Rett Syndrome (RTT) is a progressive X-linked neurodevelopmental disorder with no cure. RTT patients show disease-associated symptoms within 18 months of age that include developmental regression, progressive loss of useful hand movements, and breathing difficulties, along with neurological impairments, seizures, tremor, and mental disability. Rett Syndrome is also associated with metabolic abnormalities, and the anti-diabetic drug metformin is suggested to be a potential drug of choice with low or no side-effects. Previously, we showed that in vitro exposure of metformin in a human brain cell line induces MECP2E1 transcripts, the dominant isoform of the MECP2 gene in the brain, mutations in which causes RTT. Here, we report the molecular impact of metformin in mice. Protein analysis of specific brain regions in the male and female mice by immunoblotting indicated that metformin induces MeCP2 in the hippocampus, in a sex-dependent manner. Additional experiments confirm that the regulatory role of metformin on the MeCP2 target “BDNF” is brain region-dependent and sex-specific. Measurement of the ribosomal protein S6 (in both phosphorylated and unphosphorylated forms) confirms the sex-dependent role of metformin in the liver. Our results can help foster a better understanding of the molecular impact of metformin in different brain regions of male and female adult mice, while providing some insight towards its potential in therapeutic strategies for the treatment of Rett Syndrome.

Background: Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by MECP2 gene mutations. MeCP2 protein binding to methylated DNA is involved in normal brain development and function. T158M is a common RTT-associated mutation, where a threonine is replaced with a methionine, affecting protein function and stability. RTT has recently been identified as a neurometabolic disorder, with metformin emerging as a potential candidate drug. Metformin is a safe and accessible drug, commonly used for Type 2 diabetes. Our team previously studied the regulatory role of metformin on the expression of RTT-related genes/proteins using in vitro and in vivo approaches. However, the phenotypical and behavioral impact of metformin in transgenic mice carrying the common T158M mutation was not explored. Methods: Wild type (WT) and mutant Mecp2^T158M (Mecp2^tm4.1Bird) male mice were subjected to daily intraperitoneal injection of metformin for 20 days. The control mice received a daily intraperitoneal injection of the solvent. The main RTT-like phenotypical criteria were assessed daily. Behavioral tests included the open field test and elevated plus maze. Results: Behavioral tests indicated no significant effect of metformin on the anxiety levels, locomotion, and exploratory behaviors in the hemizygous male Mecp2^T158M mice, despite our observation of increased anxiety levels in the WT counterparts. In hemizygous male Mecp2^T158M mice, metformin treatment showed beneficial effects on RTT-like phenotypes, including breathing irregularities, gait abnormalities, hindlimb clasping, and overall total score. The positive effect of metformin was also observed on the body weight in the hemizygous male Mecp2^T158M mice. Conclusions: Our findings provide evidence for potential therapeutic effects of metformin for MeCP2-associated neurological disorders.