The NO Answer for Autism Spectrum Disorder

Abstract Autism spectrum disorders (ASDs) include a wide range of neurodevelopmental disorders. Several reports showed that mutations in different high‐risk ASD genes lead to ASD. However, the underlying molecular mechanisms have not been deciphered. Recently, they reported a dramatic increase in nitric oxide (NO) levels in ASD mouse models. Here, they conducted a multidisciplinary study to investigate the role of NO in ASD. High levels of nitrosative stress biomarkers are found in both the Shank3 and Cntnap2 ASD mouse models. Pharmacological intervention with a neuronal NO synthase (nNOS) inhibitor in both models led to a reversal of the molecular, synaptic, and behavioral ASD‐associated phenotypes. Importantly, treating iPSC‐derived cortical neurons from patients with SHANK3 mutation with the nNOS inhibitor showed similar therapeutic effects. Clinically, they found a significant increase in nitrosative stress biomarkers in the plasma of low‐functioning ASD patients. Bioinformatics of the SNO‐proteome revealed that the complement system is enriched in ASD. This novel work reveals, for the first time, that NO plays a significant role in ASD. Their important findings will open novel directions to examine NO in diverse mutations on the spectrum as well as in other neurodevelopmental disorders. Finally, it suggests a novel strategy for effectively treating ASD.

# Corresponding Author: Haitham Amal, PhD. Email: haitham.amal@mail.huji.ac.il Supplementary tables are uploaded as Excel/Word files: Table 1: MTT assay of 7-NI in primary cortical neuronal culture. Table 2: Clinical characteristics of the participants (TD and ASD). Table 3: 3-Nitrotyrosine relative value in plasma TD and ASD. Table 4: System biology analysis of the plasma samples of TD and ASD children.

Panel C & D:
Open field test-Statistical analysis of the distance traveled and velocity. No significant differences were observed in the distance traveled or velocity among the three male test groups. WT2 (n=12), M2 (n=20), and M2+7-NI (n=18).

Panel E & F:
Open field test-Statistical analysis of the distance traveled and velocity. No significant differences were observed in the distance traveled or velocity among the male test groups. WT (n=12), and WT+SNAP (n=10). In all tests, the data is presented as mean ± SD.
Statistical significance was determined using a Two-way ANOVA with Bonferroni's multiple comparisons tests or two-tailed t-test. ns = non-significant. Three-chamber sociability test. Both the WT2 (n=12, **P=0.0031) and the WT2+7-NI (n=5, ***P<0.0001) mice spent significantly increased time interacting with the stranger mouse than with the empty cage. In all tests, the data is presented as mean ± SD. Statistical significance was determined using a Two-way ANOVA with Bonferroni's multiple comparisons tests or two-tailed t-test. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns= nonsignificant. between the M2 (n=6) and M2+SNAP (n=6) was observed. In all tests, the data is presented as mean ± SD. Statistical significance was determined using a Two-way ANOVA with Bonferroni's multiple comparisons tests or two-tailed t-test. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns=non-significant. . In all tests, the time is presented as the percentage of the total time. The data is presented as mean ± SD. Statistical significance was determined using a Two-way or One-way ANOVA with Bonferroni's multiple comparisons tests. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns=non significant. presented as the percentage of the total time. The data is presented as mean ± SD. Statistical significance was determined using a Two-way or One-way ANOVA with Bonferroni's multiple comparisons tests. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns=non-significant. presented as the percentage of the total time. In all tests, the data is presented as mean ± SD.

Fig S10. Discrimination index of NOR and Sociability tests of male mice
I.For the NOR test the DI was calculated as the difference in exploring the novel and the familiar object divided by the total time exploring both objects: II. DI in the three-chamber sociability test was calculated as: significance was determined using One-way ANOVA with Bonferroni's multiple comparisons tests or two-tailed t-test. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns= nonsignificant.  Panel I: DI of the preference of the mice to interact with a novel mouse over a familiar one in the WT and WT+SNAP. Statistical significance was determined using One-way ANOVA with Bonferroni's multiple comparisons tests or two-tailed t-test. *P < 0.05, **P < 0.001, ***P < 0.0001, and ns= non-significant The data is presented as mean ± SD. A one-way ANOVA test with the Tukey post hoc test was used for multiple comparisons in all groups *P < 0.05, **P < 0.001, ***P < 0.0001, and ns=non-significant.