1521-0081/69/2/141–160$25.00 http://dx.doi.org/10.1124/pr.116.013342PHARMACOLOGICAL REVIEWS Pharmacol Rev 69:141–160, April 2017Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: LYNETTE C. DAWS

Synaptic Vesicle-Recycling Machinery Components asPotential Therapeutic Targets

Ying C. Li and Ege T. Kavalali

Departments of Neuroscience (Y.C.L., E.T.K.) and Physiology (E.T.K.), University of Texas Southwestern Medical Center, Dallas, Texas

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

A. Rationale Behind Targeting Presynaptic Vesicle-Recycling Machinery . . . . . . . . . . . . . . . . . . . 142II. An Overview of the Synaptic Vesicle Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

A. Modes of Synaptic Vesicle Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1431. Synchronous Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432. Asynchronous Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443. Spontaneous Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1444. Endocytic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

B. Vesicular Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146III. Fusion Machinery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

A. SNARE Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146B. Cleavage of SNAREs by Clostridial Toxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146C. Effects of Other Drugs on SNARE Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147D. SNARE-Associated Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

1. Munc18-1 (STXBP1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472. Synaptotagmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483. Complexin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

E. Noncanonical SNAREs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1481. Vti1a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482. VAMP7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

IV. Synaptic Vesicle Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149A. Synaptotagmin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149B. Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149C. Dynamin I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149D. Amphiphysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150E. Synaptojanin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

V. Stability and Maintenance of the Synaptic Vesicle-Recycling Machinery . . . . . . . . . . . . . . . . . . . . . 151A. Cysteine-String Protein a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151B. Synucleins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

VI. Active Zone Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151A. Munc13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151B. Rab3-Interacting Molecule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

VII. Other Synaptic Vesicle Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152A. Rab3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152B. Synaptic Vesicle Glycoprotein 2A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152C. Synapsin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153D. Synaptophysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153E. Rab5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

This work was supported by the National Institutes of Health National Institute of Mental Health [Grant MH066198].Address correspondence to: Dr. Ege T. Kavalali, Department of Neuroscience, 5323 Harry Hines Boulevard, University of Texas

Southwestern Medical Center, Dallas, TX 75390. E-mail: ege.kavalali@utsouthwestern.edudx.doi.org/10.1124/pr.116.013342.

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VIII. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Abstract——Presynaptic nerve terminals are highlyspecialized vesicle-traffickingmachines. Neurotransmitterrelease from these terminals is sustained by constantlocal recycling of synaptic vesicles independent fromthe neuronal cell body. This independence placessignificant constraints on maintenance of synapticprotein complexes and scaffolds. Key events duringthe synaptic vesicle cycle—such as exocytosis andendocytosis—require formation and disassemblyof protein complexes. This extremely dynamicenvironment poses unique challenges for proteostasisat synaptic terminals. Therefore, it is not surprisingthat subtle alterations in synaptic vesicle cycle-associated proteins directly or indirectly contribute topathophysiology seen in several neurologic andpsychiatric diseases. In contrast to the increasing

number of examples inwhich presynaptic dysfunctioncauses neurologic symptoms or cognitive deficitsassociated with multiple brain disorders, synapticvesicle-recycling machinery remains an underexploreddrug target. In addition, irrespective of the involvementof presynaptic function in the disease process,presynaptic machinery may also prove to be a viabletherapeutic target because subtle alterations in theneurotransmitterreleasemaycounterdiseasemechanisms,correct, or compensate for synaptic communicationdeficits without the need to interfere with postsynapticreceptor signaling. In this article, we will overviewcritical properties of presynaptic release machineryto help elucidate novel presynaptic avenues for thedevelopment of therapeutic strategies against neurologicand neuropsychiatric disorders.

I. Introduction

A. Rationale Behind Targeting Presynaptic Vesicle-Recycling Machinery

Chemical synapses are the major channels of infor-mation transfer and processing in the central nervoussystem (CNS). They consist of two functionally and struc-turally distinct compartments: presynaptic terminalsand postsynaptic specializations. Presynaptic terminalsstore and release neurotransmitter substances in mem-branous organelles named synaptic vesicles, whereaspostsynaptic structures contain signaling molecules re-sponsible for generation of neuronal responses to releasedneurotransmitters. Neurotransmission at the presynap-tic terminal involves synaptic vesicle exocytosis, endo-cytosis, and reuse of synaptic vesicles. Synaptic vesiclerecycling is essential to the function of neurons. Theseprocesses rely on the complex interactions of a multitudeof synaptic proteins and lipids. In this review, we aim tohighlight recent advances in our understanding of themolecular determinants of synaptic vesicle cycle, theirphysiologic functions, pathologic roles, and their phar-macological potential as drug targets for amelioration ofdisease states. Defects in presynaptic function underlie awide variety of neurologic and psychiatric disorders.However, the synaptic vesicle-recycling machinery is anunderexplored area for drug development as much focushas been placed on ion channels, G protein–coupled recep-tors, and other mainly postsynaptic targets.The presynaptic machinery is an attractive therapeu-

tic target because it allows for dynamic modulation ofsynaptic transmission. Targeting regulators of exocytosis

and endocytosis provide a range of outputs, from com-plete abolition of neurotransmitter release to subtlemod-ifications of neuronal signaling and neuronal firing.Manipulation of the diverse vesicular proteins can selec-tively alter different forms of neurotransmitter release.Recent studies demonstrate that nonsynchronous formsof neurotransmitter release are important to the regula-tion of synaptic plasticity, memory processing, and anti-depressant action (Autry et al., 2011; Xu et al., 2012;Nosyreva et al., 2013; Cho et al., 2015). The precision ofthe synaptic message is maintained at the postsynapticlevel as variations in presynaptic release differentiallyaffect receptors and downstream targets (Atasoy et al.,2008;Autry et al., 2011; Sara et al., 2011; Stepanyuk et al.,2014). This differential postsynaptic signaling is enabledby presynaptic segregation of vesicle-trafficking pathwaysthat mediate spontaneous and synchronous evoked re-lease (Kavalali, 2015). In some cases, this segregationmayalso extend to mechanisms and signaling targets of asyn-chronous release. The parallel signaling by kineticallydiverse release processes may enable isolation of neuro-trophic, homeostatic, or other functions of released neu-rotransmitter substances from their critical role in precisepresynaptic action potential-driven information transfer.Presynaptic terminals, therefore, present awide spectrumof novel targets for CNS drug design where synaptic com-munication can be altered in subtle ways that can allevi-ate disorder symptoms with limited side effects.

Due to space constraints, we will focus on componentsof the synaptic vesicle-recycling pathway in the CNSwith therapeutic potential. Although G protein–coupled

ABBREVIATIONS: Ab, amyloid-b; AD, Alzheimer disease; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ApoER2,apolipoprotein E receptor; CNS, central nervous system; CSP, cysteine-string protein; hsc70, heat shock protein 70; KO, knockout; Mnb/Dyrk1A, minibrain kinase/dual-specificity tyrosine phosphorylation–regulated kinase; NMDA, N-methyl-D-aspartic acid; PKA, protein kinaseA; RIM, Rab3-interacting molecule; SNARE, N-ethylmaleimide–sensitive factor attachment protein receptor; SV2, synaptic vesicleglycoprotein 2; syb, synaptobrevin; syt, synaptotagmin; t-SNARE, target-SNARE; VAMP4, vesicle-associated membrane protein 4; VLDLR,very-low-density-lipoprotein receptor; v-SNARE, vesicle-SNARE; vti1a, Vps10p-tail-indicator-1a.

142 Li and Kavalali

receptors are important for regulation of presynapticfunction, compounds targeting G protein–coupled re-ceptors are some of themost widely prescribed drugs forthe treatment of neurologic and psychiatric disordersand will not be discussed in this review. Similarly,vesicular transporters and voltage-gated ion channelsare also well-studied targets for pharmaceuticals andhave been covered in other reviews (Chaudhry et al.,2008; Wulff et al., 2009; Mantegazza et al., 2010;Zamponi et al., 2015; Bermingham and Blakely, 2016).

