Abstract
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.
Highlights
Alpha-synuclein is a critical protein whose aggregation and transmission from cell to cell has been implicated in the neurodegenerative process in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is a highly expressed brain protein whose endogenous function is not well understood but likely includesUnderwood et al acta neuropathol commun (2021) 9:13 regulation of synaptic transmission [1,2,3]
At 3 or 6 months post injection, mice underwent behavioral testing. Their brains were sectioned by microtome and processed for immunohistochemical staining. b WT mice were given a unilateral injection of associated virus (AAV)-14-3-3θ/GFP or AAV-GFP in the substantia nigra (SN) at 8 weeks of age. 4 weeks after AAV inoculation, mice were given an ipsilateral stereotactic injection of αsyn monomer or preformed fibril (PFF) in the STR at 12 weeks of age
Quantification of win rate of 14-3-3θ mice injected with monomer, 14-3-3θ mice injected with PFFs, or WT mice injected with PFFs in the tube test matched against monomer-injected WT mice at 6 mpi
Summary
Alpha-synuclein (αsyn) is a critical protein whose aggregation and transmission from cell to cell has been implicated in the neurodegenerative process in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is a highly expressed brain protein whose endogenous function is not well understood but likely includesUnderwood et al acta neuropathol commun (2021) 9:13 regulation of synaptic transmission [1,2,3]. We recently observed that the chaperone-like protein 14-3-3θ is a critical regulator of the release, oligomerization, and toxicity of αsyn in several cellular models [4]. Given 14-3-3s’ roles in protein folding and trafficking, we recently examined the impact of 14-3-3s on αsyn cell-to-cell transmission and toxicity in two separate cellular models: the paracrine αsyn model and the in vitro αsyn fibril model [4]. We found that 14-3-3θ reduces αsyn transfer and toxicity by inhibiting αsyn oligomerization, seeding, and internalization, whereas 14-3-3 inhibition accelerates the αsyn seeding and cell-to-cell transmission in these cellular models [4]. We observed that 14-33θ overexpression reduced social dominance deficits, delayed αsyn inclusion formation, and rescued reductions in tyrosine hydroxylase (TH)-positive neuronal counts, while pan 14-3-3 inhibition with the peptide inhibitor difopein accelerated behavioral deficits, αsyn inclusion formation, and reductions in neuronal counts in the PFF model
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