Date Published: June 12, 2012
Publisher: Public Library of Science
Author(s): Oi Wan Wan, Kenny K. K. Chung, Philipp J. Kahle. http://doi.org/10.1371/journal.pone.0038545
α-synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson’s disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation.
Parkinson’s disease (PD) is a common neurodegenerative disorder that is marked by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies (LBs) –. LBs are cytoplasmic eosinophilic protein aggregates with α-synuclein (α-syn) as one of the major components , . α-syn is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of PD –. The familial PD (FPD) linked point mutations and gene triplications in α-syn suggest that abnormal structure or excessive accumulation of α-syn can enhance its toxicity and lead to the degeneration of dopaminergic neurons in PD. In different studies, both wild type (WT) and mutant forms of α-syn have been shown to have a high propensity for forming oligomers and fibrils when incubated in vitro, while in α-syn transgenic animal model, the presence of α-syn aggregation is associated with neuronal degeneration. , –. These results suggest that the process of oligomerization, fibrillization and aggregation of α-syn are the culprits behind the neurodegeneration seen in PD , , –. However, some studies have also suggested that α-syn aggregates might be protective and oligomers and pre-fibrilliar α-syn are the toxic species responsible for neurodegeneration . For instance, small molecule that facilitates α-syn inclusion formation or histone deacetylase inhibition that enhances enlarged α-syn inclusion formation provides protection in cell against α-syn induced toxicity –. A recent study has also shown that α-syn mutants that have reduced propensity to form fibrils and aggregates have increased toxicity . In this study, we used another approach to determine if α-syn aggregation is directly related to its cellular toxicity by generating α-syn mutants that have a higher propensity to form intracellular aggregates. We used a 16 amino acids peptide called CL1, which has been shown to destabilize GFP for proteasomal degradation and enhance the GFP aggregation , . We generated α-syn mutants by attaching the CL1 peptide to the C-terminal and studied how the enhancement of α-syn aggregation affected its toxicity in cellular and animal models. Our results provide new insights into how α-syn-induced toxicity is related to its aggregation.
Previous studies have suggested that oxidative stress or post-translational modification such as nitration and phosphorylation enhance the oligomerization and aggregation of α-syn which can subsequently cause the degeneration of dopaminergic neurons –, –. However, the primary toxic species of α-syn has not been well defined. Initial studies suggested that prominent α-syn aggregates such as Lewy bodies and neurites are the components that cause the neuronal degeneration, but studies have also suggested that protein aggregates might be protective –. The understanding of the role of α-syn aggregation in PD is crucial for the development of novel treatments that target α-syn aggregation. In this study, we addressed the role of α-syn aggregation and cytotoxicity by using the characteristic features of the CL1 peptide. Attachment of CL1 sequestered both WT and mutant α-syn to the detergent-insoluble fraction of the cells and facilitated their aggregation. With this approach, we were able to demonstrate a direct correlation between α-syn aggregation and reduced cell viability. Interestingly, while WT-CL1 α-syn had a shorter half-life as determined by the pulse-chase experiment, the steady state protein level remained comparable to WT α-syn (Figure 1 A & B). This might suggest that the WT and WT-CL1 expression levels were different and might contribute to the difference in α-syn aggregation and cell viability. However, we also found that coexpression of HSP70 or treatment of MG132, which affected α-syn aggregation and cell viability, did not affect the overall expression levels of WT and WT-CL1 α-syn (Figure S1 A & B), suggesting that the expression levels were not a contributing factor in our study. In addition, the CL1-enhanced aggregation and increased toxicity of α-syn was specific as attachment of CL1 to β-syn and Htt-Q23 enhanced their aggregation but did not affect their cytotoxicity. From the SEC, we found that attachment of CL1 enhanced α-syn oligomerization and this was further increased in cells treated with MG132. In contrast, coexpression of HSP70 significantly reduced the oligomerization of α-syn. Interestingly, we observed that coexpression of HSP70 could reduce the size of WT-CL1 as determined by SEC, suggesting that HSP70 could possibly modulate the overall conformation of WT-CL1.