingly, statins upregulate SV2C expression, suggesting that promotion of proper vesicular and neuronal function via SV2C enhancement may underlie this neuroprotection130. Additionally, extensive SV2C loss in the brains of PD patients compared to controls has been reported131. This may be reflective of general synapse loss in PD or a more specific disruption in SV2C and other dopaminergic synapse proteins. Clinically, SV2C variants are associated with altered response to dopaminergic therapeutics in Parkinson’s disease, both in the therapeutic effects132 and in adverse effects133. These data further support the potential of ACS Paragon Plus Environment


Page 16 of 33

16 SV2C as a therapeutic target for PD and suggest that a specific SV2C modulator, perhaps one derived from LEV to target SV2C rather than SV2A, may provide an opportunity for improved PD treatment, either as an alternative monotherapy or to potentiate desired effects of traditional PD therapeutics.

SV2C in Huntington’s disease (HD). HD is a neurodegenerative disease characterized by hyperkinetic movement, cognitive decline, and ultimately death. HD is caused by an autosomal dominant mutation in the Huntingtin gene, which encodes the synaptic vesicle protein, huntingtin134. Huntingtin is ubiquitously expressed in neurons but, curiously, causes pathology primarily in the striatum135. A major hypothesis of early HD pathogenesis is that mutant huntingtin causes deficits in striatal GABA, leading to disinhibition and enhanced release of dopamine, which drives hyperkinetic movement136. Interestingly, recent work demonstrated that mutant huntingtin selectively downregulates SV2C expression in neuroblastoma cells and HD transgenic mice137, which could contribute to aberrant dopamine release due to reduced proteoglycan adsorption of dopamine and subsequent enhanced neurotransmission kinetics.

SV2s in addiction and neuropsychiatric conditions. As SV2C is preferentially expressed in the basal ganglia, its potential to impact reward behavior is readily apparent. However, little is known about the impact of SV2C in appetitive behavior. A gene array study comparing differences in rats bred for high and low drug preference found high consuming rats had significantly reduced SV2C expression in the frontal cortex138. Knockdown of SV2C expression by stereotaxic lentiviral miRNA injection into the midbrain of adult mice reduced cocaine place preference compared to controls, whereas global SV2C knockout mice show no alteration in preference13. Targeting the synaptic vesicle to reduce the rewarding effect of stimulants has been explored repeatedly, and several therapeutics (most notably lobeline and its derivatives) show promising efficacy139-144. However, all of these therapeutics target the vesicular monoamine transporter 2 (VMAT2), which is responsible for packaging monoamines such as dopamine into vesicles, protecting it from metabolism and readying the neuron for appropriate release. Historically, the VMAT2-targeted drug tetrabenazine has shown promise in treating a number ACS Paragon Plus Environment

Page 17 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17 of diseases, but non-desirable effects presumably from concomitant depletion of other monoamines (serotonin, norepinephrine, histamine) has limited the applicability of targeting the non-discretely expressed protein145-150. Recent evidence, however suggests that these effects are actually off-target, the byproduct of interaction of the  enantiomer with D2 receptors. Isolation of the VMAT2-targeting enantiomer drastically improves both the tolerability and efficacy of the drug in treating tardive dyskinesia151, suggesting improved outcomes for other disease treatments. Regardless, SV2C represents a novel, unexplored, and discrete therapeutic target for the treatment of addiction. In addition to its potential as a target for the treatment of addiction, SV2C also interacts with antipsychotic therapeutics. Specifically, SV2C is down-regulated by methylphenidate152 and variants in SV2C may help to predict response to atypical antipsychotic drugs153. These results suggest that SV2C may play a role in psychiatric conditions and may be an untapped target for the treatment of such conditions. A role for SV2A in neuropsychiatric behavioral alterations has also been proposed. Recent work by Serrano, et al. (2019) showed that heterozygous SV2A knockout animals demonstrate altered habituation to novel environments, increased anxiety levels, and spatial memory deficits. No changes were observed in fear conditioning or locomotor behavior154.

SV2s in peripheral nervous and neuroendocrine disorders. SV2 paralogues have been implicated in spinal muscular atrophy(SMA), the leading genetic cause of infant mortality155. SMA is caused by deficiency in the survival motor neuron protein, a known RNA metabolizing protein156. Models of SMA in zebrafish157,158 and mouse159,160 show dramatic reductions in SV2 and synaptotagmin expression. The pathology of the mouse is also interesting and potentially informative to SV2 function. By P7 in the slow-twitch transverse abdominis muscle, SMAΔ7 mutant mice display attenuated evoked endplate potentials and reduced neurotransmitter release, very interestingly coupled with increased cumulative fusion events161. These changes in neurotransmission are coupled with a pan-SV2 reduction160. Conversely, the fast-twitch levator auris longus muscle, at the same time point, demonstrates no severe pathology; neurotransmitter release and fusion event number are normal161. SV2A/B ACS Paragon Plus Environment


Page 18 of 33

18 expression is reduced in the fast-twitch muscle160. However, SV2C expression is normal160, suggesting the paralogue dictating the phenotype in slow-twitch muscle may be SV2C. Less is known about the role of SV2 in neuroendocrine disorders but, interestingly, many gastrointestinal tumors express SV2s, which is thought to be evidence of a neurosecretory phenotype in gut carcinomas162-164. Pharmacological tools to selectively target SV2 paralogues. By far, the best known and only FDA approved compounds targeting SV2 for treating neurological diseases are leviteracetam and brivaracetam. From these molecules, several PET imaging compounds have been generated, and used to interrogate synapse loss in various disease models and humans100,123,165. When SV2A was discovered as the molecular target of LEV, UCB Pharma quickly synthesized LEV-based derivatives that selectively target SV2B and SV2C. These compounds did not display anti-seizure efficacy and, thus, were not pursued as pharmacological targets for epilepsy. However, the selectivity of the compounds has been demonstrated, as they were used to generate PET ligands166. However, they still exist and may be incredibly valuable for other neurological disorders. Conclusions Synaptic release is an elegantly orchestrated phenomenon, in which many proteins work together in synchrony to accomplish the single goal of neurotransmission. It is clear that SV2s play a significant role in this process, though the details of its exact role have yet to be unveiled. Evidence suggests that SV2s anchor vesicular structure,

