Cysteine string proteins.

Cysteine string protein (CSP) was discovered by use of a synapse-specific, monoclonal antibody to screen a cDNA expression library in Drosophila. A vertebrate CSP homolog was later identified and shown to co-purify with synaptic vesicles. CSP-α is now recognized as a membrane constituent of many regulated secretory organelles. Knockout of the csp gene in Drosophila produced temperature-sensitive paralysis reflecting a loss of evoked (but not spontaneous) transmitter release. However, CSP's role in regulated exocytosis remains ambiguous. Fruit flies lacking the csp gene also exhibited nerve terminal degeneration as did mice deficient in the csp-α gene. This phenotype has been ascribed to the depletion of a functional pool of the t-SNARE, SNAP-25. However, recent studies showing that an endosomal pool of CSP-α contributes to a novel, protein-export pathway argues that CSP's role in neurodegeneration is more complex. Clients of this later pathway include tau and α-synuclein, proteins linked to neurodegeneration. Additionally, mutations in the csp-α gene cause an adult-onset, neuronal ceroid lipofuscinosis and diminished CSP-α expression is an early event in Alzheimer's disease. Collectively, these findings indicate that much remains to be learned about the role of CSPs in cellular secretory pathways and human disease.

Fast, synchronous neurotransmitter release: Past, present and future.

This mini-review starts with a summary of the crucial contributions Ricardo Miledi made to our understanding of how the action potential triggers fast, synchronous transmitter release. It then transitions to the discovery of synaptotagmin and its role as the exocytotic Ca2+ sensor at nerve terminals. The final section confronts the array of unique models that have been proposed to explain the membrane fusion step of exocytosis. More than a dozen different hypotheses seek to explain the terminal steps of the exocytotic cascade. It will be an interesting challenge for the field to distinguish among these possibilities. Nevertheless, with ongoing technological advances, perhaps we will have a better picture of this process by the end of the coming decade. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Insights into the structure and molecular topography of the fatty acylated domain of synaptotagmin-1.

Abundant attention has focused on synaptotagmin's C2 domains, but less is known about the structure and function of its other regions. Here, we synthesized the N-acetylated, C-end amidated and Cys-palmitated peptide (VLTCCFCICK KCLFKKKNKK K) which includes the fatty acylated cysteine residues in the membrane-affiliated domain of synaptotagmin-1. Fourier-transform infrared spectrometry indicated that this peptide's conformation is influenced by environmental polarity. In artificial bilayer membranes, this peptide exhibited abundant ß-structure. Electron microscopy revealed that this peptide also promoted the stacking of liposome membranes. Together these results suggest that the fatty acylated region of synaptotagmin-1 is likely to adopt ß-structure in biological membranes. This preference for ß-structure versus α-helix has functional implications for the role of synaptotagmin-1 in synaptic vesicle exocytosis.

A Membrane-Fusion Model That Exploits a ß-to-α Transition in the Hydrophobic Domains of Syntaxin 1A and Synaptobrevin 2.

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals. This "dyad model" advanced a novel proposal concerning the membrane disposition of the palmitoylated, cysteine-rich region of these synaptotagmins. Unexpectedly, it now emerges that a similar principle can be exploited to reveal how the hydrophobic, carboxyl-terminal domains of syntaxin 1A and synaptobrevin 2 can perturb membrane structure at the interface between a docked synaptic vesicle and the plasma membrane. These "ß-to-α transition" models will be compared and contrasted with other proposals for how macromolecules are thought to intervene to drive membrane fusion.

The Structure of the Synaptic Vesicle-Plasma Membrane Interface Constrains SNARE Models of Rapid, Synchronous Exocytosis at Nerve Terminals.

Contemporary models of neurotransmitter release invoke direct or indirect interactions between the Ca2+ sensor, synaptotagmin and the incompletely zippered soluble, N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex. However, recent electron microscopic (EM) investigations have raised pragmatic issues concerning the mechanism by which SNAREs trigger membrane fusion at nerve terminals. The first issue is related to the finding that the area of contact between a "fully primed" synaptic vesicle and the plasma membrane can exceed 600 nm2. Approximately four-thousands lipid molecules can inhabit this contact zone. Thus, renewed efforts will be needed to explain how the zippering of as few as two SNARE complexes mobilizes these lipids to achieve membrane fusion. The second issue emerges from the finding that "docking filaments" are sandwiched within the area of vesicle-plasma membrane contact. It is challenging to reconcile the location of these filaments with SNARE models of exocytosis. Instead, this commentary outlines how these data are more compatible with a model in which a cluster of synaptotagmins catalyzes exocytotic membrane fusion.

Fast, Ca2+-dependent exocytosis at nerve terminals: shortcomings of SNARE-based models.

