A unified theory of presynaptic chemical neurotransmission

A unified theory of presynaptic chemical neurotransmission

Z theo~ BioL (1985) 112, 513-534 A Unified Theory of Presynaptic Chemical Neurotransmission NATHAN MOSKOWITZ AND SAUL PUSZKIN Department of Patholog...

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Z theo~ BioL (1985) 112, 513-534

A Unified Theory of Presynaptic Chemical Neurotransmission NATHAN MOSKOWITZ AND SAUL PUSZKIN

Department of PathologyILaboratory of Molecular Pathology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, New York 10029 (Received 23 December 1983, and in revised form 26 June 1984) The mechanism of neurotransmission and its modulation involves the direct role of calcium on membranes, and calcium's ability to activate synergistically and simultaneously a host of interdependent enzymatic cascades in synaptic and coated vesicles and the presynaptic plasma membrane. Enzymatic products formed can either amplify or depress synaptic vesicle exocytosis and synaptic vesicle regeneration via the coated pit/vesicle system. Rate amplification produced by a series of parallel, multistepped, interconnected enzymatic cascades as well as the optimal geometric spatial orientation of synaptic vesicles induced by presynaptic structures is hypothesized to explain how neurotransmitter is released within 200 i~sec upon calcium entry into the axon terminal.

1. Introduction Chemical neutransmission is the predominant means of communication from one neuron to the next in central and peripheral synapses (Kuftter & Nicholls, 1976). The presynaptic neuron transmits information by releasing neurotransmitters that cross the synaptic cleft to interact with the postsynaptic membrane, inducing excitation or inhibition via electrochemical membrane alterations. A typical neuron has between 1000-10 000 synapses and receives information from approximately 1000 other neurons (Stevens, 1979). It is evident, therefore, that modulation o f transmitter release at one synapse can directly affect transmission o f information to more than 1000 neurons each of which subsequently can affect more than 1000 other neurons, and so forth. Therefore, modulation of presynaptic neurotransmission could affect local brain functions such as information processing, learning, memory and behavior (Vizi, 1979; Kandel, 1976). Elucidation of neurotransmission's chemical nature (Loewi, 1921; Dale, 1937), and calcium's role in releasing neurotransmitters (Del Castillo & Stark, 1952; Mitchell, Neal & Spinivasan, 1968; Katz & Meledi, 1967; 513 0022-5193/85/030513+22 $03.00/0

© 1985 Academic Press Inc. (London) Ltd

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Harvey & Macintosh, 1940) corroborated the work of Del Castillo & Katz (1954) who enunciated the quantal theory of synaptic transmission. Shortly thereafter, small vesicles were observed using electron microscopy in axon terminals by De Robertis & Bennett (1955). Del Castillo & Katz (1956) suggested that these synaptic vesicles (SV) were morphological counterparts of the quanta, hypothesizing that the SV could quantitatively release their contents into the synaptic cleft by exocytosis. Finally, the demonstration by Whittaker, Michaelson and Kirkland (1964) that SV contained the neurotransmitter acetylcholine provided physiological, morphological and biochemical observations unified into a theory of transmitter storage and release called "the vesicle hypothesis". The quantal nature of transmitter release has been established subsequently in a variety of vertebrate and invertebrate synapses (Martin, 1966; Dudel & Kuflter, 1961; Bittner & Harrison, 1970) and SV have been found in many types of chemical synapses (Elfvin, 1976). Despite criticism of this hypothesis (Tauc, 1979), physiological (Anderson & Stevens, 1973; Ito & Miledi, 1977), rapid freezing and free-fractured electron microscopy (Model, Highstein & Bennett, 1979; Heuser et al., 1974; Ciccarelli et al., 1979), and immunological (Von Wedel, Carlsson & Kelly, 1981) data have confirmed it. Because this hypothesis has withstood the test of time, we have used it as our starting point and the basis of our theory. Retrieval of SV occurs by the coated pit/coated vesicle (CP/CV) endocytotic pathway (Fried & Blaustein, 1976; Heuser & Reese, 1973; Py.sh & Wiley, 1974). Coated pits in axon terminals are invaginated areas of the presynaptic plasma membrane which by electron microscopy appear to have a fuzzy coat (Kanaseki & Kadota, 1964), hence the term coated pits. The CP selectively retrieve SV membrane, pinch off and become CV, viz., enclosed membrane bilayers surrounded by what appears morphologically as a coat and which has been demonstrated to be composed of icosahedral lattices of clathrin molecules (Pearse, 1976). Thus, CV in axon terminals participate in the regeneration of SV. Our goal is to present a unified theory for the mechanism of calciummediated neurotransmission and neuromodulation. The theory is based on a synthesis of many previous studies and on work we conducted over the past several years. Our hypothesis invokes not only the direct role of calcium on membranes but also the ion's ability to synergistically and simultaneously activate a host of enzymatic cascades, dependent and regulating one another, the products of which either can amplify or depress SV exocytosis and SV regeneration via the CP/CV system. An attempt also has been made to explain how these phenomenon may lead to release of neurotransmitter within 200 ~sec.