II. An Overview of the Synaptic Vesicle Cycle

Synapses are basic structural units for communica-tion between neurons and are essential for neuronalfunction. Neuronal signals travel along axons and triggerthe opening of voltage-gated calcium (Ca2+) channels inpresynaptic terminals. The influx of Ca2+ initiates aseries of events leading to the fusion of synaptic vesiclesto thepresynapticmembraneat active zones.This resultsin the release of neurotransmitters into the synaptic cleftand the propagation of signals downstream via the ac-tions of various postsynaptic receptors. Synaptic vesiclesin the presynaptic terminals are retrieved from themembrane, reacidified, and refilled with neurotransmit-ters for reuse. This dynamic process of synaptic vesiclerecycling is critical for maintaining normal synapticfunction. Precise release of neurotransmitters dependson the equilibrium between vesicular fusion during exo-cytosis and membrane retrieval during endocytosis (seeFigs. 1 and 2).In the full-collapse fusion model of synaptic vesicle

recycling, vesicles fuse with the presynaptic membraneat the active zone and completely collapse onto themembrane (Heuser and Reese, 1973; Südhof, 1995;Cremona and De Camilli, 1997). Subsequently, clathrinand its adaptor proteins are recruited to the membraneand form clathrin-coated vesicles that pinch off theplasma membrane through the scissioning action ofdynamin. Endocytosis of clathrin-coated vesicles may

also occur through larger structures such as membraneinfoldings or endosomal cisternae that form upon accu-mulation of fused synaptic vesicles (Koenig and Ikeda,1996; Takei et al., 1996). Vacuolar-type ATPases pumpprotons into these newly reformed vesicles, and neuro-transmitter transporters use this gradient to refill vesicleswith neurotransmitter. Kiss-and-run is an alternativemodel of synaptic vesicle fusion and retrieval that involvesfaster kinetics. In this pathway, vesicles contact presyn-aptic membranes and create transient pores for neuro-transmitter release, but do not fully collapse (Ceccarelliet al., 1973; Alabi and Tsien, 2013). The connection be-tween these two forms of synaptic vesicle recycling is stillbeing explored; it seems that stimulation intensity andCa2+ levels may induce shifts from one form to the other(Gandhi and Stevens, 2003; Zhang et al., 2009a; Leitz andKavalali, 2011, 2014). In both models, the tight couplingbetween exocytosis and endocytosis points to the fusionmachinery itself as a keymediator of the balance betweenexo- and endocytosis (Deak et al., 2004).

A. Modes of Synaptic Vesicle Exocytosis

1. Synchronous Fusion. Molecularly, the best-characterized pathway of vesicle fusion is synchronousfusion (Südhof, 2013). Vesicles fuse and neurotransmit-ters are released in a precise time-locked manner withstimulation and ensuing Ca2+ influx. This rapid and re-liable exocytosis depends on many complex protein andlipid interactions. Soluble N-ethylmaleimide–sensitivefactor attachment protein receptor (SNARE) proteinsand its binding partners are essential for this fastexocytosis. The canonical SNARE complex is composedof synaptobrevin (syb)2, on the synaptic vesicle, andsyntaxin-1 and SNAP-25, both on the target plasmamem-brane (see reviews: Südhof, 2004; Rizo and Rosenmund,2008; Südhof and Rothman, 2009). The a-helical SNAREmotifs of these proteins facilitate the formation of a tightcomplex that brings vesicles close to the presynap-tic membrane. Munc18-1 is a Sec1/Munc18 proteinessential for neurotransmitter release (Verhage et al.,

Fig. 1. Presynaptic vesicle fusion machinery. An overview of molecules involved in synaptic vesicle exocytosis. Various synaptic vesicle-bound proteinsinteract with cytosolic modulators as well as presynaptic membrane-bound target proteins. The SNARE complex forms the central mechanism forvesicle fusion by bringing vesicles and the presynaptic membrane together, while a whole host of other molecular players are involved in mediating thisprocess. The diversity of vesicle-associated proteins can allow for different effectors to specifically regulate synaptic vesicle fusion, subsequentneurotransmitter release, and downstream signaling. Levetiracetam targets synaptic vesicle-bound SV2, although its mechanism of action is not yetfully understood.

Presynaptic Drug Targets 143

2000). It interacts with syntaxin-1 and the SNAREcomplex to regulate SNARE complex assembly andconsequently synaptic vesicle exocytosis (Rizo andSüdhof, 2012). Complexins are small, hydrophilic pro-teins that bind with high affinity to assembled SNAREcomplexes via its a-helical motif (McMahon et al., 1995).Synaptotagmin 1 (Syt1) functions as the Ca2+ sensor forsynchronous neurotransmission by coupling Ca2+ influxwith SNARE-mediated SV fusion (Brose et al., 1992;Geppert et al., 1994a; Fernandez-Chacon et al., 2001).Ca2+ binding to syt1 promotes its interaction with thetarget-SNAREs (t-SNAREs), syntaxin-1 and SNAP-25,to facilitate membrane fusion and subsequent neuro-transmitter release (Chapman et al., 1995; Davis et al.,1999; Bai et al., 2004). Its function as a regulator ofendocytosis will be discussed below.2. Asynchronous Fusion. The precise molecular

mechanisms underlying asynchronous neurotransmitterrelease are unclear. Asynchronous neurotransmitter re-lease is kinetically delayed release, persisting afterstimulation-induced Ca2+ influx has ceased (Barrettand Stevens, 1972; Goda and Stevens, 1994). Duringtrains of action potentials, intracellular Ca2+ builds upand asynchronous release becomes more prominent(Hagler and Goda, 2001; Kirischuk and Grantyn, 2003;Wen et al., 2013). Asynchronous release also appears tobe more resistant to depression of evoked activity thansynchronous release and may serve to maintain longer-lasting tonic release (Lu and Trussell, 2000; Otsu et al.,2004; Iremonger and Bains, 2016).There are an increasing number of studies focusing on

asynchronous release and attempting to parse out itsphysiologic significance. The balance between synchro-nous and asynchronous release may change dependingon the output demands of different neuron types and atvarious developmental stages (Kaeser andRegehr, 2014).In some hippocampal interneurons, asynchronous vesiclefusion is the predominant form of neurotransmitterrelease (Lu and Trussell, 2000; Hefft and Jonas, 2005;Ali and Todorova, 2010; Daw et al., 2010). In corticalinterneurons, asynchronous release may play an impor-tant role in regulating epileptoform activity (Manseauet al., 2010; Jiang et al., 2012; Medrihan et al., 2015). Inexcitatory synapses, asynchronous release can generate

larger and prolonged postsynaptic responses and per-haps play a role in potentiation and plasticity (IremongerandBains, 2007; Peters et al., 2010; Rudolph et al., 2011).During development, asynchronous releasemay allow forbroadly tuned coincidence detection that becomes morenarrowly tuned inmature synapses for phase-locked high-fidelity synaptic transmission (Chuhma and Ohmori,1998). Regulation of asynchronous release has also beenobserved retrogradely as synapse-associated protein 97 inthe postsynapse can act through N-cadherin to enhancepresynaptic asynchronous release (Neff et al., 2009).

A major challenge in studying the underlying mech-anisms of asynchronous neurotransmission is the diffi-culty in separating it from itsmore dominant synchronouscounterpart. Recent work has identified potential molec-ular determinants involved inasynchronous release,whichhas allowed a better definition of this process beyond asimple kinetic distinction. Although vesicle-SNARE(v-SNARE) syb2 is involved in rapid Ca2+-dependentsynchronousneurotransmission, vesicle-associatedmem-brane protein 4 (VAMP4) seems to selectively maintainbulk Ca2+-dependent asynchronous release (Raingoet al., 2012). VAMP4 did not show robust traffickingunder resting conditions, although it was shown thatVAMP4-enriched vesicles can respond to elevated pre-synaptic Ca2+ signals and promote release (Raingo et al.,2012; Bal et al., 2013). In addition, syt7 has recentlyemerged as a key Ca2+-sensing synaptic protein thatmaintains asynchronous neurotransmitter release in-dependently of syt1 (Wen et al., 2010; Bacaj et al., 2013;Jackman et al., 2016).

3. Spontaneous Fusion. Spontaneous neurotrans-mitter release was originally thought to occur due torandom low-probability conformational changes in thevesicle fusion machinery. However, accumulating evi-dence suggests that spontaneous release has specificmolecular determinants that distinguish it from actionpotential-driven release as well as divergent postsyn-aptic effects (Ramirez and Kavalali, 2011; Kaeser andRegehr, 2014; Kavalali, 2015). One form of segregationoccurs at the v-SNARE level as spontaneous fusion canpersist in the absence of syb2 (Deitcher et al., 1998;Schoch et al., 2001; Deak et al., 2004; Sara et al., 2005).The specific molecular mechanisms that underlie the

Fig. 2. Presynaptic vesicle retrieval machinery. An overview of molecules involved in synaptic vesicle endocytosis. The relative lack of understandingof the mechanisms underlying synaptic vesicle retrieval is reflected by the paucity of molecular targets. Dynamin is well-established as beingresponsible for scission of nascent vesicles from the presynaptic membrane. The dephosphorylation of proteins by calcineurin is an importantregulatory step in synaptic vesicle endocytosis.

