Protein array analysis of oligomerization-induced changes in alpha-synuclein protein–protein interactions points to an interference with Cdc42 effector proteins

Abstract

Aggregation of α-synuclein may contribute to neuropathology in Parkinson's disease patients and in transgenic animal models. Natively unfolded α-synuclein binds to various proteins and conformational changes due to α-synuclein misfolding may alter physiological interactions. In the present study, we used protein arrays spotted with 5000 recombinant human proteins for a large scale interaction analysis of monomeric versus oligomeric α-synuclein. Monomeric α-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by α-synuclein oligomerization. Incubation with recombinant α-synuclein oligomers lead to the identification of several GTPase activating proteins and Cdc42 effector proteins as binding partners. Protein database searches revealed a Cdc42/Rac interactive binding domain in some interactors. To demonstrate in vivo relevance, we analyzed brainstem protein extracts from α-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous α-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with α-synuclein from brainstem lysates and were colocalized with α-synuclein aggregates in brain sections by double immunostaining. By two-dimensional gel electrophoretic analysis of synaptosomal fractions from transgenic mouse brains we detected additional isoforms of septin 6, a downstream target of Cdc42 effector proteins.Small GTPases have recently been identified in a genetic modifier screen to suppress α-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by α-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration.