α-Synuclein triggers cofilin pathology and dendritic spine impairment via a PrPC-CCR5 dependent pathway

Abstract

Cognitive dysfunction and dementia are critical symptoms of Lewy Body dementias (LBD). Specifically, alpha-synuclein (αSyn) accumulation in the hippocampus leading to synaptic dysfunction is linked to cognitive deficits in LBD. Here, we investigated the pathological impact of αSyn on hippocampal neurons. We report that either αSyn overexpression or αSyn pre-formed fibrils (PFFs) treatment triggers the formation of cofilin-actin rods, synapse disruptors, in cultured hippocampal neurons and in the hippocampus of synucleinopathy mouse models and of LBD patients. In vivo, cofilin pathology is present concomitantly with synaptic impairment and cognitive dysfunction. Rods generation prompted by αSyn involves the co-action of the cellular prion protein (PrP^C) and the chemokine receptor 5 (CCR5). Importantly, we show that CCR5 inhibition, with a clinically relevant peptide antagonist, reverts dendritic spine impairment promoted by αSyn. Collectively, we detail the cellular and molecular mechanism through which αSyn disrupts hippocampal synaptic structure and we identify CCR5 as a novel therapeutic target to prevent synaptic impairment and cognitive dysfunction in LBD.

Fig. 1. αSyn overexpression induces cofilin-actin rod formation in hippocampal neurons.

A Representative images of DIV14 hippocampal neurons expressing GFP or αSyn and immunostained with αSyn (red). Scale bar: 20 μm. B, C Representative western blot (B) and respective quantification (C) of α-Syn and GFP levels in DIV14 hippocampal neurons expressing GFP or αSyn. Vinculin was used as loading control. Data represent mean±SEM (n = 3 independent samples/condition). ***p < 0.001 by Student’s t test. D Representative images of GFP- or αSyn-expressing hippocampal neurons immunostained for cofilin (red) and β3-tubulin (blue). Scale bar: 20 μm. Insets and arrowheads indicate cofilin-actin rod structures in αSyn-expressing neurons. E Quantification of the percentage of neurons with rods (shown as fold change relative to control) relative to D. Data represent mean±SEM (n = 3 independent experiments with ≥100 neurons/condition/experiment). **p < 0.01 by Student’s t test. F Representative images of dendrites of GFP- or αSyn-expressing hippocampal neurons immunostained for cofilin (red). Scale bar: 5 μm.

Fig. 2. Hippocampal cofilin pathology is recapitulated in Thy1-aSyn mice with cognitive impairment and in DLB patients.

A Representative images of brain sections from 6-month-old WT and Thy1-aSyn mice immunostained for αSyn pS129 (red). DAPI (blue). Scale bar: 20 μm. B, C Western blot analysis (B) and respective quantification (C) of αSyn and αSyn pS129 levels in hippocampus from WT and Thy1-aSyn mice. GAPDH was used as loading control. Data represent mean±SEM (n = 3–5 animals/genotype). *p < 0.05, ***p < 0.001 by Student’s t test. D–I Morris Water Maze (MWM) test in 6-month-old animals. D Schematic representation of the MWM test. E Latency to target in the learning phase. Data represent mean±SEM (n = 5–7 animals/genotype). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. F–I Probe trial with analyses of the total distance (F), distance in target (G), target crossings (H), and a representative track of the WT and Thy1-aSyn mice during the probe test (I). Data represent mean±SEM (n = 5–7 animals/genotype). *p < 0.05 by Student’s t test. J, K Western blot analysis (J) and respective quantification (K) of PSD-95 levels in the hippocampus of 6-month-old WT and Thy1-aSyn mice. GAPDH was used as loading control. Data represent mean±SEM (n = 3–5 animals/genotype). *p < 0.05 by Student’s t test. L Representative images of the hippocampal region of brain sections from 6-month-old WT and Thy1-aSyn mice immunostained for PSD-95 (white). Scale bar: 5 μm. M PSD-95 puncta analysis per μm² in the hippocampus region relative to L. Data represent mean±SEM (n = 9 animals/genotype). *p < 0.05 by Student’s t test. N Representative images of brain sections from 6-month-old WT and Thy1-aSyn mice immunostained for cofilin (red). DAPI (blue). Scale bar: 200 μm. N’ Zoom-ins from N. Scale bar: 20 μm. Arrowheads indicate cofilin-actin rod structures. O Representative images of brain sections from Control and DLB patients immunostained for cofilin (red). DAPI (blue). Scale bar: 50 μm. P Hippocampal cofilin pathology evaluated by the number of cofilin aggregates and rods per mm² (fold change relative to WT) relative to N. Data represent mean±SEM (n = 4–7 animals/genotype). *p < 0.05 by Student’s t test. Q Hippocampal cofilin pathology evaluated by the number of cofilin aggregates and rods per mm² relative to O. Data represent mean±SEM (n = 2–3 patients/condition).

Fig. 3. αSyn pre-formed fibrils induce cofilin-actin rods in hippocampal neurons.