144 Li and Kavalali

segregation of the evoked and spontaneous neurotrans-mission are beginning to be elucidated (Hua et al., 2011;Ramirez et al., 2012; Bal et al., 2013). VAMP7 (alsoknown as tetanus-insensitive VAMP) and Vps10p-tail-indicator-1a (Vti1a; vesicle transport through interac-tion with t-SNAREs homolog 1A) have been identifiedas alternative v-SNAREs that drive spontaneous re-lease (Ramirez et al., 2012; Bal et al., 2013). Thesevesicular proteins tag vesicles that display divergenttrafficking activity from syb2-enriched vesicles andmayfunction to maintain separate vesicle populations thatrecycle independent of presynaptic action potentials.The Ca2+ sensitivity of spontaneous release is another

area of functional divergence from stimulation-dependentneurotransmitter release. Spontaneous release frequencyis much less dependent on changes in Ca2+ levels thanevoked release. The source of Ca2+ and its different iontransients also complicate efforts to investigate the Ca2+

dependence of spontaneous fusion. Both syt1 and cytosolicprotein doc2 have been proposed as Ca2+ sensors for spon-taneous release (Xu et al., 2009; Groffen et al., 2010).However, doc2 may also modulate spontaneous neuro-transmission through a Ca2+-independent mechanism(Pang et al., 2011). Molecular interactions of the V0a1subunit of the vacuolar-type ATPase may also regulateCa2+-dependent spontaneous release (Wang et al., 2014).Loss of complexin, a cytoplasmic protein that bindsSNARE complexes, results in increased spontaneousrelease (Huntwork and Littleton, 2007; Yang et al.,2013; Lai et al., 2014). This growing list of molecularplayers that regulate spontaneous neurotransmissionprovides specific molecular manipulations that can beused to selectively probe the mechanism and function ofspontaneous neurotransmission (Kavalali et al., 2011;Kavalali, 2015).There is also evidence that spontaneous release

distinguishes itself from evoked release in regard toits postsynaptic receptor targets. Selective blockade ofpostsynaptic N-methyl-D-aspartic acid (NMDA) anda-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA) receptors activated by spontaneously releasedglutamate does not affect receptor-mediated responsesafter evoked release (Atasoy et al., 2008; Sara et al.,2011; Reese and Kavalali, 2016). This suggests thatspontaneous and evoked release activate nonoverlap-ping populations of postsynaptic receptors. Spontane-ous release leads to distinct postsynaptic changes andplays a role in synaptic homeostasis and plasticity (Saraet al., 2005; Sutton et al., 2006; Atasoy et al., 2008;Nosyreva et al., 2013).4. Endocytic Pathways. Our relative lack of insight

into the mechanisms underlying endocytosis is due inpart to the technical challenges involved in studying themembrane retrieval process. Much of our understand-ing of exocytic pathways have come from electrophysi-ological patch-clamp experiments, which offer hightemporal resolution. However, this method does not

provide any information on endocytosis as it reports in-tegrated postsynaptic responses. Although direct mea-sures of membrane capacitance have provided usefulreal-time data on endocytosis, its application is limited tolarge synapses such as the calyx of Held (Sun and Wu,2001). An optical approach has proved important forvisualizing membrane trafficking in small central syn-apses by using fluorescent membrane dyes (Betz andBewick, 1992) and, more recently, genetically encodedpH-sensitive fluorescent proteins (Miesenbock et al., 1998).Tagging synaptic vesicle proteins with pH-sensitive greenfluorescent protein provides the ability to look at singlevesicle exo- and endocytic events (Balaji and Ryan, 2007;Leitz and Kavalali, 2011).

Clathrin-mediated endocytosis is a well-studied path-way of synaptic vesicle retrieval. It involves adaptorprotein recruiting clathrin triskelia, which link togetheraround budding vesicles. Accessory protein amphiphy-sin is also drawn to the nascent vesicle and recruitsdynamin, which pinches off the new vesicle via GTPhydrolysis (Schmid and McMahon, 2007). Clathrin-mediated endocytosis is characterized by well-definedmorphologicmarkers such as coated pits and endosomalintermediates. Kiss-and-run refers to an alternativefaster pathway of vesicle recycling during which synap-tic vesicles retain their identity and do not intermixwith the plasma membrane or endosomal compart-ments (Fesce et al., 1994). This pathway appears to beclathrin-independent and may not necessarily requirethe same fissionmachinery (Palfrey andArtalejo, 1998).However, limited experimental access to this processhasmade functional characterization and determinationof themolecularmechanism difficult. Bulk endocytosis isanother pathway of endocytosis that is observed afterhigh-frequency stimulation as it allows for the retrievalof vesicles from the excess presynaptic plasmamembrane(Cheung and Cousin, 2013). Deep plasma membraneinfoldings form characteristic intracellular endosome-like intermediates. It is dependent on activation ofcalcineurin, which dephosphorylates dynamin I as wellas clathrin (Takei et al., 1996; Ferguson et al., 2007; Wuet al., 2009a). In addition, recent studies have identified anew form of ultrafast endocytosis characterized withflash-and-freeze electron microscopy and membrane ca-pacitance measures (Watanabe et al., 2013; Delvendahlet al., 2016). This process is observed at physiologic tem-peratures (;37°C) and appears to bemediated by dynaminand actin but is clathrin-independent.

In loss-of-function experiments, compromising syn-aptic vesicle endocytosis machinery gives rise to rapiddepression and fatigue of synaptic responses duringhigh-frequency stimulation. For instance, imparing thefunction of dynamin—a GTPase that pinches synapticvesicles from the plasma membrane during synapticvesicle endocytosis—using theDrosophila temperature-sensitive dynamin mutant shibire resulted in a fastdecrease in neurotransmitter release with no detectable

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sustained release during activity (Delgado et al., 2000).Mouse hippocampal synapses deficient in Dynamin1 also showed strong synaptic depression presumablydue to loss of vesicle endocytosis and recycling (Fergusonet al., 2007). Furthermore, interference with dynaminSrc homology 3 domain interactions (Shupliakov et al.,1997), genetic deletion of synaptojanin 1, an abundantpresynaptic polyphosphoinositide phosphatase (Cremonaet al., 1999; Luthi et al., 2001), or manipulation ofdifferentially spliced isoforms of syt7 (Virmani et al.,2003) all lead to activity-dependent changes in the rate ofsynaptic depression. Although these observations sug-gest that recycled synaptic vesicles can be rapidly reusedduring activity (Pyle et al., 2000; Sara et al., 2002), theymay also indicate unavailability of release sites and sup-pression of exocytosis due to slow clearance of fused ves-icles as a consequence of impaired endocytosis (Kawasakiet al., 2000).The dephosphorylation–phosphorylation cycle of dyna-

min as well as other endocytic proteins has been shown tobe critical for their function in activity-dependent regula-tion of synaptic vesicle endocytosis (Robinson et al., 1994).In particular, the dephosphorylation of dynamin isthought to be a critical trigger for endocytosis duringactivity (Marks and McMahon, 1998). Molecular manip-ulations of the synaptic vesicle-recycling machinery areimportant in uncovering vesicle-trafficking mechanismsas well as providing an extremely valuable setting tostudy the kinetics and physiologic significance of synapticvesicle reuse during synaptic activity (Fig. 2). Further-more, recent studies have provided several examples inwhich synaptic vesicle-recycling process and subsequentdynamics of neurotransmitter release can be modulatedby small molecules that target dynamin-dependent endo-cytosis or myosin light chain kinase-dependent vesicletransport (Chung et al., 2010; Maeno-Hikichi et al., 2011;Linares-Clemente et al., 2015). From a neurotherapeuticsperspective, targeting the synaptic vesicle-recycling ma-chinery may present a key advantage as it inducesfrequency-dependent changes in the efficacy of neuro-transmission to counter or correct disease processes, incontrast to typical blockers of neurotransmission thattrigger global suppression or augmentation of neurotrans-mitter release that may potentially yield broader sideeffects (Kavalali, 2006).

B. Vesicular Heterogeneity

Accumulating evidence indicates that presynapticterminals contain a molecularly heterogeneous popula-tion of vesicles that drive distinct forms of neurotrans-mission with different Ca2+ dependence and divergentexo- and endocytic kinetics (Rizzoli and Betz, 2005; Saraet al., 2005; Fredj and Burrone, 2009). The route ofsynaptic vesicle recycling may differentially affectneurotransmission by generating vesicles with diver-gent propensities for fusion (Virmani et al., 2003;Voglmaier et al., 2006; Kavalali, 2007; Clayton et al.,

2010). These vesicles are released in one of the differentmodes (synchronous, asynchronous, or spontaneous) andgo through different routes of endocytosis that may leadto segregation into distinct vesicular pools (Crawford andKavalali, 2015). Specific targeting of vesicular proteinsmay enable it to selectively modulate different forms ofrelease pharmacologically (Figs. 1 and 2).

III. Fusion Machinery

A. SNARE Proteins

SNARE proteins are a large family of proteins in-volved in intracellular vesicle trafficking and secretion.In neurons, the canonical SNARE complex, consisting ofthe synaptic vesicle protein syb2 and the presynapticplasma membrane-associated proteins SNAP-25 andsyntaxin-1, mediates synaptic vesicle exocytosis. As-sembly of the SNARE complex is aided by a number ofcritical priming factors and provides a key substratethat the synaptic vesicle and presynaptic membraneclose together, which allows for vesicle fusion (Südhof,2004). Deletion of syb2 or SNAP-25 in mice leads toseverely impaired neurotransmission and lethality atbirth (Schoch et al., 2001; Washbourne et al., 2002).

