Abstract
α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity.
Highlights
␣-Synuclein (␣-syn)6 is a key factor in the pathogenesis of Parkinson disease (PD)
This study presents several lines of evidence that ␣-syn cycling on and off synaptic vesicles is coupled to exocytosis in partnership with the Rab3a recycling machinery that includes Rab-specific GDP dissociation inhibitor (GDI) and the 90-kDa heat shock protein (Hsp90)
Proteins were transferred to nitrocellulose (Life Sciences) and probed by Western blotting using the following antibodies: ␣-syn; a rabbit polyclonal raised to a 24-mer ␣-syn-specific peptide (LWS1, 1:1000); synaptophysin; synphilin-1; Rab3a; GDI; Hsp90; tetra-His (Qiagen); syntaxin; and glyceraldehyde-3phosphate dehydrogenase (GAPDH)
Summary
Transgenic Mice—Mice expressing human wild-type, A30P, or A53T ␣-syn were created as described previously [9] and crossed with SNCA null mice [2] from The Jackson Laboratory to create transgenic lines lacking endogenous murine ␣-syn. Following three washes with Tris-lysis buffer, the resulting bead-bound proteins were removed through incubation with 50 l of 2ϫ Sample Buffer (20% v/v glycerol, 0.1 M Tris, pH 6.8, 4% SDS, 0.008% bromphenol blue, and 2.5% -mercaptoethanol) at 95 °C for 5 min. For fractionation of membrane and cytosolic proteins, cells were hypotonically lysed in swelling buffer (10 mM HEPES and 18 mM KOAc, pH 7.2) and centrifuged at 20,000 ϫ g for 5 min and separated into supernatant and pellet. Proteins were transferred to nitrocellulose (Life Sciences) and probed by Western blotting using the following antibodies: ␣-syn (monoclonals, 211 from Neomarkers and Syn-1 from BD Transduction Laboratories, both used at 1:1000); a rabbit polyclonal raised to a 24-mer ␣-syn-specific peptide (LWS1, 1:1000); synaptophysin (monoclonal antibody at 1:10,000, Biodesign International); synphilin-1 (goat polyclonal anti-carboxyl-terminal at 1:2000, Research Diagnostics, Inc.); Rab3a (polyclonal 3335; Abcam); GDI (monoclonal 81.2; Synaptic Systems); Hsp (polyclonal 44721; Abcam); tetra-His (Qiagen); syntaxin (monoclonal HPC-1; Sigma); and glyceraldehyde-3phosphate dehydrogenase (GAPDH) (monoclonal 6C5; Meridian Life Science). Statistical comparisons were done with GraphPad InStat software using Student’s t test for comparisons between two groups or ANOVA (Bonferroni test) for multiple comparisons
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