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2. Synaptic Vesicle/Synaptic Plasma Membrane Fusion and Neurotransmitter Release

The enzymatic cascades thought to be involved in neurotransmitter release are illustrated in Fig. 1. At the resting state of the neuron, axon terminal calcium concentration ranges between 10-8-10 -7 M (DiPolo et aL, 1976). The electric field crossing the plasma membrane upon its depolarization changes the conformation of the calcium channel monomers and redistributes the charge of their inner surfaces making them negative (Llinas, Steinberg & Walton, 1976). In this way membrane depolarization allows calcium to move through the calcium channels into the axon terminal. Because the calcium channels are concentrated at the presynaptic terminal, calcium enters the terminal at the active site and is distributed throughout the 200 A layer of cytoplasm closest to the presynaptic membrane (Kelly et al., 1979). If calcium enters only at the active zones and is not uniformly distributed throughout the cytoplasm but rather is preferentially associated with negatively charged surfaces, the internal calcium concentration at the release site during the peak of neurotransmitter release reaches 10-6-10 -3 M (Kelly et al., 1979). The incoming calcium current translocates SV to the presynaptic membrane. Because SV cannot move freely in the viscous gel matrix (Crick, 1950) a propelling force must operate to translocate them the required 50-100 ,~ toward the membrane; such a force is actomyosin (Berl, Puszkin & Nicklas, 1973). Incoming calcium binds to and activates calmodulin (CAM) which is soluble and/or associated with SV (DeLorenzo et al., 1979; Moskowitz, Schook & Puszkin, 1982a; Moskowitz et al., 1982b). Calmodulin is the major calcium-binding protein in eukaryotic cells by which calcium exerts many of its physiological ef[ects (Cheung, 1980); it also is involved in the release of neurotransmitter from SV in the brain (DeLorenzo et al., 1979). Activated CaM thus may activate myosin light chain kinase leading to phosphorylation of myosin. Only when myosin is phosphorylated can actin activate myosin Mg2+-ATPase. Hydrolysis of ATP by this ATPase supplies the energy for actomyosin associated with SV (Tashiro & Stadler, 1978) to contract and subsequently bring about propulsion of SV to the presynaptic membrane (Fig. 1, Enzyme Cascade I). The actomyosin system may work in conjunction with the presynaptic densities, grid and synaptopores to orient the SV in a precise manner with respect to the synaptic plasma membrane (SPM). Presynaptic densities are found in peripheral nervous tissue and the presynaptic grid is ground in the central nervous system. Both structures have been implicated in orienting SV to the specific site of exocytosis.

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Presynaptic densities occur as thin transverse bands directly above the postsynaptic fold (Cauteaux & Pecot-Dechauassine, 1970). Akert (1973) found in the central nervous system that the presynaptic grid is on the cytoplasmic side o f the presynaptic m e m b r a n e and consists o f an assembly of dense projections which are arranged in a triagonal lattice and are interconnected by filamentous cross bridges. The holes o f the grid have roughly the dimensions required to a c c o m m o d a t e SV. Thus Akert maintains that the grid provides the most economical geometry of placing transmitter storage vesicles as closely as possible to their expected release sites. Freeze cleavage o f presynaptic m e m b r a n e s in the vertebrate central nervous system revealed specialized structures called synaptopores (Akert et aL, 1972), i.e., protruberances extending toward the presynaptic cytoplasm which are loci in direct contact with SV and exocytotic release. Akert et al. (1972), postulated that these sites must have specific properties to permit vesicle and plasma m e m b r a n e interaction. Invariably, images of SV fusion are lined up between these zones. Rarely has a vesicle opening been observed as far away as 500.3, from these structures. Thus SV exocytosis is strictly localized to the active zone. It is likely that the vesicle is propelled toward and orientated in the grid so that a certain highly specific pole or region of the SV is orientated and aligned with a highly specialized region of the SPM for fusion to occur between these two membranes. In stimulated axon terminals, SV do not fuse with each other (Heuser et al., 1974). We demonstrated that SV, when extracted from the axon terminal, associate with each other, aggregate, and possibly fuse (Moskowitz et al., 1982c). The fact that SV do not fuse with each other in oivo implies that they are prohibited from self-association by the viscous matrix separating them, and actomyosin contraction m a y direct the SV toward the synaptic grid-synaptopore region and not to other SV. Although in vitro SV-SV aggregation occurs, it does not do so within a ~sec or millisecond time range (Moskowitz et al., 1982c). This implies that FIG. I. Scheme of events involved in SV-SPM fusion and subsequent neurotransmitter release from axon terminals. Roman numerals represent individual enzyme cascades referred to in text. Abbreviations used: CaM=calmodulin; ATP=adenosine triphosphate; ADP= adenosine diphosphate; sv = synaptic vesicle; spin = synaptic plasma membrane. Cascade I: MLCK = myosin light chain kinase. Cascade I1: calcium/CaM PK = calcium/CaM-dependent protein kinase; MP=membrane proteins. Cascade III: cAMP=3'-5' cyclic adenosine monophosphate; PDE=phosphodiesterase; AMP=5'-adenosine monophosphate; PK= protein kinase; MP=membrane proteins; LM=lipomodulin. Cascade IV: PLA2= phospholipase A2; aa=arachidonic acid; PG=prostaglandin; W=change in; LL= lysolecithin; PC = phosphatidylcholine; NT= neurotransmitter; PG syn = prostaglandin synthetase; AT=acetyltransferase. Cascade VI: MT I and lI=methyltransferases I and II; PE =phosphatidylethanolamine. Cascade VII: ICPK=ion channel protein kinase; NPK= nucleus-related protein kinase; IC = ion channel; NP= nucleus proteins.