Investigations over the last two decades have made major inroads in clarifying the cellular and molecular events that underlie the fast, synchronous release of neurotransmitter at nerve endings. Thus, appreciable progress has been made in establishing the structural features and biophysical properties of the calcium (Ca2+) channels that mediate the entry into nerve endings of the Ca2+ ions that trigger neurotransmitter release. It is now clear that presynaptic Ca2+ channels are regulated at many levels and the interplay of these regulatory mechanisms is just beginning to be understood. At the same time, many lines of research have converged on the conclusion that members of the synaptotagmin family serve as the primary Ca2+ sensors for the action potential-dependent release of neurotransmitter. This identification of synaptotagmins as the proteins which bind Ca2+ and initiate the exocytotic fusion of synaptic vesicles with the plasma membrane has spurred widespread efforts to reveal molecular details of synaptotagmin's action. Currently, most models propose that synaptotagmin interfaces directly or indirectly with SNARE (soluble, N-ethylmaleimide sensitive factor attachment receptors) proteins to trigger membrane fusion. However, in spite of intensive efforts, the field has not achieved consensus on the mechanism by which synaptotagmins act. Concurrently, the precise sequence of steps underlying SNARE-dependent membrane fusion remains controversial. This review considers the pros and cons of the different models of SNARE-mediated membrane fusion and concludes by discussing a novel proposal in which synaptotagmins might directly elicit membrane fusion without the intervention of SNARE proteins in this final fusion step.

Synaptotagmins 1 and 2 as mediators of rapid exocytosis at nerve terminals: the dyad hypothesis.

The dyad model was developed to explain the extremely rapid kinetics of synaptic vesicle exocytosis. In contrast to most hypotheses which invoke interactions among synaptotagmins, SNAREs and other regulatory molecules, the dyad model features a quartet of synaptotagmins arrayed at the synaptic vesicle-plasma membrane interface. Ca(2+)-triggered movements of these synaptotagmins initiate a sequence of events culminating in the fusion of the vesicular and plasma membranes. The relative simplicity of this model and its amenability to empirical testing provide a useful template for future investigations of the molecular events underlying the exocytotic cascade.

Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

Cysteine string protein beta is prominently associated with nerve terminals and secretory organelles in mouse brain.

Cysteine string proteins (CSPs) are associated with regulated secretory organelles in organisms ranging from fruit flies to man. Mammals have three csp genes (alpha, beta and gamma), and previous work indicated that expression of the csp-beta and -gamma genes was restricted to the testes. For the current investigation, antibodies specific for CSP-beta were developed. Unexpectedly, immunoblot analysis indicated that CSP-beta was prominently expressed throughout mouse brain. Upon sub-cellular fractionation, CSP-beta was enriched in synaptosomes and synaptic vesicle fractions. Interestingly, CSP-beta existed almost exclusively as part of a high mass complex both in testis and brain. This complex required aggressive denaturation to release monomeric CSP-beta. By Northern analysis CSP-beta mRNA was present at very low abundance as a approximately 1.0kb species in mouse brain. Collectively, the enrichment of CSP-beta in synaptosomes and the association of CSP-beta with synaptic vesicles suggest that CSP-beta, like CSP-alpha, may be an important component of the regulated secretory machinery in mouse brain.

A role for myosin 1e in cortical granule exocytosis in Xenopus oocytes.

Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis. Myosin 1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin 1e relocates to the surface of cortical granules. Overexpression of myosin 1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin 1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin 1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin 1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin 1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis.

Myosin-1c couples assembling actin to membranes to drive compensatory endocytosis.

Compensatory endocytosis follows regulated exocytosis in cells ranging from eggs to neurons, but the means by which it is accomplished are unclear. In Xenopus eggs, compensatory endocytosis is driven by dynamic coats of assembling actin that surround and compress exocytosing cortical granules (CGs). We have identified Xenopus laevis myosin-1c (XlMyo1c) as a myosin that is upregulated by polyadenylation during meiotic maturation, the developmental interval that prepares eggs for fertilization and regulated CG exocytosis. Upon calcium-induced exocytosis, XlMyo1c is recruited to exocytosing CG membranes where actin coats then assemble. When XlMyo1c function is disrupted, actin coats assemble, but dynamic actin filaments are uncoupled from the exocytosing CG membranes such that coats do not compress, and compensatory endocytosis fails. Remarkably, there is also an increase in polymerized actin at membranes throughout the cell. We conclude that XlMyo1c couples polymerizing actin to membranes and so mediates force production during compensatory endocytosis.

Interaction between constitutively expressed heat shock protein, Hsc 70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes.

In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by approximately 80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.

Lithium enhances secretion from large dense-core vesicles in nerve growth factor-differentiated PC12 cells.