regulate

neurotransmitter

loading,

participate

in

neuronal

calcium

dynamics,

stabilize

neurotransmission, and support bioenergetic demands. Given these important physiological contributions, the potential of SV2 as an effective therapeutic target is unquestionable. Further application of SV2 targeting drugs is needed both as pharmacological tools to interrogate SV2 function and for unmet therapeutic need. Drugs that stabilize neurotransmission are needed for a plethora of disorders beyond epilepsy, including: addiction, neurodegeneration, motor disorders, mood disorders, and schizophrenia.


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Sudhof, T. C. Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80, 675-690, doi:10.1016/j.neuron.2013.10.022 (2013). Farsi, Z., Jahn, R. & Woehler, A. Proton electrochemical gradient: Driving and regulating neurotransmitter uptake. Bioessays 39, doi:10.1002/bies.201600240 (2017). Sudhof, T. C. The synaptic vesicle cycle. Annu Rev Neurosci 27, 509-547, doi:10.1146/annurev.neuro.26.041002.131412 (2004). Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr. Major facilitator superfamily. Microbiology and Molecular Biology Reviews 62, 1-34 (1998). Omasits, U., Ahrens, C. H., Muller, S. & Wollscheid, B. Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics 30, 884-886, doi:10.1093/bioinformatics/btt607 (2014). Gregory, S. G. et al. The DNA sequence and biological annotation of human chromosome 1. Nature 441, 315-321, doi:10.1038/nature04727 (2006). Zody, M. C. et al. Analysis of the DNA sequence and duplication history of human chromosome 15. Nature 440, 671-675, doi:10.1038/nature04601 (2006). International Human Genome Sequencing, C. Finishing the euchromatic sequence of the human genome. Nature 431, 931-945, doi:10.1038/nature03001 (2004). Bajjalieh, S. M., Frantz, G.D., Weimann, J.M., McConnell, S.K., Scheller, R.H. Differential expression of synaptic vesicle protein 2 (SV2) isoforms. The Journal of Neuroscience 14, 5223-5235 (1994). Farwell, K. D. et al. Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions. Genet Med 17, 578-586, doi:10.1038/gim.2014.154 (2015). Zerbino, D. R. et al. Ensembl 2018. Nucleic Acids Res 46, D754-D761, doi:10.1093/nar/gkx1098 (2018). Lindberg, J. et al. The mitochondrial and autosomal mutation landscapes of prostate cancer. Eur Urol 63, 702-708, doi:10.1016/j.eururo.2012.11.053 (2013). Dardou, D., Monlezun, S., Foerch, P., Courade, J-P., Cuvelier, L., De Ryck, M., Schiffmann, S.N. A role for SV2C in basal ganglia functions. Brain Research 1507, 61-73, doi:10.1016/j.brainres.2013.02.041 (2013). Janz, R., Sudhöf, T.C. SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family. The Journal of Neuroscience 94, 1279-1290 (1999). Mutch, S. A. et al. Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision. The Journal of Neuroscience 31, 1461-1470, doi:10.1523/JNEUROSCI.3805-10.2011 (2011). Crowder, K. M., Gunther, J.M., Jones, T.A., Hale, B.D., Zhang, H.Z., Peterson, M.R., Scheller, R.H., Chavkin, C., Bajjalieh, S.M. Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A). Proceedings of the National Academy of Sciences 96, 15286-15273 (1999). Xu, T., Bajjalieh, S.M. SV2 modulates the size of the readily releasable pool of secretory vesicles. Nature Cell Biology 3, 691-698 (2001). Dunn, A. R. et al. Immunochemical analysis of the expression of SV2C in mouse, macaque and human brain. Brain Res 1702, 85-95, doi:10.1016/j.brainres.2017.12.029 (2019). Dunn, A. R. et al. Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease. Proc Natl Acad Sci U S A 114, E2253-E2262, doi:10.1073/pnas.1616892114 (2017). Portela-Gomes, G. M., Lukinius, A. & Grimelius, L. Synaptic vesicle protein 2, A new neuroendocrine cell marker. Am J Pathol 157, 1299-1309, doi:10.1016/S0002-9440(10)64645-7 (2000). Vogl, C. et al. Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses. European Journal of Neuroscience 41, 398-409, doi:10.1111/ejn.12799 (2015). ACS Paragon Plus Environment