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intraaxonal mechanisms outside the SV machinery are necessary for ~sec fusion to occur and a specific orientation of different regions is necessary for ~sec fusion. In vivo fusion of the SV with SPM does not occur in regions of the presynaptic membrane other than at the synaptopore (Heuser et al., 1974). We will develop below the concept that the rate of enzymatic cascades involved in the fusion process may be amplified when concerned enzymes are geographically positioned optimally. Propulsion of SV by actin-myosin interaction into the presynaptic grid, a morphological structure of considerable strength, holds the vesicle in place and in contact with the synaptopore throughout the duration of the fusion process for a maximal rate of fusion to occur. Once the SV and SPM are optimally juxtaposed, a series of interconnected enzymatic cascades, all simultaneously stimulated by calcium/CaM, operate to regulate SV fusion to the presynaptic plasma membrane. Calcium/CaM activation of both SV and SPM phospholipase A2 (PLA2 (Moskowitz et al., 1982c, 1983a)) leads to hydrolysis of phosphatidylcholine and subsequent formation of lysolecithin and arachidonic acid (Fig. 1, Cascade IV). Lysolecithin incorporates into the bilayers of SPM and SV producing a transition from the bimolecular leaflet to radially oriented molecules (Haydon & Taylor, 1963) possibly leading to fusion of these separate membranes. There are several ongoing enzymatic processes which modulate the amount of lysolecithin produced and therefore the amount of SV-SPM fusion and neurotransmitter release. Acetyltransferase catalyzes the transfer of fatty acid to lysolecithin leading to formation of phosphatidylcholine (Webster, 1965). The concentration of acetyltransferase in axon terminal membranes, its properties and possible modulators presently are unknown. The free fatty acids released from SV and SPM as a consequence of PLA2 hydrolysis can be metabolized by endogenous SV prostaglandin (PG) synthetase to PGF2~ and PGE2 (Moskowitz & Puszkin, 1983a). These eicasanoid products may continually monitor neurotransmitter release by modulating SV and SPM-PLA2 activities. Prostaglandin E2 can feedbackinhibit and PGF2~ can feedback-stimulate PLA2 (Moskowitz et al., 1982c, 1983a). Prostaglandin E2 links the PLA2-PG synthetase cascade (Fig. 1, Cascade IV) to another simultaneous and ongoing enzymatic cascade, the adenylate cyclase/cAMP protein kinase cascade (Fig. 1, Cascade III) which can independently induce neurotransmitter release (Castelluci et al., 1980; Veda, 1981). Adenylate cyclase has been detected in presynaptic membranes (Weller, 1977). Calcium/CaM and released PGE2 may activate presynaptic membrane adenylate cyclase (in the inner surface of the plasma membrane) leading to conversion of ATP to cAMP (Nathanson, 1977). Calcium/CaM

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further controls cAMP levels by activating cytoplasmic phosphodiesterase which converts cAMP to AMP (Cheung, 1980). Cyclic AMP at sufficient concentration reciprocally interconnects enzyme cascades III and IV (Fig. 1) by two different mechanisms: (1) cAMP may directly activate SV-PLA2 (Moskowitz et al., 1982c) and directly inhibit SPM-PLA2 (Moskowitz et al., 1983a); (2) cAMP may activate cAMP-dependent protein kinase in both SV and SPM (Moskowitz et al., 1983b; Moskowitz & Puszkin, 1983a). Phosphorylation of SV lipomodulin could lead to relaxation of lipomodulin PLA2 inhibition (Hirata, 1981) and subsequent stimulation of SV PLA2. Thus PGE2 potentiates formation of cAMP which, in turn, can potentiate or inhibit the formation of PGE2. Overall inhibition or stimulation would depend on which of the above-mentioned cAMP-dependent pathways is more dominant. Phosphorylation of SV and SPM proteins by cAMP-dependent protein kinase leads to changes in their conformations (Greengard, 1976; Williams & Rodnight, 1977) which are conducive to SV-SPM fusion, thus leading to neurotransmitter release (DeLorenzo et ai., 1979). A protein phosphatase could modulate neurotransmitter release by dephosphorylation of these proteins ( M a e n o & Greengard, 1972) and subsequent inhibition of fusion. Simultaneous activation of SV and SPM calcium/CaM-dependent protein kinases (DeLorenzo et al., 1979; Krueger, Forn & Greengard, 1977) also leads to the phosphorylation of SV and SPM proteins, altering their conformational changes and inducing fusion (Fig. l, Cascade II). Again, a protein phosphatase could modulate this process by dephosphorylation of proteins and hence inhibition of SV fusion (Maeno & Greengard, 1972). Our results have indicated that inhibition of calcium/CaM kinase activity by specific antibodies directed against the calcium/CaM protein kinase also inhibited to a certain extent the cAMP-dependent kinase, implying that these kinases are dependent somehow on one another or interconnected (Moskowitz et al., 1983b). Thus we see that the calcium/CaM protein kinase cascade (II) is reciprocally connected to the cAMP protein kinase cascade (III) which, in turn, is reciprocally connected to the PLA2 cascade (IV). Therefore, modulation of any of these three cascades can simultaneously affect the other two cascades to varying degrees. Stimulation of [Ca2+-Mg2+]ATPase in SV, CV and SPM by calcium/CaM (Moskowitz et al., 1982a; Moskowitz & Puszkin, 1983b; Breer, Morris & Whittaker, 1977; Blitz, Fine & ToselIi, 1975; Blaustein, Ratzlatt & Kendrick, 1978; Kuo et aL, 1979) constantly monitors the levels of calcium in the axon terminal (Cascade V) thus modulating cascades I-IV and hence neurotransmitter release. It should be noted that cascades I, II, III and V control the levels of axon terminal ATP and, as a consequence, modulate SV- and SPM-PLA2

