Perineuronal nets (PNNs) are specialized forms of extracellular matrix that arise during development and control synaptic plasticity and functions. Overexpression of matrix metalloproteinases like MMP-9 degrades PNNs around GABAergic parvalbumin-positive (PV+) neurons in the developing auditory cortex (AC) of Fmr1KO mice. Pharmacological or genetic restoration of PNNs in the AC significantly ameliorated FXS-associated hyperresponsivity to acoustic stimuli, suggesting that PNN and MMP-9 represent new FXS therapeutic targets. We conducted an automated analysis of PNN+ and PV+ cells in the entire mouse brain at P60 and found a reduction of PNNs and PV+ in Fmr1 KO brains compared to controls. Moreover, Fmr1KO mice and controls of both sexes at different stages of development were subjected to a behavioral test battery, and their brains were analyzed to study PNN formation. Meanwhile we are screening compounds with known and putative effect on MMP-9 activity using an in vitro assay as well as human FXS and control fibroblasts; the most efficient compounds identified through the in vitro screening will be then tested in Fmr1 KO mice and human FXS iPSCs-derived neurons. We also aim to analyze the selected compounds’ effects on the intrinsic neuronal excitability, glutamatergic and GABAergic synaptic transmission in Fmr1KO brain slices by patch-clamp recordings. The final goal is to identify specific brain areas and corresponding time windows of intervention that could be therapeutically relevant for treating Fragile X Syndrome.