SNAREs are the targets of bacterial clostridial neuro-toxins, which inhibit neurotransmission and are respon-sible for botulismand tetanus (see discussion inCleavageof SNAREs byClostridial Toxins). Although the essentialnature of the synaptic SNARE proteins in neurotrans-mission limits their physiology, there are few severe loss-of-function mutations in human disease. Studies showaltered expression of syb2 in Alzheimer disease (AD)and Huntington’s disease (Shimohama et al., 1997;Morton et al., 2001). Deletion of the SNAP-25 gene inmice results in a hyperactive phenotype similar to atten-tion deficit hyperactivity disorder (Hess et al., 1996;Brophy et al., 2002). Many studies have shown DNAvariations in the SNAP-25 gene associated with at-tention deficit hyperactivity disorder (Brophy et al.,2002; Mill et al., 2002; Hawi et al., 2013). SNAP-25reductionwasalso observed in thehippocampi ofpatientswith schizophrenia (Thompson et al., 2003). Single-nucleotide polymorphisms in syntaxin-1a associatedwith schizophrenia (Wong et al., 2004). SNARE complexassembly is impaired in human brain tissue from pa-tients with AD and Parkinson’s disease (Sharma et al.,2012). Importantly, SNAREs can be a key point of vul-nerability in maintenance of synaptic proteostasis, assustained activity requires continual assembly anddisassembly of SNARE complexes, which in the absenceof chaperone activity may yield accumulation of mis-folded proteins (Chandra et al., 2005).

B. Cleavage of SNAREs by Clostridial Toxins

Anaerobic Clostridium bacteria produce potent neu-rotoxins, including several botulinum toxins and teta-nus toxin. The toxins cleave SNARE proteins involved

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in synaptic vesicle fusion and abolish neurotransmitterrelease (Schiavo et al., 2000). Botulinum toxins are takenup by motor neurons and block acetylcholine release atneuromuscular junctions and cause skeletal muscleweakness. Tetanus toxin is preferentially targeted tospinal cord interneurons, where it blocks the release ofinhibitory neurotransmitters to cause hyperexcitation ofskeletalmuscle and tetanic contractions as a result of theloss of synaptic inhibition on spinal motor neurons. Thedifferent serotypes A–G of botulinum toxin proteolyzedifferent components of the SNARE complex (Jahn andNiemann, 1994).Botulinum toxin injections have a long history of

being used for a variety of muscle disorders, includingstrabismus, blepharospasm, hemifacial spasm, cervicaldystonia, and cosmetic procedures (Jankovic, 2004).Recently, there has been more evidence suggesting thatthe action of botulinum toxin is not limited to the periph-eral nervous system. In addition to acting directly at theneuromuscular junction, peripheral botulinum toxin Ainjections may also alter sensory inputs to the CNS byindirectly inducing secondary central changes (Curra et al.,2004). Botulinum toxin A is retrogradely transported bycentral neurons and transcytosed to afferent synapses,cleaving SNAP-25 in the contralateral hemisphere afterunilateral botulinum toxin A delivery, demonstrating long-distance retrograde effects of botulinum toxin A (Antonucciet al., 2008).Although botulinum toxin can cause a deadly disease,

utilization of certain serotypes has therapeutic poten-tial. Intrahippocampal injection of botulinum toxin Eresulted in inhibition of seizure activity in rat models ofepilepsy (Costantin et al., 2005; Bozzi et al., 2006).Botulinum toxin A2 reduces incidence of seizures inmouse models of temporal lobe epilepsy (Kato et al.,2013). Botulinum toxin A2 injection into rat striatumameliorates pathologic behavior in a rat Parkinson’sdisease model (Itakura et al., 2014). In contrast, ratintrahippocampal infusion of botulinum toxin B isproconvulsant and can be used as a focal epilepsymodel.This may be due to serotype B targeting syb2 andinhibiting GABA release (Broer et al., 2013). The differ-ent botulinum serotypes have different receptor andaffinities that determine neuronal specificity and canbe used for therapeutic benefit. The nontoxic recombi-nant heavy chain of botulinum neurotoxin A coupled todextran could be an efficient drug delivery vehicle forbotulism countermeasure. The atoxic part helps to targetbotulinum neurotoxin-sensitive cells and promotes in-ternalization of the complex (Zhang et al., 2009b).Tetanus toxin binds to peripheral neurons and un-

dergoes retrograde and trans-synaptic transfer to centralinhibitory neurons, where it cleaves syb2, and blocksneurotransmitter release. Tetanus toxin entry is medi-ated through synaptic vesicle glycoprotein 2 (SV2) bind-ing and synaptic vesicle endocytosis (Matteoli et al.,1996; Yeh et al., 2010). This endogenous pathway can be

exploited to bypass the blood brain barrier for drugdelivery. The nontoxic C-terminal domains of tetanustoxin can be used to penetrate the nervous system; afragment of tetanus toxin has been shown to inhibit 5-HTreuptake and prevent serotonin from being transportedthrough the presynaptic membrane (Najib et al., 2000).Recombinant atoxic mutants, made by Escherichia coli,can be engineered quickly and efficiently as usefulvehicles for delivery to central neurons (Li et al., 2001).Using protein stapling technology, a chimera of botulinumneurotoxin A and the tetanus binding domain was cre-ated to specifically target CNS function and spare theneuromuscular junction (Ferrari et al., 2013). By limitingmuscle paralysis, this may be useful for the futuretreatment of epilepsy or chronic pain.

C. Effects of Other Drugs on SNARE Proteins

Small cell-permeable peptides patterned after the Nterminus domain of SNAP-25 inhibit SNARE complexassembly and regulate exocytosis. They protected againstglucose deprivation–induced neurodegeneration and mayattenuate dysfunctional exocytosis (Blanes-Mira et al.,2003). Plant extracts andpeptide library screens are beingused to identify compounds that alter SNARE complexformation (Blanes-Mira et al., 2004; Riley et al., 2006;Jung et al., 2009). An extract fromAlbizzia julibrissinwasfound to reduce the level of SNARE complex formation;further studies are required to determine whether this isresponsible for the observed effects ofA. julibrissin extractin alleviating stress, insomnia, and depression (Rileyet al., 2006). It did produce anxiolytic effects in elevatedplus maze in rats, possibly due to upregulation of 5-HT1Areceptors (Kim et al., 2004; Jung et al., 2005).

D. SNARE-Associated Proteins

1. Munc18-1 (STXBP1). The efficient functioning ofSNARE complexes in synaptic transmission relies oninteractions with a variety of other proteins. Munc18-1is a neuron-specific protein of the Sec1/Munc18-like fam-ily of membrane-trafficking proteins. It serves as a keycomponent of the synaptic vesicle fusion machinery, asdeletion ofMunc18-1 leads to complete loss of neurotrans-mitter release (Verhage et al., 2000). Munc18-1 binds tothe closed form of syntaxin-1 and blocks SNARE complexformation. Syntaxin-1 is opened byMunc13, andMunc18-1 translocates to bind the formed SNARE complex andprevent its dissociation (Rizo and Südhof, 2012).

Rare mutations in the gene that encodes Munc18-1,syntaxin-binding protein 1 (STXBP1), have been iden-tified in patients with various types of epilepsy. Loss-of-function heterozygous de novo mutations in STXBP1have been linked to neonatal focal seizures, early onsetepileptic encephalopathy with suppression bursts, andinfantile spasms (Vatta et al., 2012; Barcia et al., 2014).Some of the patients also have mental retardation,ataxia, and dyskinetic movements (Deprez et al., 2010).Treatment with levetiracetam in patients effectively

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managed seizures that were refractory to other antiep-ileptic drugs (Vatta et al., 2012; Dilena et al., 2016). Thismay be due to levetiracetam’s unique mechanism of ac-tion involving SV2A. Modulation of SV2A may compen-sate for the haploinsufficiency of STXBP1 by affectingthe synaptic vesicle-recycling pathway thatMunc18-1 ispart of. The interaction between levetiracetam andSV2A will be discussed in more detail below; however,more research is needed to further explore the specificmechanism of action of levetiracetam in Munc18-1–related epilepsies.Development of a protein–protein interface inhibitor

is a potential therapy for STXBP1 haploinsufficiency-associated epileptic disorders. Advances in our struc-tural understanding of SNARE protein complexes anddrug design techniques have paved the way for target-ing Munc18-1–binding partners (Hussain, 2014). How-ever, understanding Munc18-1’s specific function in thepathophysiology of these epilepsies would be a neces-sary first step.2. Synaptotagmin. Syts are a family of integral

membrane proteins with two calcium binding domains,C2A and C2B. Syt1 is an abundant isoform that is local-ized to synaptic vesicles and serves as a calcium sensorfor vesicle exocytosis. It is required for the calciumtriggering of synchronous neurotransmitter release, butis not essential for asynchronous release (Brose et al.,1992; Geppert et al., 1994a). Calcium binding to syt1promotes its interaction with the t-SNAREs, syntaxin-1and SNAP-25, and to phospholipids, thereby facilitatingsynaptic vesicle and presynaptic membrane fusion(Davis et al., 1999; Bai et al., 2004; Pang et al., 2006).Recently, a de novo syt1 missense mutation has been

identified in an individual with severe motor and cog-nitive impairments (Baker et al., 2015). Expression ofthis mutant syt1 in mouse neurons revealed slowedsynaptic vesicle fusion kinetics and faster endocytosis.Therefore, syt-specific protein–protein interaction in-hibitors may be an effective way to target the synchro-nous neurotransmitter release pathway selectively.3. Complexin. Complexin I and II are highly homol-

ogous small, hydrophilic proteins enriched in neurons.They bind with high affinity to assembled SNAREcomplexes via an a-helical motif (McMahon et al.,1995). Deletion of complexin I and II in mouse neuronresults in reduced neurotransmitter release efficiency(Reim et al., 2001). Complexin is an important regulatorof synaptic vesicle exocytosis; however, the specificmolecular mechanism of complexin function remainscontroversial. It appears to have a dual function as botha promoter and inhibitor of vesicle fusion (Xue et al.,2010; Yang et al., 2010).Changes in complexin I and II expression are seen in

several neurodegenerative and psychiatric disorders. Pro-gressive and selective loss of complexin II was observed ina transgenic mouse model for Huntington’s disease(Morton and Edwardson, 2001). Expression of mutant

huntingtin caused a decrease in complexin II levels anddefects in neurotransmission, which were rescued byoverexpression of complexin II in vitro (Edwardsonet al., 2003). Additionally, decreases in complexin I andIIwere observed in postmortembrain tissue frompatientswith AD, schizophrenia, and bipolar disorder (Harrisonand Eastwood, 1998; Tannenberg et al., 2006).

