CTNNB1 Syndrome is a developmental disorder characterized by intellectual disabilities, microcephaly, global developmental delays (motor, language) and motor disabilities (truncal muscle hypotonia, distal hypertonia with spastic gait). It is caused by CTNNB1 (Beta-catenin) haploinsufficiency due to partial or complete deletion mutations. CTNNB1 is a significant risk gene for intellectual disabilities. Several other human gene mutations also cause reduced Beta-catenin levels or functions and similar developmental disorders. Beta-catenin plays key roles in two pathways critical for proper neural and neuromuscular development and function- the canonical Wnt signal transduction pathway and cadherin-based synaptic adhesion complexes. Treatments for CTNNB1 Syndrome are lacking due to limited knowledge of the underlying pathophysiological mechanisms, limited studies of CTNNB1 heterozygous mice and as yet no human cell models. We propose in vivo mouse and in vitro human cell studies to address these critical gaps. Preliminary studies of our CTNNB1 germline heterozygous mouse show phenotypes relevant to this disorder, impaired associative and motor learning, and reduced muscle grip strength, compared with control littermates. Our Aim 1 studies will provide novel mechanistic insights by identifying in vivo molecular and functional changes in three tissue types relevant to CTNNB1 syndrome features: forebrain, spinal cord and skeletal muscle. We will use multi-disciplinary quantitative approaches, mass spectrometry proteomics, electrophysiological recordings, and further behavioral testing for altered cognitive and motor capabilities. Our Aim 2 studies will utilize the in vivo mouse model to test our hypothesis that drug treatments that normalize Beta-catenin levels will improve or remedy the phenotypes caused by Beta-catenin haploinsufficiency. We will test drug treatments that have been shown to increase Beta-catenin levels and improve learning and motor deficits in mouse models of other disorders with similar phenotypes to CTNNB1 syndrome. To increase translational relevance, Aim 3 studies will generate human cell models of CTNNB1 heterozygous loss-of-function in multiple cell types relevant to CTNNB1 syndrome features: cortical glutamatergic neurons, spinal motoneurons and skeletal myotubes. We will also generate isogenic revertant controls with the mutated allele corrected. We will define molecular changes caused by reduced Beta-catenin, using mass spectrometry proteomics. We will test the efficacy of drug treatments for correction of Beta-catenin and the molecular changes. Our findings will identify both shared and unique molecular alterations between the in vitro human cells and the corresponding in vivo mouse tissue types. Shared changes will identify core pathophysiological mechanisms. We may also identify novel components of the Beta-catenin network in the different tissues. Our studies will provide critical proof-of-concept in two preclinical mode...