difopein and Parkinson-Disease

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

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3θ overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3θ overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3θ further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3θ's effects on LRRK2 kinase activity. 14-3-3θ overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3θ to reduce LRRK2 kinase activity required direct binding of 14-3-3θ with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3θ can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.

Topics: 14-3-3 Proteins; Animals; Cell Enlargement; Cells, Cultured; HEK293 Cells; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neurites; Neurons; Parkinson Disease; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Proteins; Serine

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