Complexin’s important role in regulation of synapticvesicle fusion and its association with a multitude ofneurologic andpsychiatric disordersmake it an attractivetarget for pharmacotherapy. Manipulating phosphoryla-tion of complexinmay be oneway tomodulate its functionin synaptic vesicle fusion. In vitro phosphorylation ofcomplexin I and II by protein kinase CK2 has been shownto enhance complexin binding to SNARE complexes(Shata et al., 2007). Activity-dependent phosphorylationof complexin by protein kinase A (PKA) enhances spon-taneous neurotransmitter release and affects synapticstructural plasticity in Drosophila (Cho et al., 2015).

E. Noncanonical SNAREs

Vesicle molecular heterogeneity makes it possible totarget different vesicular proteins to selectively regu-late spontaneous or asynchronous neurotransmitterrelease without significantly altering fast synchronousneurotransmitter release. Fast synchronous release iscritical for information coding and processing in thebrain; any manipulation sparing this type of synaptictransmissionwould be expected to have fewer side effectscompared with changes of global regulation of neuro-transmission (Ramirez and Kavalali, 2012; Crawfordand Kavalali, 2015). This has important implications forthe development of novel treatment strategies as sug-gested by recent work implicating spontaneous neuro-transmission inmediating the fast antidepressant effectsof NMDA receptor antagonists (Autry et al., 2011).

1. Vti1a. Vti1a is localized to synaptic vesicles andparticipates in a novel SNARE complex, independent ofsyntaxin-1 and SNAP-25 (Antonin et al., 2000). Vti1acoimmunoprecipitates with VAMP4, syntaxin-6, andsyntaxin-16 (Kreykenbohm et al., 2002). Vti1a exhibitsrobust trafficking under resting conditions, and loss ofvti1a function selectively reduced high-frequency spon-taneous release. Taken together, these data suggestthat vti1a is localized to a vesicle pool that maintainsspontaneous neurotransmission (Ramirez et al., 2012).

2. VAMP7. VAMP7, also known as tetanus toxin–insensitiveVAMP, formsSNAREcomplexeswithSNAP-23and syntaxin-3 (Coco et al., 1999). VAMP7 is localizedto synaptic vesicles that recycle spontaneously but areunresponsive to stimulation (Hua et al., 2011). Micelacking VAMP7 do not exhibit any striking develop-mental or neurologic defects, but behavioral charac-terization revealed an increased anxiety phenotype(Danglot et al., 2012).

The heterogeneity of synaptic vesicle-associatedSNAREs allows for the selective modulation of action

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potential-independent neurotransmission, providing anattractive target for future therapeutic development.Glycoprotein reelin selectively augments spontaneoussynaptic transmission bymobilizing a VAMP7-dependentvesicle pool (Bal et al., 2013). However, VAMP7 is not adirect target of Reelin, as the effect requires calcium,phosphatidylinositol 3-kinase, apolipoprotein E receptor2 (ApoER2), and very-low-density-lipoprotein receptor(VLDLR). As we begin to elucidate the molecular mech-anisms underlying these different vesicle pools, we candevelop new tools for specific manipulation of differentforms of neurotransmitter release.

IV. Synaptic Vesicle Endocytosis

A. Synaptotagmin

Syt has a recognized role as the calcium sensor for fastsynaptic vesicle exocytosis, but recent evidence impli-cates its role in endocytosis. Biochemical experimentsshow that syt1 interacts with a variety of endocytosis-associated proteins, including clathrin adaptor protein(Zhang et al., 1994; Haucke and De Camilli, 1999; vonPoser et al., 2000). Multiple syt1 loss-of-functionmodelsshow impaired endocytosis after stimulation (Marekand Davis, 2002; Poskanzer et al., 2003; Nicholson-Tomishima and Ryan, 2004; Yao et al., 2011).Various studies have shown changes in syt1 expres-

sion in animal stroke models; however, it is unclear howsyt1 is involved in the pathophysiology of stroke-relatedbrain injury (Yokota et al., 2001; Chen et al., 2013a).In vivo knockdown of syt1 in a rat model of ischemicstroke prevented much of the ischemic damage ofhippocampal neurons, making syt1 an attractive targetfor neuroprotective therapy (Iwakuma et al., 2003). Thiseffect may be related to syt1’s exocytic function contrib-uting to excitotoxicity; however, the endocytic functionof syt1 in relation to themassive increase in presynapticcalcium may also play a role. Specific deletion of syt1and other endocytic machinery components such asAP-2 and dynamin protected Caenorhabditis elegansneurons from hypoxia-induced necrotic cell death(Troulinaki and Tavernarakis, 2012). Neuronal endo-cytic pathways are clearly disrupted in stroke models,and further investigation is needed to understand howthis pathway functions in both normal and diseasestates (McColl et al., 2003; Vaslin et al., 2007).

B. Calcineurin

Calcineurin is a calcium/calmodulin-dependent pro-tein phosphatase that regulates synaptic transmissionand plasticity (Rusnak and Mertz, 2000; Groth et al.,2003). Calcineurin dephosphorylates a set of proteins,in a calcium-dependent manner, involved in synap-tic vesicle endocytosis, including dynamin, amphiphy-sin, and synaptojanin (Cousin and Robinson, 2001).Calcineurin inhibitors abolish synaptic vesicle endocy-tosis (Marks and McMahon, 1998; Cousin et al., 2001).

Calcineurin inhibitor, FK506, treatment increases den-dritic branching and dendritic spine density and ame-liorates dendritic spine loss in an Alzheimer mousemodel in mouse brains (Rozkalne et al., 2011; Spires-Jones et al., 2011).

Schizophrenia is associated with a genetic variationin the 8p21.3 gene, PP3CC, which encodes the calci-neurin g subunit, leading to decreased calcineurinexpression (Gerber et al., 2003; Eastwood et al., 2005).Calcineurin knockout (KO) mice exhibit multiple ab-normal behaviors related to schizophrenia, such asincreased locomotor activity, decreased social interac-tion, and working memory impairment (Zeng et al.,2001; Miyakawa et al., 2003; Cottrell et al., 2013).Biochemically, there is increased hyperphosphorylationof synaptic vesicle-recycling proteins known to benecessary for high-frequency firing. These findingssupport a model in which impaired synaptic vesiclerecycling represents a critical node for disease pathol-ogies underlying the cognitive deficits in schizophrenia.

The current Food and Drug Administration–approvedcalcineurin inhibitors, FK506 and cyclosporine A, areused for immunosuppression. Meta-analysis of treat-ment of FK506 as a neuroprotective drug in animalmodels of stroke suggests that it is effective and safe(Macleod et al., 2005). However, these existing drugsare nonspecific, and long-term use has undesirableside effects. Therefore, new drugs need to be developedwith better blood brain penetration and higher spec-ificity. High-throughput screens for novel calcineurininhibitors are already underway (Margassery et al.,2012; Mukherjee et al., 2015). There is also a pat-ent filed for identifying calcineurin activators for thediagnosis and treatment of schizophrenia (Gerberet al., 2011).

C. Dynamin I

Dynamin is a GTPase that is important for endocyticmembrane fission in eukaryotic cells. Dynamin I and IIIare predominantly expressed in the brain, whereasdynamin II is ubiquitously expressed (Ferguson andDe Camilli, 2012). Dynamin 1 KO mice appear normalat birth, but die within 2 weeks. Synaptic vesicleendocytosis was severely impaired during strong stim-ulation, but resumed efficiently after the end of stimu-lation (Ferguson et al., 2007). Dynamin III KO mice donot have an obvious pathologic phenotype, but dynaminI and III KO mice have a more severe phenotype thanthe dynamin I KO mice (Raimondi et al., 2011). In-hibitory neurons appear to be more sensitive to the lossof dynamin 1 and experience endocytic defects. Silenc-ing transmission relieves the endocytic defect, suggest-ing that the high intrinsic level of tonic activity ininhibitory neurons makes themmore vulnerable to lackof dynamin 1 (Hayashi et al., 2008).

