Absence of junctional glutamate receptor clusters in Drosophila mutants lacking spontaneous transmitter release.

Little is known about the functional significance of spontaneous miniature synaptic potentials, which are the result of vesicular exocytosis at nerve terminals. Here, by using Drosophila mutants with specific defects in presynaptic function, we found that glutamate receptors clustered normally at neuromuscular junctions of mutants that retained spontaneous transmitter secretion but had lost the ability to release transmitter in response to action potentials. In contrast, receptor clustering was defective in mutants in which both spontaneous and evoked vesicle exocytosis were absent. Thus, spontaneous vesicle exocytosis appears to be tightly linked to the clustering of glutamate receptors during development.

Cysteine string proteins are associated with cortical granules of Xenopus laevis oocytes.

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II-VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.

Lithium ions Up-regulate mRNAs encoding dense-core vesicle proteins in nerve growth factor-differentiated PC12 cells.

We recently reported that lithium ions induced an up-regulation of cysteine string protein (CSP) gene expression in nerve growth factor (NGF)-differentiated PC12 cells but not in undifferentiated cells. Concomitantly, expression of two other proteins of regulated secretory pathways, synaptophysin (SY) and SNAP-25, was unaffected by lithium. To assess further the specificity of this effect of lithium, we used cDNA arrays. Our data indicate that lithium ions increase the level of mRNA for proteins such as secretogranin II and vesicular monoamine transporter 1 that are preferentially associated with large densecore secretory vesicles (LDCVs) without affecting mRNAs for proteins predominantly affiliated with small synaptic-like vesicles, including the vesicular acetylcholine transporter and SY. This action of lithium is detected in NGF-differentiated PC12 cells but not in undifferentiated cells. These observations suggest that lithium ions modulate the turnover of LDCVs, and this may play a role in mediating the therapeutic action of lithium in manic-depressive illness.

Lithium ions enhance cysteine string protein gene expression in vivo and in vitro.

Lithium is a well established pharmacotherapy for the treatment of recurrent manic-depressive illness. However, the mechanism by which lithium exerts its therapeutic action remains elusive. Here we report that lithium at 1 mM significantly increased the expression of cysteine string proteins (CSPs) in a pheochromocytoma cell line (PC12 cells) differentiated by nerve growth factor. These cells concomitantly exhibited increased expression of CSPs in their cell bodies and boutons. Enhanced CSP expression was also observed in the brain of rats fed a lithium-containing diet, which elevated serum lithium to a therapeutically relevant concentration of approximately 1.0 mM. However, both in vitro and in vivo, the expression of another synaptic vesicle protein, synaptophysin, and the t-SNARE, synaptosomal-associated protein of 25 kDa (SNAP-25), was not significantly altered by lithium. These observations indicate that lithium-induced changes of CSP gene expression may contribute to the therapeutic efficacy of this monovalent cation.

Antibodies against cysteine string proteins inhibit evoked neurotransmitter release at Xenopus neuromuscular junctions.

Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we introduced anti-CSP antibodies into the cell bodies of Xenopus spinal motor neurons that form synapses with embryonic muscle cells in culture. These antibodies produced a rapid (within 3-6 min), and in most cases complete, inhibition of stimulus-dependent neurotransmitter secretion. However, spontaneous neurotransmitter release was stable (both in frequency and amplitude) throughout the period of antibody exposure. Several control experiments validated the specificity of the anti-CSP antibody effects. First, the anti-CSP antibody actions were not mimicked either by antibodies against another synaptic vesicle protein SV2, or by nonspecific immunoglobins. Second, heat treatment of the anti-CSP antibodies eliminated their effect on evoked secretion. Third, immunoblot experiments showed that the anti-CSP and anti-SV2 antibodies were highly selective for their respective antigens in these Xenopus cultures. We conclude from these results that CSPs are vital constituents of the pathway for regulated neurotransmitter release in vertebrates. Moreover, the selective inhibition of evoked, but not spontaneous transmitter release by anti-CSP antibodies indicates that there is a fundamental difference in the machinery that mediates these secretory processes.