Page 20 of 33

20 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Morgans, C. W. et al. Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina. PLoS One 4, e5230, doi:10.1371/journal.pone.0005230 (2009). von Kriegstein, K. & Schmitz, F. The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Cell and Tissue Research 311, 159-173, doi:10.1007/s00441-002-0674-0 (2003). Von Kriegstein, K., Schmitz, F., Link, E. & Sudhof, T. C. Distribution of synaptic vesicle proteins in the mammalian retina identifies obligatory and facultative components of ribbon synapses. European Journal of Neuroscience 11, 1335-1348 (1999). Chakkalakal, J. V., Nishimune, H., Ruas, J. L., Spiegelman, B. M. & Sanes, J. R. Retrograde influence of muscle fibers on their innervation revealed by a novel marker for slow motoneurons. Development 137, 3489-3499, doi:10.1242/dev.053348 (2010). Iezzi, M., Theander, S., Janz, R. Loze, C., Wollheim, C.B. SV2A and SV2C are not vesicular Ca++ transporters, but control glucose-evoked granule recruitment. Journal of Cell Science 118, 5647-5660, doi:10.1242/jcs (2005). Fishilevich, S. et al. Genic insights from integrated human proteomics in GeneCards. Database (Oxford) 2016, doi:10.1093/database/baw030 (2016). Gillard, M., Chatelain, P. & Fuks, B. Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein. European Journal of Pharmacology 536, 102-108, doi:10.1016/j.ejphar.2006.02.022 (2006). Harada, S. et al. Inhibition of Ca(2+)-regulated exocytosis by levetiracetam, a ligand for SV2A, in antral mucous cells of guinea pigs. European Journal of Pharmacology 721, 185-192, doi:10.1016/j.ejphar.2013.09.037 (2013). Kaminski, R. M. et al. Proepileptic phenotype of SV2A-deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam. Epilepsia 50, 1729-1740, doi:10.1111/j.15281167.2009.02089.x (2009). Klitgaard, H. & Verdru, P. Levetiracetam: the first SV2A ligand for the treatment of epilepsy. Expert Opinion on Drug Discovery 2, 1537-1545, doi:10.1517/17460441.2.11.1537 (2007). Lynch, B. A., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proceedings of the National Academy of Sciences 101, 9861-9866, doi:10.1073/pnas.0308208101 (2004). Nowack, A., Malarkey, E.B., Jia, Y., Bleckert, A., Hill, J., Bajjalieh, S. M. Levetiracetam Reverses Synaptic Deficits Produced by Overexpression of SV2A. PLoS One 6, doi:10.1371/journal.pone.0029560.g001 (2011). Bajjalieh, S. M., Peterson, K., Shinghal, R. & Scheller, R. H. SV2, a brain synaptic vesicle protein homologous to bacterial transporters. Science 257, 1271-1273 (1992). Feany, M. B., Lee, S., Edward, R.H., Buckley, K.M. The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell 70, 861-867 (1992). Gingrich, J. A. et al. Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system. FEBS Letters 312, 115-122 (1992). Dong, M. et al. SV2 is the protein receptor for botulinum neurotoxin A. Science 312, 592-596, doi:10.1126/science.1123654 (2006). Yeh, F. L. et al. SV2 mediates entry of tetanus neurotoxin into central neurons. PLoS Pathogens 6, e1001207, doi:10.1371/journal.ppat.1001207 (2010). Madeo, M., Kovács, A.D., Pearce, D.A. The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae. The Journal of Biological Chemistry 289, 3306633071 (2014). ACS Paragon Plus Environment

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21 40 41 42 43 44 45 46

47 48 49 50

51 52 53 54 55 56 57 58

Chang, S., He, H. Q., Shen, L. & Wan, H. Understanding peptide competitive inhibition of botulinum neurotoxin A binding to SV2 protein via molecular dynamics simulations. Biopolymers 103, 597-608, doi:10.1002/bip.22682 (2015). Mahrhold, S. et al. Only the complex N559-glycan in the synaptic vesicle glycoprotein 2C mediates high affinity binding to botulinum neurotoxin serotype A1. Biochem J 473, 2645-2654, doi:10.1042/BCJ20160439 (2016). Yao, G. et al. N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat Struct Mol Biol 23, 656-662, doi:10.1038/nsmb.3245 (2016). Fu, Z., Chen, C., Barbieri, J. T., Kim, J.-J. P. & Baldwin, M. R. Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F. Biochemistry 48, 5631-5641 (2009). Mahrhold, S. et al. Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E. Biochemical Journal 453, 37-47, doi:10.1042/BJ20130391 (2013). Peng, L., Tepp, W. H., Johnson, E. A. & Dong, M. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors. PLoS Pathogens 7, e1002008, doi:10.1371/journal.ppat.1002008 (2011). Rummel, A. et al. Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor. The Journal of Neurochemistry 110, 1942-1954, doi:10.1111/j.14714159.2009.06298.x (2009). Davies, J. R., Liu, S. M. & Acharya, K. R. Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics. Toxins (Basel) 10, doi:10.3390/toxins10100421 (2018). Ferguson, S. M. & Blakely, R. D. The choline transporter resurfaces: new roles for synaptic vesicles? Molecular Interventions 4, 22-37, doi:10.1124/mi.4.1.22 (2004). Alvarez de Toledo, G., Fernandez-Chacon, R. & Fernandez, J. M. Release of secretory products during transient vesicle fusion. Nature 363, 554-558, doi:10.1038/363554a0 (1993). Reigada, D., Diez-Perez, I., Gorostiza, P., Verdaguer, A., Gomez de Aranda, I., Pineda, O., Vilarrasa, J., Marsal, J., Blasi, J., Aleu, J., Solsona, C. Control of neurotransmitter release by an internal gel matrix in synaptic vesicles. Proceedings of the National Academy of Sciences 100, 3485-3490, doi:10.1073/pnas.0336914100 (2003). Scranton, T. W., Iwata, M. & Carlson, S. S. The SV2 protein of synaptic vesicles is a keratan sulfate proteoglycan. The Journal of Neurochemistry 61, 29-44 (1993). Silbert, J. E. Structure and metabolism of proteoglycans and glycosaminoglycans. J Invest Dermatol 79 Suppl 1, 31s-37s (1982). Budzinski, K. L. et al. Large structural change in isolated synaptic vesicles upon loading with neurotransmitter. Biophys J 97, 2577-2584, doi:10.1016/j.bpj.2009.08.032 (2009). Lohr, K. M. et al. Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo. Proc Natl Acad Sci U S A 111, 9977-9982, doi:10.1073/pnas.1402134111 (2014). Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831-846, doi:10.1016/j.cell.2006.10.030 (2006). Harlow, M. L., Szule, J. A., Xu, J., Jung, J. H., Marshall, R. M., McMahan, U. J. Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking. PLoS One 8, e69410, doi:10.1371/journal.pone.0069410 (2013). Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M. & McMahan, U. J. The architecture of active zone material at the frog's neuromuscular junction. Nature 409, 479-484, doi:10.1038/35054000 (2001). Baldwin, M. R. & Barbieri, J. T. Association of botulinum neurotoxins with synaptic vesicle protein complexes. Toxicon 54, 570-574, doi:10.1016/j.toxicon.2009.01.040 (2009). ACS Paragon Plus Environment


