14-3-3 mitigates alpha-synuclein aggregation and toxicity in the in vivo preformed fibril model

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.

Keywords: 14-3-3s; Alpha-synuclein; Amygdala; Cortex; Dementia with Lewy Bodies; Mouse; Parkinson’s disease; Substantia nigra.

Fig. 1

14-3-3θ overexpression reduces and 14-3-3 inhibition increases social dominance deficits induced by fibrillar αsyn. a Transgenic difopein-eYFP expressing or 14-3-3θ-overexpressing transgenic mice and matching WT littermates were given a unilateral stereotactic injection of mouse αsyn monomer or PFFs in the striatum (STR) at 8–12 weeks of age. At 3 or 6 months post injection (mpi), mice underwent behavioral testing. Mice were then perfused, and their brains were sectioned by microtome and processed for immunohistochemical staining. b WT mice were given a unilateral injection of 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 PFFs in the STR at 12 weeks of age. Mice underwent behavioral testing at 6 mpi. Mice were then perfused, and their brains were sectioned by microtome and processed for immunohistochemical staining. c 14-3-3θ overexpression increased average win rate in comparison to WT PFF-injected mice. 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. Each mouse was matched against 3 separate WT mice injected with monomer over 3 individual rounds, and the win rate per mouse was determined across these 3 trials. n = 17–25 mice per group. *p < 0.05 (Tukey’s multiple comparison test). Error bars represent SEM. d Difopein transgenic expression in the cortex and amygdala decreased average win rate in comparison to PFF-injected WT mice. Quantification of difopein mice injected with monomer, difopein mice injected with PFFs, and WT mice injected with PFFs at 3 mpi in the tube test matched against monomer-injected WT mice. Mice were evaluated over 3 individual rounds. n = 14–16 per group. *p < 0.05 (Tukey’s multiple comparison test). Error bars represent SEM. e Quantification of difopein mice injected with monomer, difopein mice injected with PFFs, and WT mice injected with PFFs at 6 mpi in the tube test matched against monomer-injected WT mice. Mice were evaluated over 3 individual rounds. n = 14–15 per group. *p < 0.05, **p < 0.01 (Tukey’s multiple comparison test). Error bars represent SEM

Fig. 2

14-3-3θ overexpression delays αsyn inclusion formation in the cortex and amygdala. a Representative images of PFF-injected WT and 14-3-3θ transgenic mice stained for pS129-αsyn demonstrate extensive αsyn inclusions in the STR and cortex in WT mice at 3 mpi. Scale bar = 300 μm. b Representative images of PFF-injected WT and 14-3-3θ transgenic mice stained for pS129-αsyn and either NeuN or GFAP demonstrate that αsyn inclusions are primarily associated with neurons. Scale bar = 10 μm. c 14-3-3θ mice show decreased pS129-αsyn positive inclusion counts at 3 mpi in the cortex and amygdala, 2 areas with 14-3-3θ overexpression, but not the SN, which lacks 14-3-3θ overexpression. Quantification of pS129-αsyn positive inclusions in the cortex, SN, and amygdala of PFF-injected WT and 14-3-3θ mice at 3 mpi. n = 7–9 per group. *p < 0.05 (Student’s unpaired t-test). Error bars represent SEM. Scale bar = 100 μm for cortex and SN; 50 μm for amygdala. d 14-3-3θ mice show increased pS129-αsyn positive inclusion counts in the cortex and amygdala but no change in the SN compared to WT mice at 6 mpi. Quantification of pS129-αsyn positive inclusions in the cortex, SN, and amygdala of PFF-injected WT and 14-3-3θ mice at 6 mpi. n = 6–11 per group. *p < 0.05, **p < 0.01 (Student’s unpaired t-test). Error bars represent SEM. Scale bar = 100 μm for cortex and SN; 50 μm for amygdala

