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. Author manuscript; available in PMC: 2023 Oct 19.
Published in final edited form as: Neuron. 2022 Sep 6;110(20):3288–3301.e8. doi: 10.1016/j.neuron.2022.08.009

Figure 4. Supplemental ChP-SOD3 expression mitigates MTX-induced oxidative stress in the CSF and the hippocampus.

Figure 4.

(A) Percent cell death in cerebellar granule neurons cultured in 20% artificial CSF or 20% adult, native CSF in basal medium and treated with vehicle or 100 μM H2O2 for 24 h, n = 3 per group. **P < 0.01 ***P < 0.001. One-way ANOVA with Tukey’s post hoc test. (B) Experimental overview for intracerebroventricular (i.c.v.) delivery of AAVs into developing brain ventricles at embryonic day (E)13.5. All subsequent experiments were performed postnatally in adult mice. (C) Lysates of LV ChP and 4V ChP of mice transduced with either AAV-GFP or AAV-hSOD3 and treated with either vehicle or 75 mg/kg MTX for 48 h were probed with the SOD3 and Vinculin antibodies. (D) Representative tiled image of a LV ChP whole mount revealing location of sustained GFP expression near free margin of larger domain at 9 weeks of age. (E) CSF lysates of mice transduced with either AAV-GFP or AAV-hSOD3 were probed with the SOD3 and Albumin antibodies. Representative blot shown, n = 3. (F) Relative SOD activity measured by colorimetric assay in CSF of mice expressing either AAV-GFP or AAV-hSOD3. n = 3 vehicle, n = 3 MTX. *P < 0.05. Unpaired t test. (G) Metabolite profiling of CSF samples from mice transduced with either AAV-GFP or AAV-hSOD3 and treated with vehicle or MTX. Heatmap of top 10 changed metabolites in the CSF following MTX treatment is shown. The heatmap represents log-transformed, Pareto-scaled levels of each of the listed metabolites in the four conditions. (H) PLSDA analysis of the metabolomics data. (I) PLSDA loading depicting the metabolites that dictate the cohort distribution in the PLSDA plot with annotated metabolites indicating the data points that most influenced the separation of samples presented in the PLSDA. (J-L) Levels of reduced (GSH, J), oxidized (GSSG, K), and the ratio of reduced to oxidized glutathione (L) in CSF from mice overexpressing GFP or hSOD3 and treated with vehicle or MTX for 48 h. All measurements were done by LC-MS. n = 7 GFP + vehicle, n = 7 GFP + MTX, n = 4 SOD3 + vehicle, n = 6 SOD3 + MTX. *P < 0.05, ***P < 0.001; NS, not significant. One-way ANOVA test with Benjamini, Krieger, Yukutieli FDR. Data represent mean ± SEM.