The presynaptic plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa) has been implicated as one of several neural-specific components that direct constitutive fusion mechanisms to the regulated vesicle trafficking and exocytosis of neurotransmitter release.There exist two alternatively spliced isoforms of a and b, which differ in a putative membrane-interacting domain. We show that these two isoforms have distinct quantitative and anatomical patterns of expression during brain development, in neurons, and in neuroendocrine cells and that the proteins localize differently in neurites of transfected PC12 pheochromocytoma cells. These findings indicate that alternative isoforms of SNAP-25 may play distinct roles in vesicular fusion events required for membrane addition during axonal outgrowth and for release of neuromodulatory peptides and neurotransmitters.In the nervous system, the regulated release of neurotransmitters and neuromodulatory peptides and the addition of proteins and other constituents required for neurite outgrowth proceed through intracellular trafficking and fusion of vesicles at the plasma membrane (for review, see refs. 1-4). This regulated fusion of donor vesicle and acceptor (target) membranes is likely mediated by the same ubiquitious ATPdependent machinery, composed of soluble N-ethylmaleimide-sensitive factor (NSF) and NSF-associated proteins (SNAPs), that is utilized in constitutive pathways of all eukaryotic cells (5). To provide specificity for the fusion process, distinct sets of related proteins have been postulated to serve as membrane receptors, or SNAREs (6). These enable correct recognition between vesicle and target membranes at different intracellular compartments, and they engage the general fusion machinery. It is likely that specific SNAREs, together with additional cell-specific auxiliary proteins, are critical for the specialized sorting, targeting, and final recycling of vesicles which are required for the Ca2+-triggered release of neurotransmitter and modulatory peptides unique to neurons and neuroendocrine cells (7-9). Although evidence suggests that membrane addition to the expanding plasmalemma of axonal growth cones is Ca2+ dependent (10), it is thought that there are independent vesicle-targeting and fusion mechanisms that chiefly support either axon outgrowth or neurotransmitter release. Recent studies do suggest, however, that at least one of the SNAREs, SNAP-25, is a key player in membrane fusion events of both developing and mature neurons.The presynaptic nerve terminal protein SNAP-25 (synaptosome-associated protein of 25 kDa; ref. 11) has been identified as a plasma membrane protein that together with syntaxin and the synaptic vesicle proteins VAMP/synaptobrevin and synaptotagmin are thought to constitute an initial SNARE docking complex for regulated exocytosis (5, 12, 13). After docking, this complex can incorporate into a 20S fusion parThe publication costs of this article were defrayed in part by page charge payment. This a...

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Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potentialdependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splicevariant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury.

Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic ␤-cell. Subcellular fractionation of isolated ␤-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with ϳ35% compared with control after glucose stimulation and with ϳ65% after depolarization with glucose and KCl. Capacitance measurements performed on single ␤-cells that expressed a dominantnegative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca 2؉ concentration. Taken together these results show that Cdk5 is present in ␤-cells and acts as a positive regulator of insulin exocytosis.Insulin is stored in secretory granules in pancreatic ␤-cells and upon stimulation with secretagogues insulin is released by exocytosis. Exocytosis has been suggested to be mediated by the same core fusion machinery that traverses intracellular membrane traffic in all cells (1-3). It was reported that the membrane fusion event required the N-ethylmaleimide sensitive factor (NSF), 1 and soluble NSF Attachment Proteins, ␣-, ␤-, and ␥-SNAP (1, 2). In addition to NSF and SNAPs, a 7 S core complex with SNAp receptors or "SNARE" proteins corresponding to the vesicle component synaptobrevin/vesicular-associated membrane protein, as well as the plasma membrane proteins SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin were necessary for neuronal exocytosis (3). The SNARE hypothesis proposes that the SNARE proteins form trans-complexes between adjacent membranes, thereby forcing them to proximity. After association of ␣-SNAP, the ATPase NSF completes the reaction by disassembling the SNARE complex leading to membrane bilayer mixing (3). More recently, trans-SNARE pairing and NSF activity has been suggested to act either prior to docking of vesicles or after membrane bilayer mixing (4 -9). Initially the SNARE proteins were regarded as neuron-specific. However, syntaxin, SNAP-25, synaptobrevin/ vesicular-associated membrane protein, and other synaptic proteins regulating neuronal exocytosis have also been identified in pancreatic ␤-cells, suggesting that the mechanism for insulin secretion is similar to that of neurotransmitter release from synaptic vesicles in neurons (10 -13). Regulated secretion of insulin from pancreatic ␤-cells has to be tightl...