In humans, de novo mutations in dynamin 1 havebeen shown to cause epileptic encephalopathies

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(EuroEPINOMICS-RES Consortium et al., 2014). Ex-pression of these epileptic encephalopathy-causingdynamin 1 mutations in vitro leads to decreased endo-cytosis activity (Dhindsa et al., 2015). Spontaneousdynamin 1 missense mutation in mice confers seizuresusceptibility that could arise from greater sensitivity ofGABAergic interneurons to endocytic perturbations.The mutant dynamin 1 does not efficiently self-assembleand results in defective synaptic vesicle recycling andslower recovery from depression after trains of stimula-tion (Boumil et al., 2010). Furthermore, this mutationdifferentially affects splice variants of dynamin, interfer-ing with the function of dynamin 1a, but upregulating theexpression of dynamin 1b, providing an additional layer ofcomplexity to future therapeutic modulation of dynaminfunction (Asinof et al., 2016). In another study, upregula-tion of dynamin 1 was observed in an acute seizure modelin rats and in human patients with temporal lobe epi-lepsy. Pharmacological inhibition of dynamin 1 in the ratmodel decreased the frequency and severity of seizures,suggesting a potential role for dynamin modulators inseizure treatment (Li et al., 2015). More investigation isneeded to reconcile the apparent differences of dynaminfunction in the etiology of epilepsy. Altered dynamin ex-pression has also been detected in schizophrenia patients(Pennington et al., 2008; Focking et al., 2011; Cottrellet al., 2013).There are many new classes of dynamin inhibitors

being developed that represent a potential field for an-tiepileptic drug development (Gordon et al., 2013;McCluskey et al., 2013; McGeachie et al., 2013; Mac-Gregor et al., 2014; Robertson et al., 2014; Abdel-Hamidet al., 2015). Practically, these agentsmay need to act onspecific isoforms or specifically target excitatory synap-ses and minimize effects on inhibitory interneurons.There is also a patent application formethods and agentsthat inhibit dynamin-dependent endocytosis for the de-velopment of treatment of epilepsy and other neurologicdisorders (Hill et al., 2005). Control of dynamin phos-phorylation may be a potential method of modulatingsynaptic vesicle endocytosis. Dephosphomimeticmutantsof dynamin 1 regulate activity-dependent acceleration ofendocytosis, providing a possible target for therapeu-tic intervention (Armbruster et al., 2013). The futurechallenge will be to ensure the specificity of action andlimit side effects as dynamin I is involved in many cellu-lar processes separate from its role in synaptic vesicleendocytosis.

D. Amphiphysin

Amphiphysin I is a hydrophilic phosphoprotein abun-dant in presynaptic terminals that binds various endocyticproteins and is involved in regulating clathrin-mediatedendocytosis (Lichte et al., 1992; Wu et al., 2009b). Amphi-physin I KO mice have learning deficits and increasedsusceptibility to seizures. Neurons of these mice onlyrevealed defects under stimulated conditions where

inhibition of synaptic vesicle endocytosis was observed(Di Paolo et al., 2002).

Autoantibodies against amphiphysin and glutamicacid decarboxylase cause stiff-person syndrome, whichis characterized by progressive stiffness and musclespasms (De Camilli et al., 1993). Inhibitory GABAergicsynapses are more vulnerable than glutamatergic syn-apses to the impaired clathrin-mediated endocytosisinduced by anti-amphiphysin antibodies (Geis et al.,2010; Werner et al., 2016).

Phosphorylation of amphiphysin I by casein kinase2 and minibrain kinase/dual-specificity tyrosinephosphorylation-regulated kinase (Mnb/Dyrk1A) in-hibits its binding to other proteins involved in endocy-tosis, clathrin, and endophilin, respectively (Doringet al., 2006; Murakami et al., 2006). High-frequencystimulation reduces phosphorylation at the Mnb/Dyrk1A site, demonstrating a possible mechanism foractivity-dependent suppression of amphiphysin func-tion. Ubiquitous protease calpain cleaves amphiphysinI after strong stimulation, resulting in nonfunctionaltruncated amphiphysin I and inhibited synaptic vesicleendocytosis (Wu et al., 2007). Calpain-dependent cleav-age of amphiphysin I attenuated kainate-induced sei-zures in mice by inhibiting excessive excitatory output.Existing kinase and calpain inhibitors can be used forfurther research into their potential therapeutic use.

E. Synaptojanin

Synaptojanin is a presynaptic lipid phosphatase thatdephosphorylates phosphatidylinositol (3,4,5)-triphos-phate to phosphatidylinositol (4,5)-biphosphate (Guoet al., 1999). Its C-terminal domain interacts with manyproteins involved in clathrin-mediated endocytosis. Ge-netic disruption of synaptojanin 1 leads to stimulation-dependent accumulation of clathrin-coated vesicles,implicating synaptojanin 1 in the dissociation of clathrinadaptors and the uncoating of nascent synaptic vesicles.Synaptojanin 1–deficient mice exhibit neurologic defectsand die shortly after birth. The functional recycling poolis also smaller in synaptojanin 1–deficient neurons;synaptic depression was enhanced during high-frequencystimulation, and recovery was delayed (Cremona et al.,1999; Kim et al., 2002).

Changes in synaptojanin 1 expression have beenlinked to Down’s syndrome and AD (Voronov et al.,2008; Di Paolo and Kim, 2011; Martin et al., 2014).Mutations in synaptojanin have also been linked tobipolar disorder and autosomal recessive, early-onsetParkinsonism (Saito et al., 2001; Krebs et al., 2013;Quadri et al., 2013).

Mnb/Dyrk1Aphosphorylation of synaptojanin1altersits binding to the Src homology 3 domains of amphi-physin, intersectin, and endophilin (Bauerfeind et al.,1997). Mnb/Dyrk1A is encoded in the Down syndromecritical region of chromosome 21 (Shindoh et al., 1996;Song et al., 1996) and is an excellent target for future

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drug development. Reduction of synaptojanin 1 amelio-rates synaptic and behavioral impairments in a mousemodel of AD (McIntire et al., 2012). A screening assayfor small-molecule inhibitors of synaptojanin I demon-strates that its pharmacological potential is alreadybeing explored (McIntire et al., 2014).

V. Stability and Maintenance of the SynapticVesicle-Recycling Machinery

A. Cysteine-String Protein a

Cysteine-string protein (CSP)a is predominately lo-calized to synaptic vesicles (Mastrogiacomo et al., 1994).CSPa associates with ATPase heat shock protein 70(hsc70) and small glutamine-rich tetratricopeptiderepeat-containing protein to form a complex that func-tions as a synaptic chaperone (Tobaben et al., 2001).This CSPa-hsc70-small glutamine-rich tetratricopeptiderepeat-containing protein complex binds toSNAP-25 andprevents aggregation to enable SNARE complex assem-bly. Deletion of CSPa results in structurally abnormalSNAP-25 that inhibits SNARE complex formation and ismore rapidly degraded (Sharma et al., 2011). CSPa KOmice begin to exhibit locomotor defects at about 2–3weeksof age anddonot survive beyond3months. Their synapsesshow major changes in structure consistent with a pre-synaptic degenerative process (Fernandez-Chacon et al.,2004). This activity-dependent neurodegeneration ismoresevere inGABAergic synapses and can be partially rescuedby decreasing network excitability with pharmaco-logical blockers of glutamatergic receptors (García-Junco-Clemente et al., 2010). Impaired SNARE complexassemblymay cause neurodegeneration, not via a decreasein neurotransmitter release, but because the impairmentin SNARE complex assembly leads to an excess of reactivesyntaxin-1 and syb2 that do not participate in SNAREcomplexes (Sharma et al., 2011).Heterozygousmutations in the CSPa gene in humans

cause autosomal-dominant adult-onset neuronal ceroidlipofuscinosis, a neurodegenerative disorder character-ized by lysosomal accumulation of misfolded proteins(Noskova et al., 2011). A palmitoyltransferase inhibitorcould be a possible way to regulate CSPa function aspalmitoylation of CSPa increases aggregation seen inneuronal ceroid lipofuscinosis (Greaves et al., 2012).Quercetin, a plant flavonoid, targets the unique cyste-

ine string region of CSPa and impairs synaptic trans-mission (Xu et al., 2010). Quercetin promotes formation ofstable CSPa dimerization and inhibits assembly of theactive chaperone complex by reducing hsc70 association.The next step would be to identify other compounds thattake advantage of the unique binding site to enhancerather than inhibit CSPa function.