Page 22 of 33

22 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

77

Baldwin, M. R. & Barbieri, J. T. Association of botulinum neurotoxin serotypes a and B with synaptic vesicle protein complexes. Biochemistry 46, 3200-3210, doi:10.1021/bi602396x (2007). Bennett, M. K., Calakos, N., Kreiner, T. & Scheller, R. H. Synaptic vesicle membrane proteins interact to form a multimeric complex. The Journal of Cell Biology 116, 761-775 (1992). Wan, Q. F. et al. SV2 acts via presynaptic calcium to regulate neurotransmitter release. Neuron 66, 884895, doi:10.1016/j.neuron.2010.05.010 (2010). Chang, W.-P., Sudhof, T.C. SV2 renders primed synaptic vesicles competent for Ca2+ -induced exocytosis. The Journal of Neuroscience 29, 960-967, doi:10.1523/ (2009). Custer, K. L., Austin, N. S., Sullivan, J. M. & Bajjalieh, S. M. Synaptic vesicle protein 2 enhances release probability at quiescent synapses. The Journal of Neuroscience 26, 1303-1313, doi:10.1523/JNEUROSCI.2699-05.2006 (2006). Janz, R., Goda, Y., Geppert, M., Missler, M. & Sudhof, T. C. SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release. Neuron 24, 1003-1016 (1999). Stockburger, C. et al. A mitochondrial role of SV2A protein in aging and Alzheimer's Disease: studies with levetiracetam. Journal of Alzheimers Disease 50, 201-215, doi:10.3233/JAD-150687 (2015). Kislinger, T. et al. Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell 125, 173-186, doi:10.1016/j.cell.2006.01.044 (2006). Nicholls, D. G., Brand, M. D. & Gerencser, A. A. Mitochondrial bioenergetics and neuronal survival modelled in primary neuronal culture and isolated nerve terminals. J Bioenerg Biomembr 47, 63-74, doi:10.1007/s10863-014-9573-9 (2015). Lazzell, D. R., Belizaire, R., Thakur, P., Sherry, D.M., Janz, R. SV2B regulates synaptotagmin 1 by direct interaction. The Journal of Biological Chemistry 279, 52124-52131, doi:10.1074/jbc.M407502200 (2004). Pyle, J. L., Kavalali, E. T., Piedras-Renteria, E. S. & Tsien, R. W. Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28, 221-231 (2000). Schivell, A. E., Batchelor, R. H. & Bajjalieh, S. M. Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin. The Journal of Cell Biology 271, 27770-27775 (1996). Schivell, A. E., Mochida, S., Kensel-Hammes, P., Custer, K. L., Bajjalieh, S. M. SV2A and SV2C contain a unique synaptotagmin-binding site. Molecular and cellular neurosciences 29, 56-64, doi:10.1016/j.mcn.2004.12.011 (2005). Zhang, N. et al. Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1. J Neurosci 35, 2492-2507, doi:10.1523/JNEUROSCI.4248-14.2015 (2015). Yao, J., Nowack, A., Kensel-Hammes, P., Gardner, R. G., Bajjalieh, S. M. Cotrafficking of SV2 and synaptotagmin at the synapse. The Journal of Neuroscience 30, 5569-5578, doi:10.1523/JNEUROSCI.4781-09.2010 (2010). Son, Y. J. et al. The synaptic vesicle protein SV2 is complexed with an alpha5-containing laminin on the nerve terminal surface. The Journal of Biological Chemistry 275, 451-460 (2000). Staal, R. G., Mosharov, E. V. & Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nat Neurosci 7, 341-346, doi:10.1038/nn1205 (2004). Chand, K. K., Lee, K. M., Schenning, M. P., Lavidis, N. A. & Noakes, P. G. Loss of beta2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction. The Journal of Physiology 593, 245-265, doi:10.1113/jphysiol.2014.284133 (2015). Nakagami, Y., Abe, K., Nishiyama, N. & Matsuki, N. Laminin degradation by plasmin regulates long-term potentiation. The Journal of Neuroscience 20, 2003-2010 (2000). ACS Paragon Plus Environment

Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95

Knight, D., Tolley, L. K., Kim, D. K., Lavidis, N. A. & Noakes, P. G. Functional analysis of neurotransmission at beta2-laminin deficient terminals. The Journal of Physiology 546, 789-800 (2003). Bennett, B., Matagne, A., Michel, P., Leonard, M., Cornet, M., Meeus, M-A., Toublanc, N. Seletracetam (UCB 44212). Neurotherapeutics 4, 117-122 (2007). Rogawski, M. A. A New SV2A Ligand for Epilepsy. Cell 167, 587, doi:10.1016/j.cell.2016.09.057 (2016). Schoemaker, R., Wade, J. R., D'Souza, J. & Stockis, A. Evaluation of brivaracetam efficacy as monotherapy in adult patients with focal seizures. Epilepsy Res 137, 95-100, doi:10.1016/j.eplepsyres.2017.09.014 (2017). Wood, M. D., Sands, Z. A., Vandenplas, C. & Gillard, M. Further evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein. Epilepsia 59, e147-e151, doi:10.1111/epi.14532 (2018). von Rosenstiel, P. Brivaracetam (UCB 34714). Neurotherapeutics 4, 84-87 (2007). Feng, G., Xiao, F., Lu, Y., Huang, Z., Yuan, J., Xiao, Z., Xi, Z., Wang, X. Down-regulation of synaptic vesicle protein 2A in the anterior temporal neocortex of patients with intractable epilepsy. The Journal of Molecular Neuroscience 39, 354-359 (2009). Gorter, J. A. et al. Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy. The Journal of Neuroscience 26, 11083-11110, doi:10.1523/JNEUROSCI.2766-06.2006 (2006). Shi, J., Zhou, F., Wang, L-K., Wu, G-F. Synaptic vesicle protein 2A decreases in amygdaloid kindling pharmacoresistant epileptic rats. Journal of Huazhon University of Science and Technology 35, 716-722 (2015). van Vliet, E. A., Aronica, E., Redeker, S., Boer, K. & Gorter, J. A. Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy. Epilepsia 50, 422-433, doi:10.1111/j.1528-1167.2008.01727.x (2009). Ohno, Y. et al. Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling. Biochemical and Biophysical Research Communications 390, 415-420, doi:10.1016/j.bbrc.2009.09.035 (2009). Contreras-Garcia, I. J. et al. Differential expression of synaptic vesicle protein 2A after status epilepticus and during epilepsy in a lithium-pilocarpine model. Epilepsy Behav 88, 283-294, doi:10.1016/j.yebeh.2018.08.023 (2018). Wang, L., Shi, J., Wu, G., Zhou, F. & Hong, Z. Hippocampal low-frequency stimulation increased SV2A expression and inhibited the seizure degree in pharmacoresistant amygdala-kindling epileptic rats. Epilepsy Research 108, 1483-1491, doi:10.1016/j.eplepsyres.2014.07.005 (2014). Douaud, M. et al. Epilepsy caused by an abnormal alternative splicing with dosage effect of the SV2A gene in a chicken model. PLoS One 6, e26932, doi:10.1371/journal.pone.0026932 (2011). de Groot, M., Aronica, E., Heimans, J.J., Reijneveld, J.C. Synaptic vesicle protein 2A predicts response to levetiracetam in patients with glioma. Neurology 77, 532-539, doi:10.1212/WNL.0b013e318228c110 (2011). Dibbens, L. M. et al. Rare protein sequence variation in SV2A gene does not affect response to levetiracetam. Epilepsy Research 101, 277-279, doi:10.1016/j.eplepsyres.2012.04.007 (2012). Lynch, J. M. et al. No major role of common SV2A variation for predisposition or levetiracetam response in epilepsy. Epilepsy Research 83, 44-51, doi:10.1016/j.eplepsyres.2008.09.003 (2009). Serajee, F. J. & Huq, A. M. Homozygous Mutation in Synaptic Vesicle Glycoprotein 2A Gene Results in Intractable Epilepsy, Involuntary Movements, Microcephaly, and Developmental and Growth Retardation. Pediatric Neurology 52, 642-646 e641, doi:10.1016/j.pediatrneurol.2015.02.011 (2015). ACS Paragon Plus Environment


Page 24 of 33

24 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113

Ohno, Y. & Tokudome, K. Therapeutic Role of Synaptic Vesicle Glycoprotein 2A (SV2A) in Modulating Epileptogenesis. CNS Neurol Disord Drug Targets 16, 463-471, doi:10.2174/1871527316666170404115027 (2017). Garcia-Perez, E., Mahfooz, K., Covita, J., Zandueta, A. & Wesseling, J. F. Levetiracetam accelerates the onset of supply rate depression in synaptic vesicle trafficking. Epilepsia 56, 535-545, doi:10.1111/epi.12930 (2015). Yang, X. F., Weisenfeld, A. & Rothman, S. M. Prolonged exposure to levetiracetam reveals a presynaptic effect on neurotransmission. Epilepsia 48, 1861-1869, doi:10.1111/j.1528-1167.2006.01132.x (2007). Yang, X. et al. Brivaracetam augments short-term depression and slows vesicle recycling. Epilepsia 56, 1899-1909, doi:10.1111/epi.13223 (2015). Finnema, S. J. et al. A single-center, open-label positron emission tomography study to evaluate brivaracetam and levetiracetam synaptic vesicle glycoprotein 2A binding in healthy volunteers. Epilepsia 60, 958-967, doi:10.1111/epi.14701 (2019). Wood, M. D. & Gillard, M. Evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein. Epilepsia 58, 255-262, doi:10.1111/epi.13638 (2017). Sugata, S. et al. Neuroprotective effect of levetiracetam on hippocampal sclerosis-like change in spontaneously epileptic rats. Brain Research Bulletin 86, 36-41, doi:10.1016/j.brainresbull.2011.05.017 (2011). Sherzai, D., Losey, T., Vega, S. & Sherzai, A. Seizures and dementia in the elderly: Nationwide Inpatient Sample 1999-2008. Epilepsy Behavior 36, 53-56, doi:10.1016/j.yebeh.2014.04.015 (2014). Chin, J. & Scharfman, H. E. Shared cognitive and behavioral impairments in epilepsy and Alzheimer's disease and potential underlying mechanisms. Epilepsy Behavior 26, 343-351, doi:10.1016/j.yebeh.2012.11.040 (2013). Horvath, A., Szucs, A., Barcs, G., Noebels, J. L. & Kamondi, A. Epileptic Seizures in Alzheimer Disease: A Review. Alzheimer Disease and Associated Disorders 30, 186-192, doi:10.1097/WAD.0000000000000134 (2016). Vossel, K. A. et al. Seizures and epileptiform activity in the early stages of Alzheimer disease. The Journal of the American Medical Association Neurology 70, 1158-1166, doi:10.1001/jamaneurol.2013.136 (2013). Chan, J., Jones, N. C., Bush, A. I., O'Brien, T. J. & Kwan, P. A mouse model of Alzheimer's disease displays increased susceptibility to kindling and seizure-associated death. Epilepsia 56, e73-77, doi:10.1111/epi.12993 (2015). Ping, Y. et al. Linking abeta42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model. PLoS Genetics 11, e1005025, doi:10.1371/journal.pgen.1005025 (2015). Siwek, M. E. et al. Altered theta oscillations and aberrant cortical excitatory activity in the 5XFAD model of Alzheimer's disease. Neural Plasticity 2015, 781731, doi:10.1155/2015/781731 (2015). Sola, I. et al. Novel levetiracetam derivatives that are effective against the Alzheimer-like phenotype in mice: synthesis, in vitro, ex vivo, and in vivo efficacy studies. Journal of Medicinal Chemistry 58, 60186032, doi:10.1021/acs.jmedchem.5b00624 (2015). Ziyatdinova, S. et al. Increased epileptiform EEG activity and decreased seizure threshold in arctic APP transgenic mouse model of Alzheimer's Disease. Current Alzheimer Research 13, 817-830 (2016). Cumbo, E. & Ligori, L. D. Levetiracetam, lamotrigine, and phenobarbital in patients with epileptic seizures and Alzheimer's disease. Epilepsy Behav 17, 461-466, doi:10.1016/j.yebeh.2010.01.015 (2010). Bakker, A. et al. Reduction of hippocampal hyperactivity improves cognition in amnestic mild cognitive impairment. Neuron 74, 467-474, doi:10.1016/j.neuron.2012.03.023 (2012). ACS Paragon Plus Environment

