Fig. 2: Cntnap4 partial deficiency damages mitochondrial function and induces α-synuclein release via ferroptosis. | Cell Death & Disease

Fig. 2: Cntnap4 partial deficiency damages mitochondrial function and induces α-synuclein release via ferroptosis.

From: Cntnap4 partial deficiency exacerbates α-synuclein pathology through astrocyte–microglia C3-C3aR pathway

Fig. 2

A DEGs between Cntnap4+/− and WT mice are shown in a volcano plot. B KEGG pathways enriched by downregulated DEGs between Cntnap4+/− and WT mice. Note that ferroptosis is highlight in red. C Representative downregulated DEGs enriched in ferroptosis and GABAergic synapse. D, E The protein expression levels of TH, FTH1, GPX4, and NCOA4 in the SN were determined by western blotting; n = 3 per group. F, G The protein expression levels of FTH1, GPX4, and NCOA4 in MN9D cells treated with NC siRNA and Cntnap4 siRNA were determined by western blotting; n = 3 per group. H, I Immunofluorescence staining and quantification of JC-1 aggregates and monomers in MN9D cells treated with NC siRNA + PBS, NC siRNA + hα-Syn, Cntnap4 siRNA + PBS, and Cntnap4 siRNA + hα-Syn. Scale bars: 20 μm. Magnified figures are shown in the right column of panel I. Scale bars: 4 μm; n = 6–9. J, K Ultrastructural analysis of mitochondria in the SNpc of WT and Cntnap4+/− mice injected with either AAV-GFP or AAV-hα-Syn. Scale bars, 2 μm, and 500 nm for magnified images. Red arrows show mitochondrial swelling and mitochondrial cristae disappearance. n = 10 per group. L, M Immunofluorescence staining and quantification of JC-1 aggregates and monomers in MN9D cells treated with Cntnap4 siRNA + hα-Syn plus ferrostatin-1. Scale bars: 20 μm. Magnified figures are shown in the right column of panel L. Scale bars: 2 μm; n = 7–9. N, O The protein expression levels of phosphorylated and mouse α-synuclein in MN9D cells treated with NC siRNA + PBS, NC siRNA + hα-Syn, Cntnap4 siRNA + PBS, and Cntnap4 siRNA + hα-Syn; n = 3 per group. P, Q Supernatant levels of phosphorylated and mouse α-synuclein from MN9D cells treated with NC siRNA + PBS, NC siRNA + hα-Syn, Cntnap4 siRNA + PBS, and Cntnap4 siRNA + hα-Syn; n = 3 per group. Results are expressed as the mean ± SEM. **p < 0.01, *p < 0.05 vs. WT or NC siRNA; ##p < 0.01, #p < 0.05 vs. AAV-hα-Syn or NC siRNA + hα-Syn; &&p < 0.01 vs. Cntnap4+/− + AAV-hα-Syn or Cntnap4 siRNA + PBS. Statistical significance was determined using Student’s t-test (E, G, M), one-way ANOVA + Tukey’s multiple comparisons test (H, O, Q), and two-way ANOVA + Bonferroni’s multiple comparisons test (K).

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