Lithium chloride in vitro treatment shows potential to rescue the neuronal phenotype caused by WDFY3 haploinsufficiency.

We provide a comprehensive phenotypic characterization of loss-of-function (LoF) variants in WDFY3 based on the largest cohort reported to date (n = 32). Our findings define a monogenic disorder marked by neuropsychiatric features (including autism and ADHD), mild to moderate neurodevelopmental delay, and variable brain growth - most commonly macrocephaly and in one case reduced head circumference. To investigate the effect of WDFY3 LoF we performed knockdown (KD) in the human neuroblastoma cell line SH-SY5Y and used a Wdfy3-haploinsufficient mouse model. WDFY3 KD impaired clearance of protein aggregates and decreased protein levels of the ubiquitin-binding protein p62. Transcriptomic analyses identified dysregulation in gene networks that are primarily involved in nervous system development, neuron projection, MAPK/ERK1/2, and WNT/β-catenin signaling. This was confirmed at protein level. Neuronal cell proliferation was increased, as evidenced by elevated cell counts and proliferation markers Ki-67 and PCNA. Conversely, neuronal cell differentiation was impaired, as indicated by reduced neurite outgrowth. We show that β-catenin is downregulated, while GSK3B is upregulated. LiCl activates WNT/β-catenin signaling by inhibiting GSK3B. We thus tested LiCl treatment, which significantly increased WDFY3 expression. Moreover, in vitro treatment improved the neurite branching capacity of Wdfy3-haploinsufficient neurons above the level of the wild type, although this specific effect appeared independent of WNT/β-catenin signaling. In sum, we expand on the phenotype related to WDFY3 LoF and provide insights into the pathophysiology using in vitro human and murine neuronal models. Our findings suggest that LiCl may serve as a potential therapeutic strategy for patients with WDFY3 LoF variants.