Article Figures & Data
Figures
- Figure 1.
AAV2-SYN overexpression induces MHCII expression in vivo. A, α-syn (α-GFP, green) results in enhanced MHCII expression (α-MHCII, red) in the SNpc (α-TH, blue) 4 weeks post-transduction. Scale bar, 125 μm in the Merge part. AAV2-GFP and AAV2-SYN Zoom insets from white boxes shown. Scale bar, 75 μm. B, Quantification of MHCII staining in the SNpc of AAV2-GFP (control) and AAV2-SYN mice at 4 weeks. C, α-Syn (α-GFP, green) results in enhanced MHCII expression (α-MHCII, red) in the SNpc 12 weeks post-transduction in AAV2-SYN-injected animals. Confocal images were captured using a Leica TCS-SP5 laser-scanning confocal microscope. Images were processed using the Leica LASAF software, exported, and processed using Adobe Photoshop. For quantification of MHCII slides were observed using a Nikon Eclipse E800M fluorescent microscope. Coded slides were scored by using a numerical scale 0 (no staining) to 4 (most intense) by a single observer blind to the treatment paradigm. Data represent the median (n = 6/group) **p < 0.0043, Mann–Whitney test.
- Figure 2.
Genetic KO of MHCII attenuates AAV2-SYN-induced reactive microgliosis in vivo. A, α-Syn (α-GFP, green) results in enhanced CD11b+ microgliosis (α-CD11b, red) in the SNpc (α-TH, blue) 4 weeks post-transduction of WT mice but not MHCII KO mice. Scale bar, 125 μm. B, AAV2-GFP and AAV2-SYN Zoom insets from white boxes shown in A. Scale bar, 75 μm. C, Quantification of CD11b microgliosis in the SNpc of WT and MHCII KO mice 4 weeks post-transduction. D, α-Syn (α-GFP, green) results in enhanced CD11b+microgliosis (α-CD11b, red) in the SNpc (α-TH, blue) 12 weeks post-transduction of WT mice but not MHCII KO mice. Data represent the median (n = 6–8/group) *p < 0.05, **p < 0.01, Kruskal–Wallis test with Dunn's multiple-comparison post hoc test.
- Figure 3.
Genetic KO of MHCII attenuates AAV2-SYN-induced IgG deposition in vivo. A, Representative images of IgG deposition (α-IgG, red) on the ipsilateral side of α-syn overexpression (α-GFP, green) in WT versus MHCII KO mice. IgG deposition is markedly attenuated in virally transduced MHCII KO animals at 4 weeks (A) and 12 weeks (B).
- Figure 4.
Genetic KO of MHCII attenuates AAV2-SYN-induced neurodegeneration in vivo. Six months post-transduction TH-immunopositive neurons were quantified to determine observed neuroprotection. A, Representative images of the ipsilateral SNpc stained for TH. B, Genetic KO of MHCII attenuates AAV2-SYN-induced neuron loss. Neuron loss is reported as a percentage of contralateral side. n = 6–8/group one way ANOVA with Bonferroni selected comparison post hoc test. *p < 0.05.
- Figure 5.
Overexpression of AAV2-SYN in mouse SNpc results in the accumulation of high molecular weight α-syn species. Western blot analysis of α-syn of midbrain homogenates obtained from mice 4 weeks post-transduction into the right substantia nigra, using an antibody that is selective for human α-syn. There is an increase in high molecular weight α-syn species (≥50 kDa) is both the “whole” and Triton-soluble (“T-X-Sol”) fractions in homogenate fractions derived from right (R), infected ventral midbrain samples compared with that of noninfected left (L) control samples. There is also appearance of detectable soluble α-syn monomers (17 kDa). Insoluble high molecular weight forms of α-syn (“T-X-100-Insol” fraction) were observed only after AAV2-SYN treatment. Actin (42 kDa) was used to normalize for gel loading.
- Figure 6.
α-Syn regulates antigen processing and presentation in vitro. Primary microglia were treated with 500 nm aggregated α-syn or vehicle control (A) for 4 h (B) or overnight (C) before a 1 h pulse with DQ-Ovalbumin. Immunofluorescence was quantified from confocal images using ImageJ software (D). Four hour aggregated α-syn pretreatment resulted in an increase in antigen processing and presentation while overnight α-syn pretreatment decreased antigen processing and presentation. These effects were not observed with monomeric α-syn (E). One way ANOVA with Bonferroni multiple-comparisons post hoc test. ***p < 0.001, **p < 0.01 compared with control. Scale bar, 25 μm.
- Figure 7.
α-Syn-induces CD4 T-cell activation and proliferation in vitro. Primary microglia (previously treated with OVA323–339 and 500 nm α-syn for 6 h) and CD4 T-cell cocultures were pulsed with EdU for 1 h before flow cytometry analysis. A, No peptide control. Vehicle represented with solid line and shading, α-syn treatment represented with a dotted line. Primary microglia and CD4 T-cell cocultures revealed little or no EdU incorporation events. OVA peptide 1 μg/ml (B) and 10 μg/ml (C) treatment results in an increase in EdU+ events 60 h after coculture. Positive events were gated by live/dead, CD4, CD5, and EdU. D–I, α-syn pretreatment (500 nm) of primary microglia 6 h before T-cell coculture resulted in a statistically significant increase in pro-inflammatory cytokines and chemokines. All cytokine and chemokine expression levels were normalized to the level of vehicle-treated primary microglia and T-cell coculture. Student's t test (n = 3–4/group, representative of 2 independent experiments). D, 10 μg/ml OVA323–339 IL-1α *p < 0.0193. E, 10 μg/ml OVA323–339 IFNγ **p < 0.0087. F, 10 μg/ml OVA323–339 IL-1β ***p < 0.0009. G, 1 μg/ml OVA323–339 TNF **p < 0.0022. H, 10 μg/ml OVA323–339 TNF *p < 0.011. I, 10 μg/ml OVA323–339 IL-10 **p < 0.0021.
