α-synuclein aggregation in Parkinson's disease: Role of oxidative stress and membranes

Jeremy L Schieler, Purdue University

Abstract

Parkinson's disease is a progressive neurodegenerative disorder resulting from the loss of dopaminergic neurons in the substantia nigra. Surviving neurons are characterized by mitochondrial complex I inhibition, oxidative damage, and intraneuronal inclusions known as Lewy bodies. The underlying mechanism and the consequences of Lewy body formation are unknown. Lewy bodies are highly enriched with fibrillar α-synuclein. α-Synuclein is unstructured in solution but adopts a β-sheet conformation in its fibrillar form. Aggregated α-synuclein found in Lewy bodies has been shown to have a number of post-translational modifications that reflect oxidative damage. Oxidative modifications have been shown to promote the aggregation of α-synuclein in test-tube models, and the formation of protein inclusions in neurons is stimulated by oxidative stress. These inclusions (potential precursors of Lewy bodies) may play a role in sequestering oxidatively damaged proteins for repair or degradation. However, it is not known whether harmful post-translational modifications occur before, during, and/or after LB formation. It is also unclear which modifications affect the rate of α-synuclein aggregation in neurons. To investigate the link between oxidative stress and sequence-specific modifications of α-synuclein, we developed a stably transfected PC12 cell line that produces high levels of histidine-tagged α-synuclein. A method was developed to purify α-synuclein from PC12 cells, cultured in the absence or presence of rotenone, an inducer of oxidative stress, using metal affinity chromatography. The protein was analyzed by mass-spectrometry to assess differences in post-translational modifications associated with α-synuclein from untreated versus rotenone-treated cells. Modifications characteristic of α-synuclein in Lewy bodies were identified in the protein samples from rotenone-treated cells. Concomitant with rotenone-induced post-translational modifications of α-synuclein using biochemical and immunofluorescence methods, we also observed an increase in membrane bound aggregates of α-synuclein. DJ1, another protein correlated to Parkinson's disease through autosomal-recessive mutations in the gene encoding the protein has been demonstrated to protect neurons from complex I-mediated oxidative stress. We found that co-expression of DJ1 with α-synuclein was able to suppress the formation of membrane-bound aggregates of α-synuclein. These data suggest that oxidatively-driven modifications of α-synuclein promote aggregation and that DJ1 is able to mitigate this effect by an unknown mechanism.

Degree

Ph.D.

Advisors

Rochet, Purdue University.

Subject Area

Neurology|Pharmacology

COinS