Abstract. cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxyterminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

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Parkin, the most commonly mutated gene known to result in familial Parkinson's disease (PD), 1 encodes an E3 ubiquitin ligase (1). Several substrates for parkin have been identified including CDCrel-1, an o-glycosylated form of ␣-synuclein ␣Sp22, Pael-R (2), and synphilin-1 (3-5). These parkin substrates have little sequence or functional similarities; however, Pael-R and ␣-synuclein have a propensity to misfold and aggregate (2, 4). This common property of known parkin substrates suggests that parkin may play a general role in the degradation of misfolded proteins, which might otherwise overwhelm the ubiquitin-proteasome system (UPS).Parkin has been demonstrated to function in the endoplasmic reticulum-associated degradation (ERAD) of misfolded ER proteins (2, 6). Parkin is up-regulated during the unfolded protein response (6). Pael-R overexpression results in ER accumulation of the protein, causing ER stress-induced cell death; parkin overexpression ameliorates these effects (2). Proteasome function is also important for normal ERAD and proteasomal dysfunction can cause ER stress (7, 8). Whereas ERAD is an important pathway for eliminating misfolded proteins in the ER, there are many misfolded aggregation-prone proteins that are translated in the cytosol including most of the polyglutamine (poly(Q)) containing proteins. Accumulation of misfolded cytosolic and ER-translated proteins can ultimately inhibit proteasomal activity (9 -11).Whereas the cytotoxicity of expanded poly(Q) proteins may be because of a variety of mechanisms (12-16), expanded poly(Q) proteins impair proteasome function (9 -11). Proteasomal dysfunction has also been demonstrated in PD brain and may play a role in the accumulation of aberrant proteins and neuronal loss that characterize several of the adult neurodegenerative diseases (17). Accumulation of aberrant proteins is a hallmark of both PD and the poly(Q) expansion diseases, which include Huntington's disease (HD) and several spinocerebellar ataxias. Overexpression of aberrant proteins has been very useful for identifying genes and proteins capable of modifying their accumulation or toxicity. Molecular chaperones such as Hsp70 improve cell viability (18 -20) and facilitate the elimination of poly(Q) proteins in cellular models and ameliorate disease phenotype in transgenic Drosophila models (18,(21)(22)(23)(24)(25). Hsp70 also improves the phenotype in a Drosophila PD model in which human ␣-synuclein is overexpressed (26). Parkin appears to interact with Hsp70 along with the ubiquitinating factor CHIP (27). The N terminus of parkin contains a domain homologous to ubiquitin called the ubiquitin-like (Ubl) domain. A similar domain in the human homologue of the yeast DNA repair factor (hhRad23) has been shown to interact with expanded poly(Q) proteins (28) and bind the proteasome (29,30). Other Ubl domain containing proteins such as Dsk2 (31, 32) and Ubp6 (33) also bind the proteasome. These findings suggest that parkin may also bind expanded poly(Q) proteins and proteasomes via its Ubl ...

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2^3 days, which converted approximately half of the SNAP-25 to an altered form lacking the final nine C-terminal residues. The distribution of toxindamaged to control SNAP-25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/ A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A-truncated SNAP-25. Return of BoNT/A-truncated SNAP-25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for s 11 weeks, while recovery from BoNT/E is complete in 6 3 weeks. This longer duration of enzymatic activity appears to account for the persistence of serotype A action.z 1999 Federation of European Biochemical Societies.