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(Cascade IV). Phospholipase A2, in turn, has been demonstrated to activate [Ca2+-Mg2+]-ATPase in red blood cells (Taverna & Hanahan, 1980). It is possible that a similar phenomenon occurs in the brain. It appears that cascades I-V---which are simultaneously activated by calcium/CaM upon increase of axon terminal calcium--are parallel and interconnected cascades which constantly modulate (amplify/depress) each other. The overall activation of cascades I-IV leads to SV-SPM fusion and subsequent neurotransmitter release. Once neurotransmitter is released into the synaptic cleft, it binds to the postsynaptic receptors and induces an eventual change in postsynaptic membrane permeability, thus establishing communication between one neuron and the next (Greengard, 1976). The newly released neurotransmitter also can modulate presynaptic neurotransmission by binding to presynaptic neurotransmitter receptors (Greengard, 1976). Neurotransmitter binding to presynaptic receptors may lead to activation of methyltransferases I and II (Cascade VI), and to conversion of SPM phosphatidylethanolamine to phosphatidylcholine (Hirata & Axetrod, 1980). This would activate SV- and SPM-PLA2 (Cascade IV) by (a) presenting more substrate to PLA2 for hydrolysis, (b) increasing membrane fluidity thus bringing phosphatidylcholine and PLA2 into closer juxtaposition; and (c) increasing membrane permeability leading to influx of calcium and hence activation of Cascades I-V (Fig. 1). Activation of methyltransferases (Cascade VI) can modulate the amount of neurotransmitter released which consequently modulates activation of PLA2 methyltransferases. In this manner the methyltransferase cascade is reciprocally related to all other enzyme cascades. Additionally, activation of PLA~ by methyltransferase could lead to a change in the lipid annulus in a variety of neurotransmitter receptors leading to inhibition of neurotransmitter receptor binding (Limbird & Lefkowitz, 1976) and inhibition of methyltransferase activities. Binding of neurotransmitter to receptors also leads to coupling of the neurotransmitter receptor complex to presynaptic adenylate cyclase (Fig. 1, Cascade VII) leading to generation of cAMP (Greengard, 1976). As a result of neurotransmitter binding to receptors, cAMP can activate protein phosphorylation of certain presynaptic membrane proteins discussed above. Moreover, cAMP activates phosphorylation of nuclear proteins and enzymes leading to inhibition of neurotransmitter synthesis and inhibition of neurotransmitter release (Vizi, 1979). Cyclic-AMP also can activate phosphorylation of certain ion channels e.g., the K ÷ channel (Adams & Levitan, 1982), leading to influx of K ÷ and continued stimulation of neurotransmitter release. Because cAMP can have a variety of stimulating and inhibiting effects, its overall effect may depend

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on the compartmentalization of the various cAMP-dependent protein kinases within the axon terminal. Automodulation of an axon terminal by its own neurotransmitter may be affected by a neurotransmitter-receptorinduced increase of cAMP in the vicinity of the nucleus thereby leading to inhibition of neurotransmitter synthesis and release (Vizi, 1979). Activation of neurotransmitter release from an axon terminal by a neurotransmitter from another axon terminal may lead to activation of adenylate eyelases/protein kinases in the vicinity of the K ÷ channel thereby leading to influx of K ÷ and an increase in neurotransmitter release (Castellucci et al., 1979). Stimulation of adenylate cyclases by calcium/CaM from the inner side of the axon terminal may lead to activation of SV and SPM protein kinases and subsequent phosphorylation of proteins leading to neurotransmitter release. It should be noted that Cascade VII (Fig. 1) also is reciprocally interconnected with all the other cascades, because its activation modulates the amount of transmitter that can be released which, in turn, can bind to presynaptic receptors and activate methyltransferases (Cascade VI) which is reciprocally interconnected with Cascades I-V. It can be observed that the same modulator, e.g., cAMP, can have different effects on the same enzyme in different membranes, e.g., it activates SV PLA2 and inhibits SPM PLA2 and leads to protein phosphorylation which can either stimulate or inhibit neurotransmitter release. The overall effect of a single modulator would be determined by which enzyme cascade is the one predominantly activated in a particular axon terminal. Prostaglandins are ubiquitous cellular lipid regulators which have variable etfects on neurotransmission (Samuelson et al., 1978; Gilbert, Davidson & Wyllie, 1978; Wolfe, 1982) in terms of stimulation or inhibition. This may be attributed in vivo to the multiple effects of PGE2 on PLA2 and adenylate cyclase. Prostaglandin E2 inhibits SV and SPM PLA2 and stimulates adenylate cyclase (Wellman & Schwabe, 1973); the latter phenomenon leads to increased cAMP levels, which leads to further activation of PLA2 and subsequent production of lysolecithin, PGE2 and PGF2,. Thus inhibition of PLA2 by PGE2 leads to inhibition of neurotransmitter release. However, the simultaneous activation of adenylate cyclase by PGE2 and subsequent formation of cAMP leads to stimulation of PLA2 by cAMP and increased synthesis of PGF2~ and lysolecithin and subsequent stimulation of neurotransmission. Because multiple interconnected enzymatic cascades are involved in neurotransmitter release it becomes apparent that a single modulator such as PGE2 may have effects on multiple systems which, in vitro, have diametrically opposed effects; in vivo, these same effects are controlled by the temporal situation and thus modulate neurotransmitter release. The multiple

