Regular ArticleThe alpha-synuclein mutation E46K promotes aggregation in cultured cells
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.
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Materials and methods
To study the phenomenon of aggregation and the effect of E46K mutation on aggregation in comparison with other known AS mutants, A30P, A53T, and E46K mutants were generated through polymerase chain reaction (PCR) and cloned into pcDNA 3.1 (Invitrogen) vector. Wild-type (WT) AS (Jakes et al., 1994), a gift from Michael Goedert was similarly sub-cloned into pcDNA 3.1 cloning vector. While cloning the E46K mutant, a glycine deletion mutation (E46KΔG) adjacent to intended lysine mutation was
Generation of AS mutants
A PCR-based approach was utilized to generate AS mutants. The clones were screened by restriction fragment length analysis and confirmed by dideoxy-mediated chain termination method. Serendipitously, during the generation of the E46K construct, a deletion of a glycine residue at the 47th position generated a novel construct (E46KΔG). Since this deletion-mutation is located within a KTKEGV motif, it allowed us to further test the importance of this motif in the aggregability of AS. An
Discussion
The underlying pathogenic mechanisms leading to PD are unknown; however, AS has been implicated in both sporadic and many familial forms of PD. Three mutations have been described leading to autosomal-dominant disease: A53T in Italian–Greek (Contursi) kindred (Polymeropoulos et al., 1997), A30P in German kindred (Kruger et al., 1998), and E46K in Spanish kindred (Zarranz et al., 2004). Each of these mutations occurs in the N-terminus in or around the KTKEGV consensus repeats. Earlier, it has
Acknowledgments
We would like to thank Dr. Michele Goedert for AS construct. We are grateful to Dr. John Trojanowski for AS antibodies. This work was supported by grants from the American Federation on Aging Research and the National Institute on Aging (K08 AG20764). Dr. Galvin is a recipient of the Paul Beeson Physician Faculty Scholar Award in Aging Research. This project was also supported by generous gifts from the Alan A. and Edith L. Wolff Charitable Trust and the Blue Gator Foundation.
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