Page 25 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 114 115 116 117 118 119 120 121 122 123 124 125

126 127 128 129 130 131

Helmstaedter, C. & Witt, J. A. Cognitive outcome of antiepileptic treatment with levetiracetam versus carbamazepine monotherapy: a non-interventional surveillance trial. Epilepsy Behavior 18, 74-80, doi:10.1016/j.yebeh.2010.02.011 (2010). Loscher, W., Gillard, M., Sands, Z. A., Kaminski, R. M. & Klitgaard, H. Synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond. CNS Drugs 30, 1055-1077, doi:10.1007/s40263-0160384-x (2016). Wu, T. et al. Clinical efficacy and cognitive and neuropsychological effects of levetiracetam in epilepsy: an open-label multicenter study. Epilepsy Behavior 16, 468-474, doi:10.1016/j.yebeh.2009.08.026 (2009). Devi, L. & Ohno, M. Effects of levetiracetam, an antiepileptic drug, on memory impairments associated with aging and Alzheimer's disease in mice. Neurobiology of Learning and Memory 102, 7-11, doi:10.1016/j.nlm.2013.02.001 (2013). Celikyurt, I. K. et al. Positive impact of levetiracetam on emotional learning and memory in naive mice. Life Science 90, 185-189, doi:10.1016/j.lfs.2011.11.003 (2012). Robinson, J. L. et al. Perforant path synaptic loss correlates with cognitive impairment and Alzheimer's disease in the oldest-old. Brain 137, 2578-2587, doi:10.1093/brain/awu190 (2014). Sze, C.-I., Bi, H., Kleinschmidt-DeMasters, B.K., Filley, C., Martin, L.J. Selective regional loss of exocytotic presynaptic vesicle proteins in Alzheimer's disease brains. Journal of the Neurological Sciences 175, 8190 (2000). Heese, K., Nagai, Y. & Sawada, T. Identification of a new synaptic vesicle protein 2B mRNA transcript which is up-regulated in neurons by amyloid beta peptide fragment (1-42). Biochemical and Biophysical Research Communications 289, 924-928, doi:10.1006/bbrc.2001.5932 (2001). Detrait, E., Maurice, T., Hanon, E., Leclercq, K. & Lamberty, Y. Lack of synaptic vesicle protein SV2B protects against amyloid-beta(2)(5)(-)(3)(5)-induced oxidative stress, cholinergic deficit and cognitive impairment in mice. Behavioural Brain Research 271, 277-285, doi:10.1016/j.bbr.2014.06.013 (2014). Bahri, M. A. et al. Measuring brain synaptic vesicle protein 2A with positron emission tomography and [(18)F]UCB-H. Alzheimers Dement (N Y) 3, 481-486, doi:10.1016/j.trci.2017.08.004 (2017). Chen, M. K. et al. Assessing Synaptic Density in Alzheimer Disease With Synaptic Vesicle Glycoprotein 2A Positron Emission Tomographic Imaging. JAMA Neurol 75, 1215-1224, doi:10.1001/jamaneurol.2018.1836 (2018). Hill-Burns, E. M., Singh, N., Ganguly, P., Hamza, T. H., Montimurro, J., Kay, D. M., Yearout, D., Sheehan, P., Frodey, K., McLear, J. A., Feany, M. B., Hanes, S. D., Wolfgang, W. J., Zabetian, C. P., Factor, S. A., Payami, H. A genetic basis for the variable effect of smoking/nicotine on Parkinson's disease. The pharmacogenomics journal, doi:10.1038/tpj.2012.38 (2012). Dardou, D., Dassesse, D., Cuvelier, L., Deprez, T., De Ryck, M., Schiffmann, S. N. Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system. Brain Research 1367, 130-145, doi:10.1016/j.brainres.2010.09.063 (2011). Coon, S. et al. Whole-body lifetime occupational lead exposure and risk of Parkinson's disease. Environ Health Perspect 114, 1872-1876, doi:10.1289/ehp.9102 (2006). Yang, M. et al. Lead exposure inhibits expression of SV2C through NRSF. Toxicology 398-399, 23-30, doi:10.1016/j.tox.2018.02.009 (2018). Alter, S. P., Lenzi, G.M., Bernstein, A.I., Miller, G. W. Vesicular integrity in Parkinson's disease. Current neurology and neuroscience reports 13, 362, doi:10.1007/s11910-013-0362-3 (2013). Schmitt, M., Dehay, B., Bezard, E., Garcia-Ladona, F.J. Harnessing the trophic and modulatory potential of statins in a dopaminergic cell line. Synapse 70, 71-86, doi:10.1002/syn.21881 (2016). Bereczki, E. et al. Synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach. Brain 141, 582-595, doi:10.1093/brain/awx352 (2018). ACS Paragon Plus Environment