Electrical and optical monitoring of alpha-latrotoxin action at Drosophila neuromuscular junctions.

Electrophysiological recording demonstrates that alpha-latrotoxin, a 125,000 mol. wt component of black widow spider venom, promotes high frequency quantal discharges at larval neuromuscular junctions of Drosophila. Concomitantly, fluorescence imaging of presynaptic calcium ion activity reveals that this toxin qualitatively elevates cytosolic ionized calcium in this preparation. These activities of alpha-latrotoxin are selectively antagonized by a monoclonal antibody, 4C4.1, that was previously shown to inhibit the action of this toxin in PC-12 cells. However, 4C4.1 does not block the release-promoting activity of gel-filtered extracts of black widow spider venom. This indicates that black widow spider venom has multiple components that promote quantal transmitter secretion in invertebrates. This investigation demonstrates that alpha-latrotoxin is among the active principles in black widow spider venom that enhance transmitter release and raise cytosolic ionized calcium in Drosophila. These results suggest that Drosophila, because of the relative ease of genetic manipulation, may be useful to study the target protein(s) that mediate the binding and action of alpha-latrotoxin at nerve endings. Moreover, the procedure that we report for loading Drosophila nerve terminals with the calcium ion-sensing dye, Calcium Crimson, may have utility for studying calcium dynamics in mutant alleles with alterations in synapse development and function in this organism.

Attenuated influx of calcium ions at nerve endings of csp and shibire mutant Drosophila.

Previous work has shown that cysteine-string proteins (csps) are synaptic vesicle proteins that are important for evoked neurotransmitter release at Drosophila neuromuscular junctions. Indirect evidence has implicated csps in a regulatory link between synaptic vesicles and presynaptic calcium (Ca) channels. In this report, we use Ca Crimson to monitor stimulus-dependent changes of cytosolic Ca at motor nerve terminals of csp mutant Drosophila. These mutants display temperature-sensitive (TS) paralysis and a presynaptic failure of evoked synaptic transmission. We show that this TS inhibition of neuromuscular transmission is correlated with a block of Ca ion entry at nerve endings of csp mutants. These data support the hypothesis that csps mediate a regulatory interaction between synaptic vesicles and presynaptic Ca channels. Moreover, these results predict that if one depletes nerve endings of synaptic vesicles, one may see a reduction of presynaptic Ca ion entry. Defects of the dynamin gene in TS shibire mutant Drosophila interfere with synaptic vesicle recycling and lead to an activity-dependent depletion of these organelles. Our results show that Ca influx is blocked at nerve terminals of shibire mutant larvae at the same time that synaptic transmission fails in these organisms. Thus, using two completely independent Drosophila mutants, we demonstrate that synaptic vesicles and csps are vital for the function of presynaptic Ca channels.

A Xenopus cysteine string protein with a cysteine residue in the J domain.

A cDNA clone encoding a Xenopus cysteine string protein (Xcsp) was isolated and sequenced. The deduced primary sequence of Xcsp is very similar to other vertebrate csps with the exception of a cysteine residue that lies outside of the cysteine-string domain. This cysteine residue replaces a serine that is highly conserved among vertebrate csps, and thus may be of functional importance. Xcsp mRNA appears as a 4.6 kb species on Northern analysis, and immunoblot of Xenopus brain membranes reveals a single, 35 kDa Xcsp that can be deacylated, like other csps.

Evidence that cysteine string proteins regulate an early step in the Ca2+-dependent secretion of neurotransmitter at Drosophila neuromuscular junctions.