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number of cascades that PG are capable of modulating may explain why exogenous PG have such variable effects on neurotransmitter release (Wolfe, 1982). Recently the term "synarchy" was introduced to describe the mutual effects o f calcium and cAMP on various metabolic and cellular events (Rasmussen, 1981). Our previous studies confirm that calcium and cAMP are synarchic with respect to activation o f SV protein kinases and PLA2 (Moskowitz et aL, 1982c, 1983a). Figure 2 illustrates the synarchy o f calcium

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FIG. 2. Calcium-cAMPsynarchy in modulation of SV protein kinase activity. M, of proteins that are phosphorylated in SV are indicated. Abbreviations used: CaM = calmodulin: PK= protein kinase; RzC2= regulatory subunits2-catalyticsubunits2, and cAMP with respect to protein kinase activation in SV. Activation of calcium/CaM- and cAMP-dependent protein kinases independently leads to phosphorylation o f SV membrane proteins which leads to neurotransmitter release. Synarchy is evident in two aspects o f these cascades. As mentioned previously the activation by calcium and cAMP of the kinases is interdependent as evidenced by the fact that antibody inhibition of the calcium kinase inhibits the cAMP kinase. Also, two of the SV substrates phosphorylated by the two protein kinases with Mrs 55 K and 53 K are identical. These two substrates may be a- and/3-tubulins whose conformational changes may lead to SV-SPM fusion (Burke & DeLorenzo, 1982). Figure 3 illustrates the synarchy between calcium and cAMP with respect to PLA2. C a l c i u m / C a M activates PLA2 leading to production of lysolecithin and arachidonic acid. The latter product subsequently can be converted to

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FIG. 3. Calcium-cAMP synarchy in modulation of SV PLA2. Abbreviations used: CaM = calmodulin; AC=adenylate cyclase; PLA2=unactivated phospholipase A2; PLA2W= activated PLA2; cAMP= 3'-5' cyclic adenosine triphosphate; ATP = adenosine triphosphate; AMP = adenosine monophosphate; PDE = phosphodiesterase; PC = phosphatidylcholine; LL= lysolecithin; aa = arachidonic acid; AT = acetyltransferase; PG = prostaglandin. PGE2 which can lead to activation o f adenylate cyclase and generation of cAMP. Cyclic AMP, in turn, can increase PLA2 activity in SV or inhibit PLA2 activity in SPM, thereby modulating its own axon terminal levels. Because cAMP inhibits purified PLA2 (Moskowitz et al., 1983c), it is likely that the universal effect of cAMP on PLA2 is inhibition. Because most PLAs~ are homologous to a certain extent (Yang & King, 1980), it is likely that SV and SPM PLAs2 are highly similar. Stimulation of SV PLA2 by cAMP in SV may be due to stimulation of cAMP-dependent protein kinases by virtue o f its interconnection with SV PLA2 via lipomodulin (Moskowitz et al., 1982c). In light o f the fact that a host of enzymatic cascades may synergistically control neurotransmitter release it is apparent that the inhibition of any one of these cascades may severely alter neurotransmitter release. It is no wonder therefore that trifluoperazine, a CaM inhibitor, has been reported to inhibit neurotransmitter release (DeLorenzo et al., 1979) in view of the fact that CaM activates many o f the enzyme cascades that lead to neurotransmitter release. It is also apparent why disruption of actomyosin by actin disrupters (Pumplin & McClure, 1974) disruption of tubulin (a substrate protein for SV c a l c i u m / C a M and cAMP-dependent protein kinases) by tubulin disrupters (Allison & Davies, 1974), inhibition of PLA2 by quinacrine and