Page 26 of 33

26 132 133 134 135 136 137 138 139 140

141 142

143 144 145 146 147 148 149

Altmann, V. et al. Influence of genetic, biological and pharmacological factors on levodopa dose in Parkinson's disease. Pharmacogenomics 17, 481-488, doi:10.2217/pgs.15.183 (2016). Redensek, S. et al. Dopaminergic Pathway Genes Influence Adverse Events Related to Dopaminergic Treatment in Parkinson's Disease. Front Pharmacol 10, 8, doi:10.3389/fphar.2019.00008 (2019). DiFiglia, M. et al. Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14, 1075-1081 (1995). Subramaniam, S. & Snyder, S. H. Huntington's disease is a disorder of the corpus striatum: focus on Rhes (Ras homologue enriched in the striatum). Neuropharmacology 60, 1187-1192, doi:10.1016/j.neuropharm.2010.10.025 (2011). Andre, V. M., Cepeda, C. & Levine, M. S. Dopamine and glutamate in Huntington's disease: A balancing act. CNS Neurosci Ther 16, 163-178, doi:10.1111/j.1755-5949.2010.00134.x (2010). Peng, C., Zhu, G., Liu, X. & Li, H. Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C. Neurosci Bull 34, 747-758, doi:10.1007/s12264-018-0230-x (2018). Higuera-Matas, A. et al. Differential gene expression in the nucleus accumbens and frontal cortex of lewis and Fischer 344 rats relevant to drug addiction. Current Neuropharmacology 9, 143-150, doi:10.2174/157015911795017290 (2011). Beckmann, J. S. et al. The effect of a novel VMAT2 inhibitor, GZ-793A, on methamphetamine reward in rats. Psychopharmacology (Berl) 220, 395-403, doi:10.1007/s00213-011-2488-9 (2012). Beckmann, J. S. et al. The Novel Pyrrolidine Nor-Lobelane Analog UKCP-110 [cis-2,5-di-(2-phenethyl)pyrrolidine hydrochloride] Inhibits VMAT2 Function, Methamphetamine-Evoked Dopamine Release, and Methamphetamine Self-Administration in Rats. The Journal of Pharmacology and Experimental Therapeutics 335, 841-851, doi:10.1124/jpet.110.172742 (2010). Damaj, M. I., Patrick, G. S., Creasy, K. R. & Martin, B. R. Pharmacology of lobeline, a nicotinic receptor ligand. The Journal of Pharmacology and Experimental Therapeutics 282, 410-419 (1997). Dimatelis, J. J., Russell, V. A., Stein, D. J. & Daniels, W. M. The effects of lobeline and naltrexone on methamphetamine-induced place preference and striatal dopamine and serotonin levels in adolescent rats with a history of maternal separation. Metabolic brain disease 27, 351-361, doi:10.1007/s11011012-9288-8 (2012). Dwoskin, L. P. & Crooks, P. A. A novel mechanism of action and potential use for lobeline as a treatment for psychostimulant abuse. Biochemical Pharmacology 63, 89-98, doi:10.1016/S00062952(01)00899-1 (2002). Harrod, S. B., Dwoskin, L. P., Crooks, P. A., Klebaur, J. E. & Bardo, M. T. Lobeline attenuates dmethamphetamine self-administration in rats. The Journal of Pharmacology and Experimental Therapeutics 298, 172-179 (2001). Jankovic, J. & Clarence-Smith, K. Tetrabenazine for the treatment of chorea and other hyperkinetic movement disorders. Expert Review of Neurotherapeutics 11, 1509-1523, doi:10.1586/ern.11.149 (2011). Jankovic, J., Glaze, D. G. & Frost, J. D., Jr. Effect of tetrabenazine on tics and sleep of Gilles de la Tourette's syndrome. Neurology 34, 688-692 (1984). Kenney, C., Hunter, C. & Jankovic, J. Long-term tolerability of tetrabenazine in the treatment of hyperkinetic movement disorders. Movement Disorders 22, 193-197, doi:10.1002/mds.21222 (2007). Kenney, C., Hunter, C., Mejia, N. & Jankovic, J. Is history of depression a contraindication to treatment with tetrabenazine? Clinical Neuropharmacology 29, 259-264, doi:10.1097/01.WNF.0000228369.25593.35 (2006). Leung, J. G. & Breden, E. L. Tetrabenazine for the Treatment of Tardive Dyskinesia. The Annals of Pharmacotherapy 45, 525-531 (2011). ACS Paragon Plus Environment

Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


27 150

Paleacu, D., Giladi, N., Moore, O., Stern, A. & Honigman, S. Tetrabenazine Treatment in Movement Disorders. Clinical Neuropharmacology 27, 230-233 (2004). 151 Muller, T. Valbenazine granted breakthrough drug status for treating tardive dyskinesia. Expert Opinion on Investigational Drugs 24, 737-742, doi:10.1517/13543784.2015.1029573 (2015). 152 Bartl, J. et al. Effects of methylphenidate: the cellular point of view. Atten Defic Hyperact Disord 2, 225232, doi:10.1007/s12402-010-0039-6 (2010). 153 Ramsey, T. L., Liu, Q., Massey, B. W. & Brennan, M. D. Genotypic variation in the SV2C gene impacts response to atypical antipsychotics the CATIE study. Schizophr Res 149, 21-25, doi:10.1016/j.schres.2013.07.008 (2013). 154 Serrano, M. E. et al. Anxiety-like features and spatial memory problems as a consequence of hippocampal SV2A expression. PLoS One 14, e0217882, doi:10.1371/journal.pone.0217882 (2019). 155 Pearn, J. Incidence, Prevalence, and Gene Frequency Studies of Chronic Childhood Spinal MuscularAtrophy. J Med Genet 15, 409-413, doi:DOI 10.1136/jmg.15.6.409 (1978). 156 Pellizzoni, L., Kataoka, N., Charroux, B. & Dreyfuss, G. A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell 95, 615-624, doi:10.1016/s00928674(00)81632-3 (1998). 157 Boon, K. L. et al. Zebrafish survival motor neuron mutants exhibit presynaptic neuromuscular junction defects. Hum Mol Genet 18, 3615-3625, doi:10.1093/hmg/ddp310 (2009). 158 Hao, L. T., Burghes, A. H. M. & Beattie, C. E. Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene. Mol Neurodegener 6, doi:Artn 24 10.1186/1750-1326-6-24 (2011). 159 Dale, J. M. et al. The spinal muscular atrophy mouse model, SMADelta7, displays altered axonal transport without global neurofilament alterations. Acta Neuropathol 122, 331-341, doi:10.1007/s00401-011-0848-5 (2011). 160 Tejero, R., Lopez-Manzaneda, M., Arumugam, S. & Tabares, L. Synaptotagmin-2, and -1, linked to neurotransmission impairment and vulnerability in Spinal Muscular Atrophy. Hum Mol Genet 25, 47034716, doi:10.1093/hmg/ddw297 (2016). 161 Ruiz, R., Casanas, J. J., Torres-Benito, L., Cano, R. & Tabares, L. Altered intracellular Ca2+ homeostasis in nerve terminals of severe spinal muscular atrophy mice. J Neurosci 30, 849-857, doi:10.1523/JNEUROSCI.4496-09.2010 (2010). 162 Bumming, P. et al. Gastrointestinal stromal tumors regularly express synaptic vesicle proteins: evidence of a neuroendocrine phenotype. Endocr Relat Cancer 14, 853-863, doi:10.1677/ERC-06-0014 (2007). 163 Jakobsen, A. M. et al. Expression of synaptic vesicle protein 2 (SV2) in neuroendocrine tumours of the gastrointestinal tract and pancreas. J Pathol 196, 44-50, doi:10.1002/path.1002 (2002). 164 Nilsson, O. et al. Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. Ann N Y Acad Sci 1014, 280-283, doi:10.1196/annals.1294.032 (2004). 165 Gillard, M. et al. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur J Pharmacol 478, 1-9, doi:10.1016/j.ejphar.2003.08.032 (2003). 166 Serrano, M. E. et al. Evaluating the In Vivo Specificity of [(18)F]UCB-H for the SV2A Protein, Compared with SV2B and SV2C in Rats Using microPET. Molecules 24, doi:10.3390/molecules24091705 (2019).



Page 28 of 33

28


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract



Figure 1 – SV2 proteins are highly homologous, as visualized here. The paralogues share around 60% sequence homology and 80% structural homology. The N-termini are the most variable regions between the three proteins; this region is substantially truncated in SV2B. The 4th interluminal loop is massive, long enough to span the entire width of a synaptic vesicle, and the site of N-glycosylation. SV2A and SV2B both have three N-linked glycosylation sites. SV2C has five sites. The proteins are also phosphorylated. The Thr84 site is phosphorylated by casein kinase family 1 proteins, which controls recruitment of synaptotagmin71. The functional consequences of the other phosphorylation sites have yet to be determined. Image was created by the authors using Protter5.


Page 30 of 33

Page 31 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2 - SV2 as a vesicular protein. Neurosecretory vesicles typically express 5 copies of SV2, with little intravesicular variation. The 4th intraluminal loop* of SV2A (at 3 sites), B (at 3 sites), and C (at 5 sites) are heavily glycosylated (orange, SV2C representative diagram shown). In acidic environments, proteoglycans become negatively charged. This allows neurotransmitters that are either positively charged (monoamines, acetylcholine) or zwitterionic (GABA) to adsorb into the charged proteoglycan matrix, removing them the concentration gradient and allowing the vesicle to package more neurotransmitter (left). Additionally, the negatively charged matrix likely interacts with integral vesicular proteins. In acidic environments, proteins generally become positively charge, promoting interaction with the matrix (right). Thus, SV2 proteins, via their glycosylated luminal domain, may form the backbone of the vesicle, orienting each protein into the correct location to enable rapid neurotransmission. * - For diagram clarity we have not displayed the 4th intraluminal loop to scale.



Figure 3 - SV2 as a cytosolic protein. SV2 proteins bind synaptotagmin (SYT1) in their N-terminus. This tethers SYT1 to the vesicle, bringing it into the appropriate orientation to participate in fusion. Calcium influx removes complexin from the SNARE complex: syntaxin (STX), SNAP25, and VAMP (synaptobrevin). The importance of the SYT1/SV2 interaction for neurotransmission remains unclear.


Page 32 of 33

Page 33 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 - SV2 as an extracellular protein. Upon full fusion with the synaptic membrane, the lumen of the vesicle becomes the extracellular compartment. This completely changes the potential binding partners of these proteins. Both botulinum (BoNT) and tetanus toxin gain access to the neuron via glycosylationdependent interaction with SV2. In yeast, SV2 acts to transport galactose from the extracellular compartment into the cell. This has not been demonstrated in higher order organisms. SV2 interaction with laminin has been reported in electric organ tissue, which may act as an extracellular anchor for full fusion.


The Synaptic Vesicle Glycoprotein 2: Structure, Function, and Disease

Recommend Documents