Fig. 3

AAV-mediated 14-3-3θ overexpression delays αsyn inclusion formation in the substantia nigra. a Representative images of immunohistochemistry for GFP (blue) and TH (brown) in brain sections containing the SN from AAV-GFP mice and AAV-14-3-3θ/GFP mice injected with αsyn monomers. Colocalization of GFP and TH staining indicates viral induction into dopaminergic nigral neurons in the targeted area. Scale bar = 100 μm for 10 × image; scale bar = 25 μm for inset. b Representative images of pS129-αsyn immunostaining in AAV-GFP mice and AAV-14-3-3θ/GFP mice injected with PFFs at 3 mpi. Scale bar = 100 μm. c Representative images of pS129-αsyn immunostaining in AAV-GFP mice and AAV-14-3-3θ/GFP mice injected with PFFs at 6 mpi. Scale bar = 100 μm. d AAV-mediated 14-3-3θ expression in PFF-injected mice decreases pS129-αsyn positive inclusion counts at 3 mpi and increases counts at 6 mpi in the SN. Quantification of nigral inclusions at 3 mpi and 6 mpi in AAV-GFP mice and AAV-14-3-3θ/GFP mice injected with PFFs. n = 13–14 per group. *p < 0.05 (Student’s t-test). Error bars represent SEM. e Representative images of PFF-injected AAV-14-3-3θ mice stained for pS129-αsyn and either NeuN or GFAP demonstrate that αsyn inclusions are primarily associated with neurons. Scale bar = 10 μm

Fig. 4

14-3-3 inhibition increases αsyn inclusion formation. a Representative images of eYFP-difopein immunostaining in the cortex of WT and difopein (“cortical” line 138) mice at 3 mpi. GFP-difopein expression is found only in difopein mice. Scale bar = 100 μm. b Representative images of pS129-αsyn immunostaining in the cortex of WT and difopein mice at 3 mpi. Scale bar = 100 μm. c Difopein expression in the cortex increases inclusion counts at 3 mpi and decreases counts at 6 mpi in the cortex. Quantification of pS129-αsyn positive inclusions at 3 mpi and 6 mpi in the cortex of PFF-injected WT or difopein mice. n = 15–16 per group at 3mpi; n = 13–14 per group at 6mpi. *p < 0.05 (Student’s t-test). Error bars represent SEM. d Representative images of eYFP-difopein immunostaining in the SN of WT and difopein (“nigral” line 166) mice at 3 mpi. GFP-difopein expression is found only in difopein mice. Scale bar = 100 μm. e Representative images of pS129-αsyn immunostaining in the SN of WT and difopein mice at 3 mpi. Scale bar = 100 μm. f Difopein expression in in the nigra increases inclusion counts at 3 mpi and decreases counts at 6mpi in the SN. Quantification of pS129-αsyn positive inclusions at 3 mpi and 6 mpi in the SN of PFF-injected WT and difopein mice. n = 7–9 per group at 3 mpi; n = 10–13 per group at 6 mpi. *p < 0.05 (Student’s t-test). Error bars represent SEM

Fig. 5

14-3-3θ overexpression mitigates reduction in nigral TH-positive neuronal counts in response to PFFs, while 14-3-3 inhibition exacerbates reductions in dopaminergic neurons. a Representative images of TH immunohistochemistry in the SN of AAV-GFP and AAV-14-3-3θ/GFP mice injected with αsyn monomers or PFFs at 6 mpi. Scale bar = 500 μm. b Stereological counts of TH-positive neurons in the SN of AAV-GFP and AAV-14-3-3θ/GFP mice injected with αsyn monomers or PFFs at 6 mpi. n = 9–14 per group. *p < 0.05 (Tukey’s multiple comparison test). n.s. = non-significant. Error bars represent SEM. c Representative images of TH immunohistochemistry in the SN of WT and difopein nigral mice (line 166) injected with αsyn monomers or PFFs at 6 mpi. Scale bar = 500 μm. d Stereological counts of TH-positive neurons in the SN of WT and difopein mice injected with αsyn monomers or PFFs at 3 mpi. n = 7–8 per group at 3 mpi. Error bars represent SEM. e Stereological counts of TH-positive neurons in the SN of WT and difopein mice injected with αsyn monomers or PFFs at 6 mpi. n = 11–14 at 6 mpi. *p < 0.05, **p < 0.01, ****p < 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM