Therapeutic GSK-3β targeting stabilizes multifunctional β-catenin to rescue neuronal and behavioral deficits in fragile X messenger ribonucleoprotein 1 knockout mice.

Fragile X syndrome (FXS) is the predominant singlegene cause of inherited intellectual disability and is strongly associated with autism spectrum disorder (ASD). FXS results from the disruption of fragile X messenger ribonucleoprotein 1 gene (FMR1) and is characterized by synaptic dysfunction manifesting as impaired cognitive function and social communication. The Wnt/β-catenin pathway plays a pivotal role in regulating synaptic structural remodeling and functional homeostasis, critically contributing to higher-order neural processes such as learning and memory. Studies have identified glycogen synthase kinase 3 beta (GSK3β), a key negative regulator of Wnt signal transduction, is abnormally activated in the pathophysiology of FXS, and demonstrated that GSK3β inhibition partially rescues cognitive and behavioral deficiencies in FXS mice. However, the spatiotemporal dysregulation of β-catenin dynamics and its synaptic consequences remain poorly understood. Here, we investigated the role and molecular mechanism of Wnt/β-catenin pathway during developmental stages in FXS using Fmr1 gene knockout (Fmr1 KO) mice. We systematically explored β-catenin homeostasis across subcellular compartments. Our results showed increased phosphorylation of β-catenin at Ser33,37, Thr41 and Ser552 residues, which fosters its degradation. This was accompanied by reduced levels of active β-catenin in the membrane, cytoplasm and nucleus within the hippocampus (Hipp) and prefrontal cortex (PFC) of Fmr1 KO mice. Confocal microscopy further demonstrated diminished co-localization of β-catenin with N-cadherin, leading to compromised intercellular adhesion in both Fmr1 KO neurons. Moreover, FXS mice showed impaired neuronal morphology and deficiencies in social and cognitive functions, which were associated with the downregulation of pre- and postsynaptic proteins targeted by Wnt pathway. Strikingly, pharmacological activation of Wnt signal transduction restored β-catenin nuclear translocation and synaptic protein expression, rescued neuronal ultrastructural abnormalities and improvd cognitive and social behaviors. Our findings establish hypoactivity of canonical Wnt signaling as a central mechanism underlying synaptic pathology in FXS, linking β-catenin destabilization to altered neuronal morphology, aberrant synaptic protein networks, and behavioral phenotypes. Consequently, bolstering Wnt pathway may represent a promising neuroprotective strategy for precision intervention in FXS.