The alpha-synuclein mutation E46K promotes aggregation in cultured cells

Abstract

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 (E46KΔG) 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-E46KΔG and 18% of AS-E46K 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 E46KΔG mutants demonstrated two distinct kinds of inclusions: Type I, which showed dense granular profile; and Type II, 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 mutations in 4th KTKEGV repeat lead to higher propensity of aggregation of AS compared to other mutants.

Introduction

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, affecting 1–2% of the population older than 65 years and 4–5% of those aged over 85 years (Eriksen et al., 2003). To date, more than 10 genes and loci have been implicated in PD (Dekker et al., 2003); however, most sporadic and familial forms of PD are linked to the aggregation of α-synuclein (AS). Symptoms of PD are largely attributed to the loss of dopaminergic neurons in substantia nigra pars compacta. The pathology of PD is characterized by the accumulation and aggregation of AS in neuronal cell bodies as Lewy bodies (LBs), and in neuronal processes as Lewy neuritis (LNs) or spheroids (Forno, 1996). Deposition of AS is common to several other neurodegenerative diseases such as Dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer’s disease (LBVAD), multiple systems atrophy (MSA), and neurodegeneration with brain iron accumulation type-1 (Galvin, 2003, Galvin et al., 2000).

We hypothesize that PD is a disease of protein misfolding due to the structural perturbations and dynamic instability of protein–protein interactions of AS protein with its interaction partners. Structure prediction algorithms and structural data demonstrate AS to be unstructured protein (Dunker et al., 2001, Uversky et al., 2001, Uversky, 2002) having extensive interactions with other proteins and co-factors (Peri et al., 2003, Uversky, 2003). Structural perturbation of the protein due to point mutations may lead to aberrant interactions initiating a cascade which ultimately leads to aggregation and fibrillization of AS into LBs and LNs. However, it has been difficult to model AS aggregation in cell culture models. Model systems using non-neuronal lines such as human embryonic kidney cells (HEK293) or adrenal pheochromocytoma cells lines (PC12) (Stefanis et al., 2001) have inconsistently demonstrated punctate aggregates without fibrillar forms, other investigators have been entirely unable to demonstrate aggregates (Iwata et al., 2001). Utilizing the catecholaminergic, neuroblastoma-derived cell line SH-SY5Y (5Y cells) as a model system, we describe the effect of three known familial AS mutations and one novel mutation on the aggregation properties of AS, and document the presence of two distinct kinds of inclusions in cultured cells expressing mutant forms of AS protein.