Abstract: Fragile X Syndrome (FXS), the most common cause of intellectual disability and autism, is caused by the loss of FMR1 gene function. Drosophila and mouse models of FXS have been developed that are based on loss of function mutations of their respective homologues of FMR1, dfmr1 and Fmr1. These models display several phenotypes that bear similarity to Fragile X patient symptoms. In previous studies, we and others have identified reduced cAMP levels and increased insulin/PI3K signaling as defects in the Fragile X animal model brains. Our work on a Drosophila FXS model demonstrated that restoration of the cAMP deficit, by treatment with PDE4 inhibitors, restores behavior and memory. We have also shown that genetic and pharmacological manipulations that restore normal insulin signaling levels rescue behavioral and memory deficits. Our findings for both pathway defects have been reproduced in the mouse FXS model. Further, we discovered that metformin treatment of the FXS Drosophila model also restores memory and behavior, a finding that too has been replicated in the mouse FXS model. In sum our studies have determined that three seemingly distinct approaches, e.g. increasing cAMP levels, decreasing insulin signaling and metformin treatment can restore behavioral and cognitive phenotypes displayed by the FXS animal models. Importantly two of these findings are being pursued clinically and have given rise to promising results. A novel PDE4 inhibitor, BPN14770, has been tested in a phase II clinical trial with Fragile X adults. The results of this study have shown that treatment with this compound can significantly improve cognitive and life skills in Fragile X adult aged 18 to 45. Also, several case studies of Fragile X patients treated with metformin have reported improvements in cognitive and social domains. These findings have led to the initiation of clinical trials with metformin. Given the clinical relevance of our findings, an important question that we will address in this study is how these three seemingly different approaches act to restore behavior and cognition in a FXS animal model. Our preliminary studies indicate that they converge on improving mitochondrial function. In recent studies we have identified robust mitochondrial deficits displayed by the Drosophila FXS model and FXS patient derived cells. We have also determined that the mitochondrial master regulator PGC-1a is significantly decreased in the Drosophila model and in FXS patient derived cells. Importantly we have determined that the mitochondrial defects and PGC-1a are improved by metformin treatment and the genetic reduction of insulin signaling. We have also demonstrated that independently increasing PGC-1a expression improves mitochondrial function and a behavioral phenotype. In our proposed studies we will perform experiments to verify that the restoration of the signaling pathway defects, as well as metformin treatment increase both PGC-1a expression and mitochond...