Cross-linking of alpha-synuclein and Lewy body formation have been implicated in the dopaminergic neuronal cell death observed in Parkinson's disease (PD); the mechanisms responsible, however, are not clear. Reactive oxygen species and advanced glycation end products (AGEs) have been found in the intracellular, alpha-synuclein-positive Lewy bodies in the brains of both PD as well as incidental Lewy body disease patients, suggesting a role for AGEs in alpha-synuclein cross-linking and Lewy body formation. The aims of the present study were to determine 1) whether AGEs can induce cross-linking of alpha-synuclein peptides, 2) the progressive and time-dependent intracellular accumulation of AGEs and inclusion body formation, and 3) the effects of extracellular or exogenous AGEs on intracellular inclusion formation. We first investigated the time-dependent cross-linking of recombinant human alpha-synuclein in the presence of AGEs in vitro, then used a cell culture model based on chronic rotenone treatment of human dopaminergic neuroblastoma cells (SH-SY5Y) over a period of 1-4 weeks, in the presence of different doses of AGEs. Cells (grown on coverslips) and cell lysates, collected at the end of every week, were analyzed for the presence of intracellular reactive oxygen species, AGEs, alpha-synuclein proteins, and intracellular alpha-synuclein- and AGE-positive inclusion bodies by using immunocytochemical, biochemical, and Western blot techniques. Our results show that AGEs promote in vitro cross-linking of alpha-synuclein, that intracellular accumulation of AGEs precedes alpha-synuclein-positive inclusion body formation, and that extracellular AGEs accelerate the process of intracellular alpha-synuclein-positive inclusion body formation.
2008 Wiley-Liss, Inc.