Alpha synuclein aggregation in Parkinson's disease: Role of primary structure, membranes, and oxidative stress

Jagadish Kumar Hindupur Ranganathan, Purdue University

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by a loss of dopaminergic neurons in the substantia nigra. Among several factors involved in the pathology of PD, oxidative stress and aggregation of the presynaptic protein α-synuclein (aSyn) play a major role. A neuropathological hallmark of PD is the presence in some surviving neurons of cytosolic inclusions named Lewy bodies (1–3), which consist largely of fibrillar aSyn (4–7). Two types of mutations involving the aSyn gene have been linked to early-onset, autosomal-dominant PD: (i) mutations that increase the copy number of the aSyn gene, including a triplication (8) and a duplication (9); and (ii) point mutations that encode the proteins A30P (10), E46K (11), and A53T (12). The purpose of this study is to investigate mechanisms involved in aSyn aggregation. In one set of experiments, we identified primary structural features of aSyn that modulate the protein’s self-assembly by comparing the aggregation behavior of chimeric variants that consist of different combinations of human and mouse residues. Mouse aSyn, which differs from human aSyn at 7 residues, fibrillizes at a faster rate compared to human aSyn. This difference in fibrillization rate can be explained by an NMR study showing that autoinhibitory, intramolecular (‘long-range’) interactions between the N-terminal and central NAC region of human wild-type aSyn are markedly diminished in the mouse protein. We found that the residues at positions 87, 121 and 122 played an important role in regulating aSyn oligomerization and fibrillization, presumably by effects on intramolecular long-range interactions and intermolecular hydrophobic interactions. The perturbation of the long-range interactions may increase flexibility between the N-terminal and NAC regions, resulting in an enhanced fibrillization rate. Using human-mouse chimera variants, we also investigated the role of primary structure in dopamine-mediated oligomerization of aSyn. We found that different variants had different propensities to form adducts with dopamine on the basis of fluorescence measurements, and the variants underwent dopamine-mediated oligomerization to different extents and at different rates as determined by western blot analyses. The second purpose of this study was to investigate the role of membranes in aSyn aggregation. aSyn has high affinity for phospholipid membranes and binds reversibly to vesicles and organelles. Membrane binding is known to stimulate aSyn aggregation. To understand the relationship between the physiological composition of membranes and aSyn aggregation, we investigated the effect of changing the lipid composition on the formation of membrane-bound aSyn oligomers. We found that the presence of sphingomyelin in combination with either anionic PG or zwitterionic PC favors the formation of aSyn aggregates at the membrane surface, whereas PE in combination with PG or PC disfavors aSyn membrane binding and aggregation. However, sphingomyelin in combination with PE is conducive to the formation of membrane bound aSyn aggregates, suggesting that sphingomyelin plays a dominant role by changing the membrane properties to enable aSyn binding and aggregation. Oxidative stress is known to play an important role in PD pathogenesis. For example, oxidized products of dopamine can form adducts with aSyn (as demonstrated by our data) and these adducts promote the accumulation of potentially toxic aSyn oligomers. In addition, membrane lipid oxidation under conditions of oxidative stress can result in changes in membrane fluidity and permeability, and in turn these changes can affect aSyn membrane binding and aggregation. Further, oxidative modifications of aSyn itself are likely to affect the protein’s aggregation properties. The antioxidant enzyme MsrA relieves oxidative stress and repairs oxidized aSyn, and these effects may decrease the formation of toxic aSyn assemblies in dopaminergic neurons. In this study, we showed for the first time that oxidized methionine residues in recombinant aSyn are reduced by recombinant MsrA and MsrA purified from cell extracts. Collectively, our data highlight the deleterious effects of various forms of oxidative stress (e.g. dopamine- or lipid-induced) in modifying aSyn and driving its aggregation. In this context a multipronged therapeutic approach capable of targeting both oxidative stress and aSyn aggregation needs to be developed.

Degree

M.S.

Advisors

Rochet, Purdue University.

Subject Area

Molecular biology|Neurosciences|Biochemistry|Pathology

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