Considerable attention has been focused on the therapeutic role of lithium (Li) in bipolar disorders. Although no consensus has emerged, Li presumably influences the behavior of neurons that regulate mood and behavior. Using PC12 cells to study cellular and molecular actions of Li, we previously reported that Li modulates the expression of proteins associated with large dense-core vesicles (LDCVs; organelles typically containing monoamines, neuropeptides and other cargo proteins). The current investigation indicates that this enhanced expression of LDCV proteins correlates with an altered secretory phenotype in Li-treated cells. Immunoblotting detects significant increases in the cellular content and secretion of the LDCV cargo proteins chromogranin B and secretogranin II. Amperometry reveals an increase of spike number elicited by K+-depolarization of Li-treated cells but no change of spike amplitude or kinetics. Electron microscopy reveals no significant change in LDCV number per unit area in Li-treated cells. However, there is a significant increase (about 15%) in the diameter of LDCVs after Li. Thus, Li induces changes in the properties of LDCVs that culminate in augmented regulated secretion in nerve growth factor-differentiated PC12 cells. These results extend our understanding of Li-dependent changes of cellular function that may be germane to the therapeutic action of Li.

Convergent effects of lithium and valproate on the expression of proteins associated with large dense core vesicles in NGF-differentiated PC12 cells.

Lithium and valproate are chemically unrelated compounds that are used to treat manic-depressive illness. Previously, we reported that lithium ions upregulate genes encoding proteins primarily associated with large dense core vesicles (LDCV) in nerve growth factor (NGF)-differentiated PC12 cells, but not in undifferentiated PC12 cells. Moreover, lithium did not alter the expression of proteins associated with small-clear, synaptic-like vesicles (SSV) in these cells. Based on these observations, we investigated whether valproate had actions similar to those of lithium in PC12 cells. Thus, undifferentiated or NGF-differentiated PC12 cells were exposed to lithium (1 mM) or valproate (1 mM) for 48 h. Extracts from these cells were submitted to semiquantitative Northern and Western analyses. In NGF-differentiated cells, both agents increased the expression of proteins associated with LDCV, the vesicular monoamine transporter 1 (VMAT1), and cysteine string protein (CSP). These same treatments did not alter the expression of proteins primarily associated with SSV, the vesicular acetylcholine transporter (VAChT), and synaptophysin (SY). Furthermore, neither drug affected the expression of these proteins in undifferentiated cells. Interestingly, secretion of (3)H-dopamine was increased in cells exhibiting the increase of VMAT1 and csp. Taken together, the convergent effects of these chemically diverse compounds suggest that altered dynamics of LDCV may play a vital role in the biochemical pathway, leading to the relief of the symptoms of manic depression.

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes.

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.

Dietary lithium induces regional increases of mRNA encoding cysteine string protein in rat brain.

Lithium salts are used to treat manic-depressive disorders; however, the mechanism by which lithium produces its therapeutic benefit remains obscure. The action of lithium may involve alterations of proteins important for regulating synaptic function. In this context, we observed recently that lithium at therapeutically relevant concentrations enhanced expression of cysteine string protein (csp) at the level of both mRNA and protein, in cell culture and in rat brain. Several lines of evidence have shown that csps are vital components of the regulated secretory pathway. We were interested whether lithium modulates expression of csp in specific brain regions. To study this issue, we analyzed the effects of chronic lithium administration (21 days) on csp mRNA levels in rat brain using in situ hybridization. Densitometric analysis revealed that lithium upregulated csp mRNA in several brain areas that are important for mood and behavior. This effect may be germane to understanding the beneficial action of lithium in mood disorders.

Lithium ions modulate the expression of VMAT2 in rat brain.

Recent work has indicated that lithium (at 1 mM, a concentration that is efficacious in the treatment of manic-depressive disorders) modulates the level of vesicular monoamine transporter 1 (VMAT1) mRNA in PC12 cells as a function of the differentiation status of these cells. To ascertain whether VMAT expression in neurons is sensitive to lithium, in vivo, rats were fed a lithium-supplemented diet for 21 days (which raised serum lithium to 0.98+/-0.1 mM). Northern analysis revealed an overall increase (199+/-27%) of the neuronal VMAT isoform (VMAT2) in rat brain after lithium. However, in situ hybridization analysis revealed regional differences in the effects of lithium. Thus, VMAT2 mRNA increased by 50-100% over control in the raphe nuclei, ventral tegmental area, and substantia nigra of rats fed the lithium diet. Concomitantly, VMAT2 mRNA declined by about 50% in the locus coeruleus. Because VMAT2 is expressed in neurons that are strongly implicated in regulating mood and behavior, these data support the hypothesis that alterations of VMAT2 expression contribute to the therapeutic effects of lithium in psychiatric disorders.

Nerve growth factor signals via preexisting TrkA receptor oligomers.

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.

Activation of protein kinase Ceta triggers cortical granule exocytosis in Xenopus oocytes.

Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel (calcium-independent) PKCs. Pharmacological investigations using Gö6976 and Gö6983 tended to exclude PKCdelta, epsilon and mu as secretory triggers. Subcellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCeta that colocalized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCdelta and eta strongly supported the conclusion that it is PKCeta that initiates cortical granule exocytosis in these cells. These observations represent an important step in identifying the mechanism of secretory triggering in this system.