alpha-synuclein and difopein

Alpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies. αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal counts in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.

Topics: 14-3-3 Proteins; alpha-Synuclein; Amygdala; Animals; Behavior, Animal; Cerebral Cortex; Disease Models, Animal; Dopaminergic Neurons; Gene Knock-In Techniques; Mice; Mice, Transgenic; Neurons; Parkinson Disease; Protein Aggregates; Protein Aggregation, Pathological; Proteins; Social Dominance; Substantia Nigra

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 alpha-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, ε, 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, ε, or γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), while 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 homolog (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, ε, 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.

Topics: 1-Methyl-4-phenylpyridinium; 14-3-3 Proteins; alpha-Synuclein; Animals; Caenorhabditis elegans; Cell Line, Tumor; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Parkinson Disease; Protein Isoforms; Proteins; RNA Interference; RNA, Small Interfering; Rotenone