Systematic Mutagenesis of α-Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities

alpha-Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, alpha-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, alpha-synuclein mutations and alpha-synuclein overexpression cause Parkinson's disease, and aggregates of alpha-synuclein are found as Lewy bodies in multiple neurodegenerative disorders ("synucleinopathies"). The relation of the physiological functions to the pathological effects of alpha-synuclein remains unclear. As an initial avenue of addressing this question, we here systematically examined the effect of alpha-synuclein mutations on its physiological and pathological activities. We generated 26 alpha-synuclein mutants spanning the entire molecule, and analyzed them compared with wild-type alpha-synuclein in seven assays that range from biochemical studies with purified alpha-synuclein, to analyses of alpha-synuclein expression in cultured neurons, to examinations of the effects of virally expressed alpha-synuclein introduced into the mouse substantia nigra by stereotactic injections. We found that both the N-terminal and C-terminal sequences of alpha-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the central region of alpha-synuclein, referred to as nonamyloid beta component (residues 61-95), as well as point mutations linked to Parkinson's disease (A30P, E46K, and A53T) increased the neurotoxicity of alpha-synuclein but did not affect its physiological function in SNARE-complex assembly. Thus, our data show that the physiological function of alpha-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of alpha-synuclein.