B. Synucleins

Synucleins are a family of small, soluble proteins pri-marily expressed in neural tissue with a highly conserved

amphiphilic a-helical lipid-binding motif (George, 2002).a-Synuclein protein is localized to presynaptic terminalsand nuclei (Maroteaux et al., 1988). a-Synuclein bindsdirectly to syb2 and promotes SNARE complex assembly(Burre et al., 2010). a-Synuclein KOmice have functionaldeficits in the nigrostriatal dopamine system, resulting inincreased dopamine release and reduced reserve pool size(Abeliovich et al., 2000; Cabin et al., 2002). Behaviorally,the a-synuclein KO mice have cognitive impairmentsand reduced working and spatial memory (Kokhan et al.,2012). Overexpression of a-synuclein, below toxic levels,decreased the readily releasable pool size and led to de-creased neurotransmitter release (Nemani et al., 2010).a-Synuclein’s function may be to maintain vesicle poolhomeostasis by regulating the size of synaptic vesiclepools (Cabin et al., 2002; Nemani et al., 2010; Scott andRoy, 2012). a-Synuclein may also promote clathrin-mediated endocytosis and regulate the kinetics of syn-aptic vesicle endocytosis (Ben Gedalya et al., 2009;Vargas et al., 2014). a-Synuclein can also inhibit vesiclemembrane fusion independent of SNARE proteinsthrough direct interactions with lipid bilayers (Darioset al., 2010; DeWitt and Rhoades, 2013)

Mutations in a-synuclein are associated with rarefamilial and sporadic forms of Parkinson’s disease.Toxic a-synuclein accumulates abnormally in Parkinson’sdisease, AD, dementia with Lewy bodies, and otherneurodegenerative diseases (Goedert, 2001; Stefanis,2012). The functional role of a-synuclein in the pathogen-esis of these neurodegenerative diseases is unclear. Thereis growing evidence to suggest the a-synuclein–mediatedaberrant synaptic vesicle recycling precedes overt neuro-pathology and may contribute to the pathophysiology ofthese a-synucleinopathies (Galvin et al., 2001; Nakataet al., 2012; Yasuda et al., 2013).

a-Synuclein ser129 is phosphorylated by many differ-ent kinases (Okochi et al., 2000; Pronin et al., 2000; Ishiiet al., 2007; Inglis et al., 2009). Experiments in yeastshow that blocking a-synuclein phosphorylation signif-icantly increased trafficking defects and a-synucleintoxicity (Sancenon et al., 2012). This hints at a role fordynamic phosphorylation on a-synuclein’s function insynaptic vesicle recycling and disease progression. Asmall-molecule screen yielded a compound that targetsvesicle-bound a-synuclein and inhibits its aggregation(Fonseca-Ornelas et al., 2014). It is yet to be seen howthis affects a-synuclein’s effect on synaptic vesiclerecycling; however, similar methods can be used to findother modulations of a-synuclein function.

VI. Active Zone Proteins

A. Munc13

Munc13, a homolog of unc-13 in C. elegans, is a largeprotein localized to presynaptic terminals (Brose et al.,1995). Deletion of munc13-1, the most abundant iso-form, in mice leads to signification reductions in the

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readily releasable pool size and neurotransmitter re-lease in glutamatergic neurons only (Richmond et al.,1999; Varoqueaux et al., 2002). Munc13-1 interacts withthe N terminus of syntaxin and primes synaptic vesiclesfor exocytosis (Betz et al., 2001). Munc13 also interactswith Rab3-interacting molecules (RIM) to tether andprime synaptic vesicles and modulate long-term synap-tic plasticity (Betz et al., 2001; Yang and Calakos, 2011).In synapses, munc13-1 is a key target for diacylgly-

cerol signaling in addition to the well-described impactof this signaling pathway on protein kinase C activity(Brose and Rosenmund, 2002). In experimental set-tings, this function of munc-13 is typically probed withapplication of phorbol esters as phorbol ester binding (tothe C1 domain) induces membrane association andenhances neurotransmitter release (Betz et al., 1998).

B. Rab3-Interacting Molecule

RIM are large scaffolding proteins localized to pre-synaptic active zones. The major isoforms RIM1a andRIM2a are made up of an N-terminal zinc-finger do-main, a central PDZ domain, and multiple C2 domains,allowing for binding to a multitude of synaptic proteins(Südhof, 2004). The N-terminal of RIM1a binds to syn-aptic vesicle GTPase rab3 and active zone proteinMunc13 to regulate neurotransmitter release (Wanget al., 1997; Betz et al., 2001; Schoch et al., 2002). TheGTP-dependent interaction between rab3A and theN-terminal of RIM1a is implicated in tethering of syn-aptic vesicles at the active zone (Wang et al., 1997). RIMinteraction with munc13 is thought to be critical forsynaptic vesicle priming (Betz et al., 2001). RIM1a KOmice have defects in synaptic transmission, short-termsynaptic plasticity, and long-term synaptic plasticity inthe hippocampus (Castillo et al., 2002; Schoch et al.,2002). In addition to their key role in synaptic vesiclepriming, RIM proteins also form a close synapticvesicle–voltage-gated Ca2+ channel scaffold to ensurerapid release (Kaeser et al., 2011).

VII. Other Synaptic Vesicle Proteins

A. Rab3

Small GTPase rab3 is an abundant synaptic vesicleprotein. Mice lacking the rab3A isoform are viable andfertile with mostly normal synaptic function, except forincreased synaptic depression after repetitive stimuli(Geppert et al., 1994b). Quadruple rab3A, rab3B, rab3C,rab3D KO mice do not survive; however, culturedhippocampal neurons from these embryos have normalspontaneous release with decreased evoked vesicle re-lease probability (Schlüter et al., 2004). Experimentswith rab3 single-, double-, and triple-KOmice show thatthe four rab3 isoforms appear to be functionally re-dundant. These studies suggest that rab3 is not anessential component of neurotransmission, but partici-pates in the subtle regulation of synaptic vesicle fusion

and activity-dependent recruitment of vesicles to theactive zone (Leenders et al., 2001).

Changes in rab3A levels were observed in Alzheimerpatients and in a mouse model for Huntington’s disease(Davidsson et al., 2001; Morton et al., 2001). Rab3Aalso associates with pathologic a-synuclein in a GTP-dependent manner (Dalfó et al., 2004; Dalfó and Ferrer,2005; Chen et al., 2013b). SpecificGTPase inhibitorsmaybe a possible mechanism to modulate rab3A activity.

B. Synaptic Vesicle Glycoprotein 2A

SV2 is a membrane glycoprotein localized to synapticvesicles and neuroendocrine secretory granules (Buckleyand Kelly, 1985). SV2 has significant amino acid se-quence identity to bacterial transporters (Bajjalieh et al.,1992). SV2A, SV2B, and SV2C isoforms are highly ho-mologous proteins with differential expression through-out the brain (Janz and Südhof 1999). SV2A KO andSV2A/SV2B double-KO mice exhibit severe seizures anddie postnatally (Crowder et al., 1999; Janz et al., 1999).Neurons lacking both SV2A and SV2B exhibited sus-tained increases in calcium-dependent synaptic trans-mission after repetitive stimulation due to presynapticcalcium accumulation (Janz et al., 1999). SV2 may func-tion as regulators of presynaptic calcium and influencesynaptic vesicle dynamics (Wan et al., 2010).

Decreased SV2A was observed in surgically removedtemporal lobe tissue and postmortem hippocampaltissue from patients with temporal lobe epilepsy (Fenget al., 2009; van Vliet et al., 2009). Levetiracetam is aunique antiepileptic drug that targets a synaptic vesicleprotein instead of other antiepileptics that block voltage-gated sodiumchannels ormodulateGABAreceptors (Fig. 1).SV2A is both necessary and sufficient for levetiracetambinding and consequent antiepileptic action (Lynch et al.,2004). Low-frequency stimulation facilitates entry oflevetiracetam into neurons perhaps through bindingexposed SV2A on the intravesicular surface. Increasedsynaptic activity leads to corresponding increases inlevetiracetam’s efficacy, which may explain the antiepi-leptic effect of levetiracetam (Meehan et al., 2011). Apossible mechanism for levetiracetam’s antiepilepticaction may be due to SV2A-dependent acceleration ofsynaptic depression in excitatory neurons during epilep-tiform activity (García-Pérez et al., 2015). Brivaracetam(UCB 34714) is an analog of levetiracetam with 10-foldgreater binding affinity for SV2 and more potent antiep-ileptic effects in animal models of epilepsy (Kenda et al.,2004;Matagne et al., 2008). A phase III study shows thatbrivaracetam treatmentwas associatedwith statisticallysignificant reductions in seizure frequency comparedwith patients that received placebo treatment (Bitonet al., 2014). These results confirm SV2 as the target oflevetiracetam’s antiepileptic effect and establish theimportance of targeted small-molecule screens.

SV2 also interacts with botulinum and tetanus neuro-toxins and facilitates their entry into central nerve

152 Li and Kavalali

terminals (Dong et al., 2006; Yeh et al., 2010). Tetanusneurotoxin is unable to cleave syb2 in SV2 KO neurons,further implicating SV2 as a new target that can beexploited to prevent tetanus or for uptake of modifiedclostridial toxins into the synapse.

