Membrane association and protein conformation of α-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations

Two missense mutations (Ala-30 → Pro and Ala-53 → Thr) in the gene encoding α-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, α-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of α-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of α-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between α-synuclein and cellular membranes. Both the N-and C-terminal regions of α-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 → Pro mutation alters the three-dimensional conformation of α-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.