Previous work indicated that the temperature-dependent block of synaptic transmission in cysteine string protein (csp) mutants of Drosophila was attributable to a failure of nerve impulses to trigger transmitter release. The current investigations were undertaken to resolve in more detail the mechanism of this transmission deficit. Our studies reveal that the spider venom toxin alpha-latrotoxin can trigger a sustained discharge of quanta at neuromuscular junctions of csp mutant larvae at nonpermissive temperature. The same is true of the calcium ionophore ionomycin. However, solutions with an elevated concentration of K or Ca ions fail to circumvent the block of quantal secretion in these mutants. Likewise, 4-aminopyridine, which augments transmitter release at permissive temperature in csp mutants, fails to reverse the inhibition of impulse-evoked transmitter release at elevated temperature. These data are consistent with the hypothesis that there is a deficit either in Ca ion entry or in the ability of Ca ions to trigger exocytosis in csp mutants at nonpermissive temperatures. In part, because of previous work showing that csps are important for the functional expression of N-type Ca channels in frog oocytes, we favor the idea that csps participate in a regulatory interaction involving presynaptic Ca channels.

Cysteine-string proteins: a cycle of acylation and deacylation?

We used tunicamycin, an inhibitor of protein fatty acylation, to examine the possibility that there is a cycle of acylation and deacylation of cysteine string proteins at nerve terminals. Using both physiological and immunoblot approaches, we obtained no evidence for a cycle of acylation and deacylation that affects these proteins. These data suggest that this lipid modification of cysteine string proteins is relatively more stable than that observed for other nerve ending proteins, like SNAP-25.

Cysteine string protein immunoreactivity in the nervous system and adrenal gland of rat.

Cysteine string proteins (csps) are a recently discovered class of cysteine-rich proteins. They have been shown to associate preferentially with synaptic vesicle fractions of Torpedo electric organ or rat brain where they have been implicated in events associated with transmitter secretion. However, to date there has been no information concerning the distribution of csps in rat tissues. We investigated the localization of csps in the rat retina and CNS using immunohistochemistry with affinity purified anti-csp antibodies. Specific csp immunoreactivity having a punctate appearance is present throughout the neuraxis. Csp immunoreactivity is particularly abundant in synapse-rich regions including those of the retina, main olfactory bulb, hippocampal formation, and cerebellum. White matter tracts are devoid of csp immunoreactivity. Neuromuscular junctions show strong csp immunoreactivity. This localization of csp immunoreactivity is compatible with a role for csps in presynaptic events at a wide variety of synapses. Immunohistochemical analysis of a non-neuronal, secretory tissue, the adrenal gland, reveals prominent csp immunoreactivity in the chromaffin cells of the adrenal medulla. However, csp immunoreactivity is not detected in adrenal cortical regions. These findings are confirmed and extended by immunoblot and Northern analyses which identify a 35 kDa and a 5 kb product, respectively, in extracts of adrenal. The presence of csps in the adrenal medulla suggests that these proteins may also participate in secretion-related events in certain non-neuronal cells.

Cysteine-string proteins as templates for membrane fusion: models of synaptic vesicle exocytosis.

Cysteine-string proteins are relatively small, cysteine-rich components of synaptic vesicle membranes. Recent investigations demonstrated that at least 11 of the 13 cysteine residues of the Torpedo cysteine-string protein are fatty acylated. This exceptional level of fatty acylation occurs along a short stretch (less than 25 residues) of amino acids which are flanked on either side by very polar amino and carboxy termini. This amphipathic structure may have unique capabilities to catalyze events at membrane interfaces. We propose two distinct pathways to explain how these capabilities might subserve membrane fusion and exocytosis.

Cysteine string proteins and presynaptic function.