A Representative images of DIV7 hippocampal neurons pre-treated with control (PBS) or αSyn PFFs for 24 h and immunostained for β3-tubulin (green) and cofilin (red). Scale bar: 20 μm. Arrowheads indicate rod structures. B Percentage of neurons with rods (fold change relative to control) of DIV7 hippocampal neurons pre-treated with control (PBS) or αSyn PFFs for 3 h, 6 h, 12 h, 18 h, and 24 h. Data represent mean±SEM (n = 3 independent experiments with ≥100 neurons/condition/experiment). *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA with Dunnett’s multiple comparisons test. C Rod index (fold change relative to control) relative to D. Data represent mean±SEM (n = 3 independent samples/condition with ≥100 neurons/sample). *p < 0.05, **p < 0.01 by Student’s t test. D Representative images of DIV14 hippocampal neurons expressing GFP or αSyn, untreated or pre-treated at DIV7 with 150 ng/mL of αSyn PFFs, and immunostained at DIV14 for β3-tubulin (white) and cofilin (red). Scale bar: 20 μm. Arrowheads indicate rod structures. E Representative images of hippocampal brain sections from control (Saline) and αSyn PFFs injected WT mice 6 months post-injection immunostained for cofilin (red). DAPI (blue). Scale bar: 200 μm. E’ Zoom-ins from E. Scale bar: 20 μm. Arrowheads indicate cofilin-actin rod structures. F Cofilin pathology evaluated by the number of cofilin aggregates and rods per mm² (fold change relative to WT) in the hippocampal region relative to E. Data represent mean±SEM (n = 3–4 animals/condition). ***p < 0.001 by Student’s t test.

Fig. 4. A PrP^C-CCR5 pathway mediates αSyn-induced rod formation.

A Rod index quantification (fold change relative to control) of DIV14 hippocampal neurons expressing GFP or αSyn either with pmRFP-N1 (empty) or cofilin-S3E. Data represent mean±SEM (n = 4–6 independent samples/condition with an average of 25 neurons/sample). **p < 0.01, ***p < 0.001 by One-way ANOVA with Sidak’s multiple comparisons test. B Quantification of the percentage of neurons with rods (fold change relative to control) in DIV7 hippocampal neurons from WT or PrP^C KO mice expressing GFP or αSyn. Data represent mean±SEM (n = 4–6 independent samples/condition with ≥100 neurons/sample). **p < 0.01 by Two-way ANOVA with Sidak’s multiple comparison test. C Quantification of the percentage of neurons with rods in DIV7 hippocampal neurons expressing GFP or αSyn and pre-treated for 24 h with 50 nM Maraviroc. Data represent mean±SEM (n = 3 independent experiments with ≥100 neurons/condition/experiment). *p < 0.05 by One-way ANOVA with Sidak’s multiple comparisons test. D Representative images of DIV7 hippocampal neurons expressing GFP or αSyn and pre-treated for 24 h with 50 pM RAP-103. Immunostaining for β3-tubulin (red) and cofilin (white). Scale bar: 20 μm. Arrowheads indicate rod structures. E Quantification of the percentage of neurons with rods (shown as fold change relative to control) relative to D. Data represent mean ± SEM. (n = 3 independent experiments with ≥100 neurons/condition/experiment). ***p < 0.001, ****p < 0.0001 by One-way ANOVA with Sidak’s multiple comparisons test. F Quantification of the rod index in DIV6 hippocampal neurons overexpressing PrP^C and pre-treated with 50 pM RAP103 for 24 h. Data represent mean±SEM (n = 3 independent samples/condition with ≥100 neurons/sample). ***p < 0.001, ****p < 0.0001 by One-way ANOVA with Tukey’s multiple comparisons test. G Representative images of differentiated SH-SY5Y cells expressing GFP or αSyn and immunostained for cofilin (red) and βIII-tubulin (blue). Scale bar: 10 μm. H qPCR results for CCR5, PRNP and GAPDH gene expression in differentiated SH-SY5Y cells. ACTB was used as a reference gene. Data represent mean ± SEM (n = 3 independent experiments). ***p < 0.001, ****p < 0.0001 by Student’s t test. I Schematic representation of the signaling pathway of αSyn-induced cofilin-actin rod formation in hippocampal neurons.

Fig. 5. Blocking CCR5 rescues dendritic spine impairment induced by αSyn overexpression.

A qPCR results for Ccr5 gene expression in DIV14 hippocampal neurons expressing GFP or αSyn. Data shown as fold change in relation to the control sample. Data represent mean ± SEM (n = 4 independent experiments). *p < 0.05 by Student’s t test. B qPCR results for CCR5 gene expression in Control and DLB patient samples. Data are shown as fold change in relation to control samples. ACTB was used as a reference gene. Data represent mean ± SEM (n = 2–3 cases/condition). C Rod index quantification of DIV14 hippocampal neurons expressing GFP or αSyn and pre-treated with RAP103 (50 pM) for 24 h. Data represent mean±SEM (n = 3 independent samples/condition with ≥100 neurons/sample). *p < 0.05 by One-way ANOVA with Tukey’s multiple comparison test. D Representative images of DIV14 hippocampal neurons expressing GFP or αSyn and pre-treated with 50 pM RAP-103 for 24 h, and immunostained for cofilin (red) and β3-tubulin (white). Scale bar: 10 μm. D’ Zoom-ins from D. GFP (white). Scale bar: 10 μm. D” Zoom-ins from D’. GFP (white). Scale bar: 5 μm. E Dendritic spine density relative to D. Data represent mean ± SEM (n = 9–13 dendrites/condition. Representative experiment). **p < 0.01, ***p < 0.001 by One-way ANOVA with Tukey’s multiple comparisons test. F Dendritic spine density by morphology relative to D. Data represent mean±SEM (n = 9–13 dendrites/condition. Representative experiment). **p < 0.01, ****p < 0.0001 by Two-way ANOVA with Tukey’s multiple comparison test.