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parabromophenacylbromide (Moskowitz et al., 1982c) and inhibition of methyltransferase (Hirata & Axelrod, 1980) all individually lead to inhibition of secretion and/or neurotransmitter release. The fact that PLA2 was demonstrated to be modulated by a variety of key cellular modulators such as PGE2, PGF2,,, cAMP and ATP (Moskowitz et al., 1982c, 1983c) and by other modulators including neurotransmitters and hormones (Ramwell et al., 1966; Juan & Lombeck, 1976) makes it a most important enzyme to be reckoned with in terms of understanding its neurotransmitter release process. The involveme:: of enzymes in neurotransmitter release has been criticized previously as not being rapid enough to account for release of neurotransmitter within 100-200 v~sec. According to Stadtman & Chock (1977), enzyme cascades serve as multiplier systems with respect to rate amplification. Based on their theoretical calculations, activation of merely an adenylate cyclase/protein kinase cascade can operate in a 10-3-sec time scale. Although this is sufficient time for neurotransmitter-induced, postsynaptic membrane-increased permeability, it is not rapid enough to account for neurotransmitter release from axon terminals. It must be borne in mind that seven types of enzymatic cascades are proceeding simultaneously within the axon terminal. Enzyme Cascades I-V (Fig. 5) occur in the activated neuron. Cascades V and VII probably occur in activated and resting neurons. Although Cascades I-V are most operative in the active neuron, in the resting state and in the absence of CaM activation there may exist a steady-state level of baseline enzymatic activity. It must be emphasized that the primary enzyme cascades involved in neurotransmitter release (Fig. 1, Cascades I-IV) can be activated synergistically and simultaneously and are interdependent, and thus may be considered parallel interconnecting cascades. Stadtman & Chock (1977) have calculated that an exponential linear relationship exists between the number of steps in an enzymatic cascade and its amplification potential. The simultaneous activation of a series of parallel enzymatic cascades could be envisioned as activation of a polycyclic enzymatic cascade with " n " number of steps. Therefore if activation of Cascade III (Fig. 1) by itself occurs within 10-3sec,---because it is interconnected to six other cascades-neurotransmitter release could occur in 10-4 sec or less, i.e., within the required time limit for neurotransmitter release. In addition to the synergistic activation of parallel cascades, a variety of non-enzymatic mechanisms also in operation could amplify the enzymatic effects. At the area of SV-SPM apposition, calcium may reduce the electrostatic surface charge of both membranes thereby bringing about more direct apposition, inducing a phase transition or phase separation in the mem-

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branes which leads to crystallization of the outer membrane bilayers. Condensation of these membrane regions would result in instability making them more susceptible to fusion (Papahadjopoulas et al., 1977). Calcium also may directly serve as an ionic link brigding ionic groups of the SV to the SPM, decrease the energetic barriers between the two membranes and/or lead to calcium-dithiolate formation which could lead to further apposition and fusion (Hubbard, Jones & Landau, 1968; Cooke, Okomoto & Quastel, 1973; Kosower & Werman, 1971). Once the SV is brought into relative proximity of the SPM, key SV and SPM glycoproteins may facilitate membrane attachment and interaction leading to apposition and fusion (Tauc & Hinzen, 1974). The fact that calcium alone does not induce neurotransmitter release (DeLorenzo et aL, 1979) supports the hypothesis that calcium/CaM stimulation of enzymes promotes neurotransmitter release. It must be recalled that fusion of SV to SPM must occur in discrete areas. Chock & Stadtman (1979) have shown that in cyclic cascades an even greater enzyme rate amplification is achieved by proper positioning of the multienzyme complex. Such a situation also occurs in mammalian pyruvate dehydrogenase (Chock & Stadtman, 1979). The function of the presynaptic grid and synaptopores must be to perfectly align the various enzymes in both SV and SPM to amplify further the enzymatic cascade for enhancement of transmitter release. Thus a series of enzymatic cascades--optimally oriented by presynaptic morphological structures to obtain maximal geographical positioning--may explain how SV-SPM fusion can occur in 10-4 sec or less. The fact that an adenylate cyclase/protein kinase cascade can operate only within 10-3 sec (Chock & Stadtman, 1979) emphasizes and corroborates the idea that more than one cascade is involved in neurotransmitter release and that multiple interconnected cascades are necessary to bring about the fastest cell-cell communication presently known in biological systems. 3. Coated Pit-coated Vesicle Retrieval of Synaptic Vesicles After SV fuse with the SPM and intermix, SV particles translocate laterally within the SPM and are subsequently retrieved by the CP/CV system. There is a soluble pool of clathrin heavily concentrated at the axon terminal (Cheng et al., 1980). These clathrin triskelions are in equilibrium with clathrin triskelions bound to CP and CV. How does clathrin recognize and insert into the SV membrane to become a CP? What governs the reversible nature of clathrin-membrane recognition? At the peak of neurotransmitter release calcium may lead to CaM activation of calcium/CaM-dependent protein kinase which phosphorylates proteins with M,s of 175 K and 55 K

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(Moskowitz et al., 1983b). Phosphorylation of these proteins would lead to transient conformationat changes, possibly allowing clathrin to recognize and insert into them, and lead to creation of a CP. Calmodulin has been implicated in the recruitment of clathrin to membranes to form CP (Salisbury et al., 1980). The calcium/CaM-dependent phosphorylation of membrane proteins may be the mechanism by which this is accomplished. Transglutaminases cross-link proteins into the CP by catalyzing the formation of E (3,-glutamyl)-lysine bonds between proteins and coupling of amines and diamines to the ~,-carbonyl residues of glutamine (Folk & Finlayson, 1977). This has been confirmed by inhibition of CP/CV-mediated endocytosis in the presence of transglutaminase inhibitors (Davies et aL, 1980). Once selected proteins are cross-linked, clathrin may invaginate the phosphorylated 175 K and 55 K Mr proteins which possibly are located at regular intervals between the specific SV proteins destined for endocytosis. Transglutaminase cross-linking would assure that all the proteins form a discrete entity. This would mean that clathrin would have to recognize only the 175 K and 55 K M~s SV proteins. The other SV proteins would be selectively retrieved in the CP by virtue of transglutaminase cross-linking. Selectivity of SV proteins included in the pit would be mediated, therefore, mostly by transglutaminase activity and, to a limited extent, by clathrin recognition of proteins. The fact that CV do not have cAMP-dependent protein kinase activity, whereas SV do, implies that the SV cAMP-dependent protein kinase is one protein complex not selectively retrieved by CV. The mechanism of transglutaminase protein selectivity remains to be resolved. Others have demonstrated that a 100 K protein is necessary for insertion of clathrin triskelions into CP/CV (Unanue et al., 1981). A transglutaminase of Mr 100 K that is calcium/CaM dependent has been demonstrated in platelets (E. Puszkin, unpublished observations). Our results indicate the existence of a CV CaM-binding protein with a Mr of 100 K (Moskowitz et al., 1982b). Conceivably, this 100 K protein may be a calcium/CaM-dependent transglutaminase and upon its activation cross-links the CP proteins stabilizing the entire cross-linked CP complex and thereby facilitating clathrin binding to the membrane. The inhibition of CV morphology by metabolic inhibitors (Palade & Fletcher, 1977) suggests that ATP plays an important role in CV function; ATP's role may be in the protein kinasemediated transfer of its gamma phosphate to coated membrane proteins. After clustering of proteins and formation of the CP, it invaginates, pinches off and becomes a CV. Although Kanaseki & Kadota's non-enzymatic hypothesis (1969) is documented by electron microscopy, to wit, that conversion of CP hexagons into pentagons causes increased curvature and

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527

creates a force sufficient to pinch off the CP from the membrane, it is uncertain exactly what force initiates this transformation. Calmodulin activation of PLA2 in the region of the CP on both sides of the membrane and subsequent release of lysolecithin could create membrane nicks and perturbations thereby leading to detachment of the CP from the rest of the plasma membrane (Moskowitz & Puszkin, 1982d). Presynaptic plasma membrane PLA2 activation may function to reseal the free ends remaining at the membrane. Coated vesicle PLA2 also may function in resealing of the detached CP into a CV. The contribution of PLA2 to this process is unknown. Activation of the CV and SPM PLAs2 leads to release of arachidonic acid and its consequent metabolism by CV-PG and thromboxane synthetase to PGE2 and PGF2~ (Moskowitz et al., 1983b). Prostaglandin E2 and PGF2~ serve to modulate PLA2 and facilitate CP formation by amplifying CV PLA2 (Moskowitz & Puszkin, 1982d). Cyclic-AMP, which is generated by SPM adenylate cyclase, and axon terminal ATP serve to inhibit this process of CP/CV formation. The regulation of plasma membrane area is modulated therefore by CV and SPM PLA2. Prostaglandins stimulate, whereas cAMP and ATP inhibit PLA2. The generation of an acidic milieu by PG and thromboxanes would increase local H + concentration. This increase in H +, along with the increase in local calcium, facilitates clathrin-clathrin interaction in the CP and CV leading to formation of the continuous coat structure (Schook et al., 1979). Electron microscopic observations have revealed that once the cV forms the coat disassembles from the CV and the denuded vesicle subsequently is transformed into a SV (Heuser & Reese, 1973). Because clathrin association and dissociation are reversible, it is possible that as calcium concentration decreases the protein kinase is no longer maximally stimulated. Moreover, a protein phosphatase might be activated to hydrolyze phosphates from previously phosphorylated CV proteins leading to the return of the protein's original conformation which does not recognize clathrin, thereby leading to clathrin dissociation from the membrane. It is interesting to note that a cytoplasmic factor in conjunction with ATP leads to dissociation of coats from vesicles (Patzer et al., 1982). Protein phosphatases are usually soluble and cytoplasmic. Recently a brain phosphatase dependent on ATP was characterized (P. Simonelli, unpublished observations). Possibly this soluble factor leading to the dissociation of coats from CV is a protein kinase. Although there are specific high-affinity neurotransmitter uptake systems (Cooper, Bloom & Roth, 1978), the invagination of CP and subsequent CV formation may lead to additional uptake of neurotransmitter from the

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FIG. 4. Scheme o f events involved in CP and CV formation. Abbreviations used: SPM = s y n a p t i c plasma membrane; A C = a d e n y l a t e cyclase; P D E = phosphodiesterase; A M P = adenosine m o n o p h o s p h a t e ; C V = coated vesicles; PLA2 = phospholipase A 2; PG = prostaglandin; T x = t h r o m b o x a n e ; AT=acetyltransferase; P K = p r o t e i n kinase; M P = m e m b r a n e proteins; c p = c o a t e d pit; C a M = calmodulin; c A M P = 3'-5' cyclic adenosine m o n o p h o s p h a t e ; A T P = adenosine triphosphate; PC = phosphatidylcholine; LL = lysolecithin; aa = arachidonic acid ; + = stimulation ; - = inhibition.

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PRESYNAPTIC

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synaptic cleft. Until neurotransmitter content in CV is determined, the contribution of this pathway to neurotransmitter conservation will remain unresolved. After clathrin dissociates from the CV, SV are regenerated. There are many unexplained phenomena in this recycling process. If CV are retrieved by SV, why do so many of the SV and CV enzymes and their properties differ? There may be several explanations for this phenomenon. By some unknown mechanism, SV that are newly transformed from CV far away from the active zone may be continually transformed so that by the time they reach the active zone they have the full characteristics of SV. If there is a limited amount of protein synthesis occurring in the axon terminal, it may function in transformation of CV to SV. It must also be recognized that CV and SV purified by standard procedures comprise a heterogeneous population. Both SV and CV are heterogeneous in that they come from a variety of axon terminals. The CV also may arise from perikaryon regions as well as from the axon terminal. Coated vesicles from these different origins may have different characteristics. Figure 4 summarizes all the enzymatic cascades presumably involved in CP/CV regeneration of SV. Figure 5 is a schematic model emphasizing the possible functional significance of vesicle protein kinase activities.

4. Concluding Remarks Interdisciplinary experiments may yield information on the true in vivo functions of various enzymatic cascades in the transmitter process. It has been demonstrated by Castellucci et al. (1980), that injection of cAMPdependent protein kinase can modulate neurotransmitter release concomitantly with behavior. Similarly, by looking at other passible presynaptic enzymatic cascades, it may be possible to understand certain general aspects of brain function. The mechanism of anesthetic action, for example, has been attributed to inactivation of PLA2 (Vanderhock & Feinstein, 1979; Feinstein et al., 1976). In light of PLA2 involvement in neurotransmitter release, this hypothesis remains tenable. Recently the pharmacological agent piracetam was used in combination with choline for memory improvement (Bartus et al., 1982) ; choline converts to acetylcholine, a t~eurotransmitter associated with memory (Fovall et al., 1980). Piracetam has many effects including the ability to enhance intercerebral neuronal activity; also, it may deplete hippocampus Ach levels by enhancing neurotransmitter release (Buresova & Bures, 1973; Wurtman, Magil & Reinstein, 1981). Piracetam is an activator of brain PLA2 (Woelk, 1979). It is quite possible that after choline is converted to acetylcholine

530

N. M O S K O W I T Z

A N D S. P U S Z K I N

Post-synaptic

membrane

FIG. 5. Model of the possible functional significance of SV and CV protein kinase activities. Enlarged details of steps A-F are inserted. Insert (A) illustrates a SV prior to calcium influx into the axon terminal. The cAMP-dependent kinase (cAMP K) and the calcium/CaMdependent kinase (30 K) as well as a variety of protein kinase substrates with M,s 75 K, 80 K, 57 K, 55 K, and 53 K are shown inserted in the lipid bilayer of the vesicle. Insert (B) illustrates an activated SV with calmodulin (Calm) bound to the 30 K protein kinase, and cAMP bound to the cAMP-dependent protein kinase. Phosphorylated protein substrates are depicted. Insert (C) illustrates a SV that has fused with the SPm. Insert (D) illustrates proteins of the SV integrated in the SPM prior to their selective reuptake. Insert (E) illustrates the insertion of clathrin triskelions (Y) into the phosphorylated CP proteins of M, 175 K and 55 K after activation of CP calcium/CaM-dependent protein kinaSe. It is postulated that clathrin inserts into the proteins only in their phosphorylated state. Insert (F) illustrates a CV formed from the invaginated CP. It is postulated that the integrity and existence of the coat depends on the phosphorylated state of the 175 K and 55 K protein substrates. During Step 1, there is an increase in calcium and cAMP concentration. During Step 2 the SV is propelled to and fuses with the presynaptic plasma membrane. During Step 3 there is a decrease in calcium and cAMP concentration and SV proteins intermix with the presynaptic plasma membrane proteins. During Step 4 there is a local increase in calcium ion concentration activation of membrane calcium/CaM-dependent protein kinase and transglutaminase activities with resultant crosslinking of specific SV proteins destined for retrieval, insertion of soluble cytoplasmic clathrin triskelions into the membrane with the resultant formation of a CP. During Step 5, as a result of several processes discussed in the text, the CP invaginates, pinches off and forms a CV. During Step 6 there is a decrease in local calcium concentration, activation of CV protein phosphatase resulting in the shedding of the clathrin triskelions from the vesicle and eventual transformation of the CV into a SV. The clathrin triskelions join the cytoplasmic soluble pool. NT = neurotransmitter.

PRESYNAPTIC

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and stored in SV, piracetam may activate SV and SPM PLA2, facilitating release of the newly-formed transmitter leading to improvement of memory. The defect in various Alzheimer patients may be releated to a lack of acetylcholine as well as to some pathology in the neurotransmitter release process which pharmacological PLA2 activation somehow may ameliorate. It is hoped our hypothesis can be used as a preliminary working model which eventually may help to elucidate abnormalities in brain function. It is possible that a pathological diversion of any one step of any one of the many multiple enzymatic cascades in the axon terminal may lead to profound alteration in brain function. The a u t h o r s t h a n k J o h n M o r g a n w h o edited this r e p o r t in m a n u s c r i p t . S u p p o r t e d by N.I.H. g r a n t # N S 12467 to S.P.

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