Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

␣-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. ␣-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, ␣-synuclein is natively unstructured but assumes an ␣-helical conformation upon binding to phospholipid membranes. We now show that this conformation of ␣-synuclein consists of two ␣-helical regions that are interrupted by a short break. The structural organization of the ␣-helices of ␣-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.In recent years, the presynaptic protein ␣-synuclein has attracted much attention because of its involvement in neurodegenerative diseases (1-3). Two independent mutations in human ␣-synuclein cause familial Parkinson's disease, and wild type ␣-synuclein is a major component of Lewy bodies, cytoplasmic inclusion bodies found in Parkinson's disease and in several forms of neurodegenerative dementia. However, independent of its role in neurodegenerative diseases, ␣-synuclein is an interesting protein in its own right. It is an abundant presynaptic protein that may regulate neurotransmitter release and may contribute to synaptic plasticity (4 -6). ␣-Synuclein is the founding member of a protein family that additionally includes ␤-and ␥-synucleins and synoretin (7-9). The sequences of all synucleins are similar, although only ␣-synuclein is implicated in disease. Synucleins are composed of six copies (␤-synuclein) or seven copies (all other synucleins) of an unusual 11-residue imperfect repeat, followed by a variable short hydrophilic tail. Synucleins are soluble, natively unfolded proteins that avidly bind to negatively charged phospholipid membranes and become ␣-helical upon binding (10). Although secondary structure predictions indicate that the synuclein repeats could form an amphipathic structure consistent with lipid binding, the ␣-helical conformation is puzzling because the synuclein repeats are punctuated by central glycine residues. Furthermore, in Lewy bodies ␣-synuclein is thought to be in a ␤-strand aggregate, but aggregation of ␣-synuclein into dimers and multimers is promoted by lipid environments that induce an ␣-helical conformation (11-13). In the present study, we have examined the conformation of ␣-synuclein in lipidic environments to understand the relation of its sequence to its physicochemical properties and to map a potential pathway of misfolding in neurodegenerative disease. EXPERIMENTAL PROCEDURESProduction of ␣-Synuclein-Recombinant ␣-synuclein was expressed in bacteria as GST-fusion proteins with a TEV protease recognition sequence preceding the N-terminal methionine and cleaved with TEV protease (Invitrogen), resulting in a single additional glycine residue at the N terminus. After TEV cleavage, ␣-synuclein was isolated as the only heat-stable component upon boiling for 15 min, purified by i...

An abundant presynaptic protein, ␣-synuclein, is centrally involved in the pathogenesis of Parkinson's disease. However, conflicting data exist about the normal function of ␣-synuclein, possibly because ␣-synuclein is redundant with the very similar ␤-synuclein. To investigate the functions of synucleins systematically, we have now generated single-and double-knockout (KO) mice that lack ␣-and͞or ␤-synuclein. We find that deletion of synucleins in mice does not impair basic brain functions or survival. We detected no significant changes in the ultrastructure of synuclein-deficient synapses, in short-or long-term synaptic plasticity, or in the pool size or replenishment of recycling synaptic vesicles. However, protein quantitations revealed that KO of synucleins caused selective changes in two small synaptic signaling proteins, complexins and 14-3-3 proteins. Moreover, we found that dopamine levels in the brains of double-KO but not single-KO mice were decreased by Ϸ20%. In contrast, serotonin levels were unchanged, and dopamine uptake and release from isolated nerve terminals were normal. These results show that synucleins are not essential components of the basic machinery for neurotransmitter release but may contribute to the long-term regulation and͞or maintenance of presynaptic function.