Abstract
14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson's disease (PD) and the ability of 14-3-3s to interact with α-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, -ɛ, and -γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, -ɛ, or -γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium, whereas other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homologue (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression – particularly of the 14-3-3θ, -ɛ, and -γ isoforms – in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD.
Highlights
Parkinson’s disease (PD) is a neurodegenerative disorder affecting more than four million older individuals in the most populous nations.[1]
In an effort to identify factors contributing to a-syn toxicity, we have previously evaluated alterations in gene expression in the substantia nigra (SN) of transgenic mice overexpressing human wild-type a-syn.[13]
The observation that [-3] expression was dysregulated in a rodent model of a-syn overexpression was of particular interest because of other evidence linking 14-3-3s to PD. 14-3-3s share structural homology with a-syn,[17] and coaggregate with a-syn in Lewy bodies in human PD18 and in a mouse PD model.[] and a-syn can be co-immunoprecipitated from mammalian brains,[11,17] and co-immunoprecipitation is increased in PD brains.20 14-3-3Z is a negative regulator of the protein parkin,[20] mutation of which leads to early onset PD.[21]
Summary
14-3-3 isoform expression in a-syn transgenic mice. We had previously examined changes in overall gene expression in the SN of a-syn transgenic mice and found that distinct [-3] isoforms were among the several hundred genes whose expression was altered in this gene array study.[13]. The reduction in inclusion formation was not secondary to reduced a-syn expression, as expression levels of synT were comparable between control and 14-3-3y-transfected cells (Figure 2f). Given our results in the a-syn inclusion assay, we evaluated whether levels of insoluble a-syn were reduced in 14-3-3y cells as compared to control cells in response to rotenone. Cells infected with 14-3-3y shRNA did not show increased rotenone toxicity compared to control cells (Supplementary Figure 1b) These results were confirmed using another 14-3-3y shRNA in the same lentiviral construct (data not shown). Transgenic C. elegans overexpressing human wild-type a-syn show a predictable loss of dopaminergic neurons as indicated by Figure 5 Overexpression of 14-3-3y protects M17 cells from rotenone toxicity. The 14-3-3y-overexpressing line was more resistant to rotenone at several different concentrations compared to control stable cells at all time points tested. Semiquantitative RT-PCR was used to confirm lack of ftt-2 expression (data not shown)
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