N-Acetylcysteine Counteracts Immune Dysfunction and Autism-Related Behaviors in the Shank3b Mouse Model of Autism Spectrum Disorder

Abstract

Autism spectrum disorder (ASD) includes a range of neurodevelopmental disabilities characterized by social interaction deficits, communication impairments, and repetitive behaviors. Previous studies have shown that pro-inflammatory conditions play a key role in ASD. Despite this, how oxidative stress and inflammation may contribute to ASD-related behaviors is still poorly understood. Here, we reported that increased levels of molecules related to inflammation are present in the cerebellum and peripheral blood (PB) of mice lacking Shank3b, an established model of syndromic ASD. In parallel, immune dysfunction was documented in the bone marrow (BM) and spleens of mutant mice. N-acetylcysteine (NAC) treatment rescued inflammation in the cerebellum and PB and impaired the production of pro-inflammatory molecules in the BM and spleen. In addition, social impairment was counteracted in NAC-treated Shank3b^-/- animals. Taken together, our results provide clear evidence of the key role of cerebellar oxidative stress and inflammation in the establishment of ASD-related behaviors. Furthermore, our findings underscore the importance of considering ASD as a systemic disorder.

Keywords: NAC; ROS; autism; cerebellum; inflammation.

Figure 1

Pro-inflammatory changes in the brain of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice. (a) TNF mRNA expression in the cerebral cortex (Cx), cerebellum (Cb), and hippocampus (Hp) of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice measured using RT-qPCR and TNF expression at the protein level (shown as mean fluorescence intensity, MFI) within cerebellar CD14⁺ cells assessed using flow cytometry. (b) IFNγ mRNA expression in the Cx, Cb, and Hp and IFNγ levels within cerebellar T cells. mRNA expression of (c) IL-6, (d) IL-1β, (e) CCL3, (f) CCL5, (g) CCL20, (h) MMP8, and (i) p21 in the Cx, Cb, and Hp. mRNA expression was normalized against the housekeeping gene β-actin. (j) TNF MFI within CD14⁺ cells, IFNγ MFI within (k) CD8⁺ T cells and (l) CD4⁺ T cells in the PB of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice. One-way ANOVA, Tukey’s post-hoc test. N = 8 per group (RT-qPCR); n = 10–12 per group (flow cytometry). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Pro-inflammatory changes in the bone marrow and spleen of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice. (a) TNF mRNA expression in the bone marrow (BM) and spleen (SPL) of Shank3b^+/+, Shank3b^+/− and Shank3b^−/− mice measured using RT-qPCR and TNF expression at the protein level (shown as mean fluorescence intensity, MFI) within CD14⁺ cells assessed using flow cytometry. (b) IFNγ mRNA expression and IFNγ levels within CD8⁺ T cells. (c) IL-6 mRNA expression and IL-6 levels within CD14⁺ cells. mRNA expression of (d) IL-1β, (e) CCL3, (f) CCL5, (g) CCL20, (h) MMP8, (i) p21, (j) IL-15, and (k) SOD3 in the BM and SPL. mRNA expression was normalized against the housekeeping gene β-actin. One-way ANOVA, Tukey’s post-hoc test. n = 8 per group (RT-qPCR); n = 10–12 per group (flow cytometry). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Behavioral tests in Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice treated with NAC. Time spent moving (s) (a) and frequency in the center (b) in the open field test in NAC-treated Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice and PBS-treated control animals. (c) Time on the rotarod (latency to fall, s) in the rotarod test. (d) Number of marbles buried in the marble burying test. (e) Sociability index (time in the sniffing zone of mouse chamber—time in the sniffing zone of empty chamber)/total time in the sniffing zones) in the three-chamber test. n = 8 (Shank3b^+/+, PBS), n = 8 (Shank3b^+/+, NAC), n = 9 (Shank3b^+/−, PBS), n = 9 (Shank3b^+/−, NAC), n = 9–11 (Shank3b^−/−, PBS), n = 10 (Shank3b^−/−, NAC). Two-way ANOVA, Tukey’s post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 4

Pro-inflammatory molecules in the cerebellum of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice treated with NAC. mRNA expression of (a) TNF, (b) IFNγ, (c) IL-6, (d) IL-1β, (e) CCL3, (f) CCL5, (g) CCL20, (h) MMP8, and (i) p21 in the cerebellum of NAC-treated Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice and PBS-treated control animals assessed using RT-qPCR. mRNA expression was normalized against the housekeeping gene β-actin. n=8 in each group. Mean fluorescence intensity (MFI) of (j) TNF within CD14⁺ cells, (k) IFNγ within T cells, and (l) IL-6 within CD14⁺ cells in the cerebellum measured using flow cytometry. (m) Cys-Gly levels in the serum of PBS- and NAC-treated animals. (n) TNF within CD14⁺ cells, (o) IFNγ within CD4⁺ T cells, (p) IFNγ within CD8⁺ T cells, and (q) IL-6 within CD14⁺ cells in the peripheral blood (PB). n = 8 (Shank3b^+/+, PBS), n = 8 (Shank3b^+/+, NAC), n = 9 (Shank3b^+/−, PBS), n = 9 (Shank3b^+/−, NAC), n = 9–11 (Shank3b^−/−, PBS), n = 10 (Shank3b^−/−, NAC); for (m) N = 5–6 in each group. Two-way ANOVA, Tukey’s post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Pro-inflammatory molecules in the bone marrow of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice treated with NAC. mRNA expression of (a) TNF, (b) IFNγ, (c) IL-6, (d) IL-1β, (e) CCL3, (f) CCL5, (g) CCL20, (h) MMP8, (i) p21, and (j) IL-15 in the bone marrow (BM) of NAC-treated Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice and PBS-treated control animals assessed using RT-qPCR. mRNA expression was normalized against the housekeeping gene β-actin. n = 8 in each group. Mean fluorescence intensity (MFI) of (k) TNF within CD14⁺ cells, (l) IFNγ within CD4⁺ T cells, (m) IFNγ within CD8⁺ T cells, and (n) IL-6 within CD14⁺ cells in the BM measured using flow cytometry. n = 8 (Shank3b^+/+, PBS), n = 8 (Shank3b^+/+, NAC), n = 9 (Shank3b^+/−, PBS), n = 9 (Shank3b^+/−, NAC), n = 9–11 (Shank3b^−/−, PBS), n = 10 (Shank3b^−/−, NAC); for (m) N = 5–6 in each group. Two-way ANOVA, Tukey’s post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Pro-inflammatory molecules in the spleens of Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice treated with NAC. mRNA expression of (a) TNF, (b) IFNγ, (c) IL-6, (d) IL-1β, (e) CCL3, (f) CCL5, (g) CCL20, (h) MMP8, (i) p21, and (j) IL-15 in the spleens of NAC-treated Shank3b^+/+, Shank3b^+/−, and Shank3b^−/− mice and PBS-treated control animals assessed using RT-qPCR. mRNA expression was normalized against the housekeeping gene β-actin. n=8 in each group. Mean fluorescence intensity (MFI) of (k) TNF within CD14⁺ cells, (l) IFNγ within CD4⁺ T cells, (m) IFNγ within CD8⁺ T cells, and (n) IL-6 within CD14⁺ cells in the spleen measured using flow cytometry. n = 8 (Shank3b^+/+, PBS), n = 8 (Shank3b^+/+, NAC), n = 9 (Shank3b^+/−, PBS), n = 9 (Shank3b^+/−, NAC), n = 9–11 (Shank3b^−/−, PBS), n = 10 (Shank3b^−/−, NAC). Two-way ANOVA, Tukey’s post-hoc test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.