A brief review is presented of investigations of a novel family of synaptic vesicle proteins, the cysteine string proteins (csps). Studies of csp mutants in Drosophila reveal that csps are crucial components of the excitation-secretion machinery at nerve terminals. Current data cannot distinguish between a primary role of csps in modulating calcium ion influx at the nerve terminal versus a more-direct role in the exocytotic cascade. In this context, the remarkable post-translational modification of csps, namely the fatty acylation of as many as 12 of the 13 cysteine residues of the Torpedo protein, suggests that csps may participate more directly in the process of membrane fusion that underlies exocytosis. This would be achieved by using the fatty acyl chains of the csps as templates for 'lipid flow' that would allow the fusion of vesicular and plasma membranes. These hypotheses provide a useful framework for empirical tests of the role of csps in nerve terminal function.

Presynaptic dysfunction in Drosophila csp mutants.

Cysteine string proteins are synapse-specific proteins. In Drosophila, csp deletion mutants exhibit temperature-sensitive paralysis and early death. Here, we report that neuromuscular transmission is impaired presynaptically in these csp mutant larvae. At 22 degrees C, evoked transmitter release is depressed relative to wild type and rescued controls, and high frequency stimulation of the nerve leads to sporadic failures. At 30 degrees C, stimulus-evoked responses decline gradually before failing completely. When the temperature is returned to 22 degrees C, evoked responses recover. Spontaneous release events persist at both 22 degrees C and 30 degrees C. Since nerve conduction and postsynaptic sensitivity are unaffected, these data indicate that csp mutations disrupt depolarization-secretion coupling. This disruption explains the cellular basis of the temperature-sensitive paralysis of these organisms.

Glia of the cholinergic electromotor nucleus of Torpedo are the source of the cDNA encoding a GAT-1-like GABA transporter.

A PCR-based strategy was used to clone DNAs encoding Na(+)- and Cl(-)-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

Extensive lipidation of a Torpedo cysteine string protein.

Cysteine string proteins are relatively low mass components of synaptic vesicle membranes. Structurally, their primary sequence is distinguished by a remarkable, cysteine-rich motif. Investigations revealed an unprecedented degree of lipidation of these cysteine residues. At least 11 of the 13 cysteines of the Torpedo protein were modified, principally by palmitoyl moieties. This fatty acylation creates a prominent hydrophobic domain flanked by polar amino and carboxyl termini. An amphipathic structure of this type is uniquely suited to mediate events at membrane interfaces. Thus, cysteine string proteins are candidates to participate in exocytotic membrane fusion.

Cysteine string proteins: a potential link between synaptic vesicles and presynaptic Ca2+ channels.

Presynaptic calcium channels are key regulators of neurotransmitter release. Oocyte expression studies suggest that cysteine string proteins are essential subunits or modulators of these channels. Subcellular fractionation revealed that cysteine string proteins copurify with synaptic vesicles. An average vesicle had eight protein monomers with both the amino and carboxyl termini detected on the cytoplasmic face. Thus, docked synaptic vesicles may regulate presynaptic calcium channels and neurotransmitter release.

Suppression cloning of the cDNA for a candidate subunit of a presynaptic calcium channel.

A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.

Expression of Ca2+ receptors in Xenopus oocytes injected with poly(A)+ mRNA from a rat calcitonin-secreting cell line.

Poly(A)+ mRNA extracted from rat calcitonin-secreting cells (rMTC 44-2) was injected into Xenopus oocytes. In mRNA-injected oocytes the intracellular Ca2+ concentration ([Ca2+]i), measured with the Ca2+ indicator dye, fura2, increased in response to an elevation of the extracellular Ca2+ ions ([Ca2+]o). In some oocytes [Ca2+]i transiently increased in high [Ca2+]o but it did not respond to the subsequent alterations of [Ca2+]o. The addition of 10 microM carbonyl cyanide m-chlorphenylhydrazone (CCCP) to the extracellular medium restored the dependence of [Ca2+]i on [Ca2+]o in such cells. It was concluded that rMTC 44-2 cells possessed a receptor which recognizes changes in [Ca2+]o and that these receptors can be functionally expressed by microinjection of messenger RNA from rMTC 44-2 cells into Xenopus oocytes.