The alpha-synuclein mutation E46K promotes aggregation in cultured cells

Parkinson's disease (PD) is characterized by the polymerization of wild-type (WT) or mutant alpha-synuclein (AS) into aggregates and fibrils, which are observed as Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients. However, inability to demonstrate aggregation in many cell Culture systems is a major drawback for effective in vitro modeling of AS aggregation. Utilizing PCR-based cloning approach, we generated A30P, A53T, and the recently reported E46K encoding mutation in the KTKEGV repeat region of AS gene. While cloning E46K mutant, a glycine deletion mutation (E46K Delta G) adjacent to the intended lysine Mutation was serendipitously generated. Expression of mutant constructs and green fluorescent protein (GFP)-tagged mutant constructs in catecholaminergic SH-SY5Y (5Y) cells revealed 40% of AS-E46K Delta G and 18% of ASE46K transfected cells formed aggregates as compared to 12% in AS-A53T, 6% in AS-WT, and 2% in AS-A30P transfected cells. Western blot analysis demonstrated the formation of high molecular weight AS aggregates. Electron microscopic analysis of 5Y cells expressing the E46K and E46K Delta G mutants demonstrated two distinct kinds of inclusions: Type 1, which showed dense granular profile; and Type 11, which were largely membranous vacuolar inclusions without granular material. These two inclusions are reminiscent of Lewy bodies and pale bodies observed in PD postmortem brain samples. Our results demonstrate that imitations in 4th KTKEGV repeat lead to higher propensity of aggregation of AS compared to other mutants. (c) 2005 Elsevier Inc. All rights reserved.