Role of alpha-synuclein primary structure and oxidative stress in Parkinson's disease pathology

Katherine E Strathearn, Purdue University

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder which affects approximately 1% of the population 65 years of age and younger. Two classic hallmarks of PD include loss of dopaminergic neurons from the substantia nigra and the presence in surviving neurons of Lewy Bodies. Lewy bodies are cellular inclusions which contain fibrillar forms of the presynaptic protein, alpha-synuclein (aSyn). aSyn mutants involved in familial PD (e.g. A53T, A30P, E46K) have a greater tendency to self-associate than the wild-type (WT) protein, suggesting that aSyn aggregation contributes to PD pathogenesis. Accordingly, understanding more about how the primary structure of aSyn relates to aggregation and neurotoxicity can provide greater insight into the pathology of PD. Whereas patients carrying the A53T mutation typically suffer from PD, aged mice that also carry this mutation do not show PD phenotypes. Comparison of the human and mouse aSyn sequences reveals seven mismatches including A53T. Moreover, 5 of the 6 additional mismatches are located in the C-terminal region (residues 100, 103, 107, 121, and 122). We chose to focus on the role of residues 121 and 122 because evidence suggests that the C-terminal region encompassing these two residues modulates aSyn self-assembly’ (or ‘aggregation’). Primary midbrain cultures were transduced with adenoviruses encoding Chimera 1 (human A53T with mouse residues at 121 and 122), Chimera 2 (mouse aSyn with human residues at 121 and 122), mouse aSyn, and A53T aSyn. Cultures were analyzed immunocytochemically to assess relative dopaminergic cell viability. Our data showed that mouse and Chimera 1 had no effect on dopaminergic cell viability compared to that in the untransduced control, whereas A53T and Chimera 2 were significantly toxic compared to mouse and Chimera 1. Taken together, these results identified two key C-terminal residues, D121 and N122, as being involved in aSyn-mediated toxicity. To further elucidate the role of the C-terminal region, we investigated the impact of oxidative stress and C-terminal post-translational modifications on aSyn aggregation and toxicity. Previous studies in the lab revealed that aSyn undergoes post-translational modifications, including phosphorylation of Ser 129 and nitration or phosphorylation of Tyr 125, 133, and 136, in neuronal cells exposed to rotenone, an inhibitor of mitochondrial complex I that causes oxidative stress. We found that the appearance of these modifications correlated with the formation of membrane-bound and cytosolic aSyn aggregates in rotenone-treated PC12 cells. To determine the effects of tyrosine modifications on aSyn aggregation, we constructed variants in which the three tyrosine residues were replaced with phenylalanine (‘3YF’, to eliminate post-translational modifications) or aspartate (‘3YD’, to mimic the introduction of a negative charge by tyrosine phosphorylation), and we examined the impact of these substitutions on aSyn aggregation. PC12 cells expressing each variant were cultured in the absence or presence of rotenone and separated into membrane and cytosolic fractions, and levels of SDS-resistant aSyn oligomers in each fraction were determined via Western blotting. Results suggested that 3YF and 3YD have reduced and increased propensities (respectively) to form membrane-bound aSyn aggregates in rotenone-treated cells. Additional experiments revealed that 3YF and 3YD exhibit reduced and enhanced dopaminergic cell killing (respectively) in primary midbrain cultures, These results imply that (i) phosphorylation and/or nitration of the three C-terminal tyrosine residues leads to an increase in aSyn self-assembly and neurotoxicity, and (ii) a build-up of membrane-bound aggregates correlates with aSyn-mediated dopaminergic cell death. Given the importance of oxidative stress in PD pathogenesis as outlined above, we hypothesized that natural products with strong antioxidant properties (e.g. phenolic compounds) may protect dopaminergic neurons from PD related-insults. To address this hypothesis, we examined the effects of extracts enriched in anthocyanins (ANC) (e.g. a blueberry (BB) extract) proanthocyanidins (PAC), phenolic acids (PA), or stilbenes on rotenone neurotoxicity in primary midbrain cultures. We found that in general dopaminergic cell death elicited by rotenone was suppressed to a greater extent by extracts enriched in ANC than those enriched in PAC, PA, or stilbenes. In other studies, we showed that a BB alleviated aSyn neurotoxicity in the primary cell culture model and inhibited aSyn fibril formation in a test-tube model. Collectively, our data indicate that the C-terminal region of aSyn, specifically residues 121 and 122, play an important role in aSyn neurotoxicity. Additionally, our results suggest that oxidative stress stimulates the formation of toxic aSyn aggregates by promoting harmful post-translational modifications in the C-terminal region, and dopaminergic neurons may be protected from PD-related insults by phenolic-rich extracts. Our findings provide insight into PD pathogenesis and suggest that antioxidant therapies may help slow disease progression.

Degree

Ph.D.

Advisors

Rochet, Purdue University.

Subject Area

Molecular biology|Neurosciences|Cellular biology

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