C. Synapsin

Synapsins are a family of four synaptic vesicle phos-phoproteins with conserved phosphorylation sites at theN-terminal domain and a high-affinity ATP-bindingmodule (Südhof et al., 1989; Hosaka and Südhof, 1998;Südhof, 2004). Most synapses express abundant levels ofsynapsin I and II, but they are not essential forneurotransmitter release (Rosahl et al., 1993). SynapsinI/II KO mice are viable and fertile, but suffer fromseizures (Rosahl et al., 1995). Disrupting synapsin func-tion results in a reduction of reserve pool of vesicles andfailure to sustain neurotransmitter release in response tohigh-frequency stimulation (Pieribone et al., 1995;Rosahl et al., 1995). Further studies with synapsin KOmice show that synapsins maintain the reserve pool ofvesicles in excitatory synapses, but regulate the size ofthe readily releasable pool in inhibitory synapses (Gitleret al., 2004). These results implicate synapsin in theregulation of network excitability and may explain theunderlying cause of epileptic seizures in synapsin KOmice (Chiappalone et al., 2009). Rab3A deletion insynapsin II KO mice appears to restore the excitatory/inhibitory balance, suggesting that the two synapticvesicle proteins coregulate synaptic activity (Felicianoet al., 2013).Genetic studies in humans with epilepsy have impli-

cated synapsin in disease etiology. A nonsensemutationin the synapsin I gene was identified in a type of familialX-linked (Garcia et al., 2004). Most epilepsies have beenlinked tomutations in voltage-gated channels; however,this is the first time a synaptic vesicle protein has beenrecognized. Single-nucleotide polymorphisms in thesynapsin II gene were found to be associated withsporadic epilepsy (Cavalleri et al., 2007; Lakhan et al.,2010). Postmortem brain studies show significant de-creases in synapsin II mRNA in prefrontal cortex ofsubjects with schizophrenia (Mirnics et al., 2001).Chronic treatment with the antipsychotic haloperidol,a dopamine D2 receptor antagonist, leads to increasesin synapsin II mRNA and protein levels in rats andhumans (Chong et al., 2006; Tan et al., 2014). SynapsinII KO mice exhibit behavioral abnormalities commonlyseen in preclinical animal models of schizophrenia,including prepulse inhibition deficits, decreased socialbehavior, and locomotor hyperactivity (Dyck et al.,2009). These results implicate synapsin II in thepathophysiology of schizophrenia and as a possibletarget for novel therapeutics with less severe sideeffects. Abnormal phosphorylation of synapsin I hasbeen linked to AD and Huntington’s disease (Parkset al., 1991; Lievens et al., 2002). However, additional

research is needed to deepen our understanding ofsynapsin’s role in the synaptic vesicle-recycling path-way and its involvement in pathogenic mechanisms ofdisease states.

Phosphorylation of synapsin may be a useful mecha-nism to regulate synaptic vesicle activity. PKA phos-phorylation of synapsin I promotes its dissociation fromsynaptic vesicles, enhancing the rate of stimulation-driven exocytosis and accelerating recovery from syn-aptic depression by recruiting vesicles from the reservepool to readily releasable pool (Menegon et al., 2006).Plant extract forskolin has anticonvulsant effects indrug-induced seizures in mice (Sano et al., 1984; BorgesFernandes et al., 2012), possibly through downstreamactivation of PKA by the cAMP pathway and conse-quent regulation of synapsin activity in synaptic vesiclerecycling. Small-molecule screens for modulators ofsynapsin I phosphorylation in primary neurons havealready identified more potential compounds (Chanet al., 2014). Cyclin-dependent kinase-5 phosphoryla-tion of synapsin I regulates the resting and recyclingpools of synaptic vesicles in an activity-dependentmanner and may be another pathway for pharmaco-logical intervention (Verstegen et al., 2014). Four pro-tein kinase inhibitors (staurosporine, quercetagetin,roscovitin, 70159800251) were found to compete againstATP for binding to the ATP binding site of synapsin I,highlighting one more potential mechanism to altersynapsin-mediated activity for therapeutic advantage(Defranchi et al., 2010). The challenge for drug devel-opment in this avenue is to find compounds that arespecific to synapsin sites to limit off-target or systemiceffects.

D. Synaptophysin

Synaptophysin is an abundant synaptic vesicle gly-coprotein that binds syb2 (Johnston and Südhof, 1990;Calakos and Scheller, 1994). This interaction regulatesthe distribution of available syb2 and prevents forma-tion of the SNARE complex (Edelmann et al., 1995;Pennuto et al., 2003). Dissociation of synaptophysinfrom syb2 allows it to form a complex with dynamin andpotentially regulate quantal size and duration of exo-cytic events (Daly and Ziff, 2002; Gonzalez-Jamett et al.,2010). Synaptophysin KO mice reveal that synaptophy-sin is not essential for neurotransmitter release; how-ever, they exhibit behavioral alterations and learningdeficits (McMahon et al., 1996; Schmitt et al., 2009).More recent studies have found that synaptophysin isrequired for syb retrieval and affects the kinetics ofsynaptic vesicle endocytosis (Gordon et al., 2011; Kwonand Chapman, 2011). Taken together, this suggestssynaptophysin may modulate the efficiency of thesynaptic vesicle cycle and have effects on higher-orderbrain functions.

Innormal states, synaptophysin is oneof themostabun-dant synaptic vesicle proteins, but its levels are reduced

Presynaptic Drug Targets 153

in AD and schizophrenia human brains (Shimohamaet al., 1997; Eastwood et al., 2000; Masliah et al., 2001).These changes in synaptophysin in neurons of thehippocampus and association cortices correlate withchanges in the cognitive decline in AD patients (Terryet al., 1991; Sze et al., 1997). Amyloid-b (Ab) peptide,which is elevated in AD brains, disrupts the interactionbetween synaptophysin and syb2 in vitro, leading to anincreased amount of primed vesicles and exocytosis(Russell et al., 2012). The effect on synaptic vesicle endo-cytosiswasnot examined;however, other studies show thatAb disrupted synaptic vesicle endocytosis perhaps due todynamin1depletion (Kelly et al., 2005;Kelly andFerreira,2007). It is possible that Ab disruption of synaptophysinbinding to syb2 results in more syb2 available to formSNARE complexes, leading to increased synaptic vesi-cle exocytosis. The defective endocytosis observed maybe due to both lack of synaptophysin-mediated syb2retrieval and also increased synaptophysin-dynamin1 complexes. This deregulation of synaptic transmis-sion may contribute to the pathogenesis of AD andperhaps explain the early cognitive loss precedingsignificant synapse loss seen in AD patients. Syb2–synaptophysin interactions are also disrupted in familialX-linked intellectual disability. Human synaptophysinX-linked intellectual disability mutants expressed insynaptophysin KO mice were dysfunctional in theirretrieval of syb2 during endocytosis, providing a pos-sible mechanistic basis for the disorder (Gordon andCousin, 2013).Synaptophysin may be a viable therapeutic target

through disruption of its interactions with syb2 anddynamin 1. Its function may be regulated by phosphor-ylation at its C-terminal domain by calcium/calmodulin-dependent protein kinase II and tyrosine kinase (Alderet al., 1995; Daly and Ziff, 2002; Mallozzi et al., 2013).

E. Rab5

Rab5 GTPase is found in high concentration on syn-aptic vesicles and mediates synaptic vesicle endocytosisas well as early endosome trafficking (de Hoop et al.,1994; Fischer vonMollard et al., 1994; Stenmark, 2009).In Drosophila studies, a loss-of-function rab5 mutantreduced exo- and endocytosis rates and decreased therecycling synaptic vesicle pool size (Wucherpfenniget al., 2003).Rab5 is associated with a few neurodegenerative

diseases, although not much is known about its role indisease pathology. Isoform rab5b has been shown tocolocalize with leucine-rich repeat kinase 2, a defectivegene causing an autosomal dominant form of Parkin-son’s disease, resulting in impaired synaptic vesicleendocytosis, and overexpression of rab5b rescued thedefect (Shin et al., 2008). In a Drosophila model ofHuntington’s disease, mutant huntingtin protein inter-acts indirectly with rab5 and inhibits its function. Rab5overexpression then attenuated aggregation and toxicity

(Ravikumar et al., 2008). Elevated rab5 levels wereobserved in brains from AD patients, suggesting thatendocytic dysfunction may underlie the pathogenesis ofAD (Ginsberg et al., 2010).

Although rab59s functions are not confined specifi-cally to synaptic vesicle recycling, it may be a viabletherapeutic target, as much is known about GTPaseinhibitors and may help in the development of modula-tors of rab5 function. The Research Foundation for theState University of New York recently filed a patent forsmall molecules thatmodulate rab5 activity by prevent-ing conversion of rab5-GTP to rab5-GDP, therebyenhancing rab5-mediated activity (Zong, 2014).

VIII. Conclusion

Studies in the last decade have greatly advanced mo-lecular characterization of synaptic vesicle proteins andour understanding of the mechanisms underlying synap-tic vesicle recycling. These efforts have also elucidated thekey roles played by the presynaptic vesicle-recyclingmachinery in the pathogenesis of several neurologicand neuropsychiatric disorders. However, despite theseadvances, systematic investigation of the presynapticmachinery components as drug targets received verylittle attention. Many presynaptic proteins function inlarger complexes and may have multiple roles in thesynaptic cycle process, making specific pharmacologicalintervention challenging. Nevertheless, the availabilityof increasingly sophisticated and specific functional as-saysmakes identification of novel drugs acting on presyn-aptic targets highly feasible. A combination of thecurrently available approaches to dynamically monitorpresynaptic function and our advanced understanding ofthe molecular details of neurotransmission will help usuncover how pathologic mutations alter presynapticfunction and also better equip us for presynapticallytargeted drug development.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Li, Kavalali.

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