WDFY3 loss-of-function leads to increased proliferation and altered differentiation of neuronal cells underlying the haploinsufficient human phenotype

Neurodevelopmental disorders (NDD) are poorly understood, and late diagnosis and interventions result in a high burden for the society and health system. WD Repeat and FYVE Domain Containing 3 (WDFY3) encodes for the autophagy adaptor protein ALFY. Loss-of-function (lof) variants in WDFY3 result in a monogenic condition characterized by mild to moderate developmental delay, neuropsychiatric disorders, and altered brain size. We present a comprehensive description of lof WDFY3-associated phenotypes in the largest cohort of patients to date (n = 26). Overall design: To dissect effects of WDFY3 lof, we knocked down (KD) WDFY3 in the human neuroblastoma cell line, SH-SY5Y. WDFY3 KD resulted in impaired clearance of aggregated Huntingtin protein and decreased protein levels of the ubiquitin-binding protein p62/SQSTM1. Through transcriptomic analyses, we delineated dysregulated gene networks primarily involved in nervous system development, neuron projection development, and signaling pathways such as MAPK/ERK1/2 and WNT/ß-catenin. The dysregulation of signaling pathways was further confirmed at protein level, as shown by increased levels of ERK1/2 and decreased levels of endogenous ß-catenin. We found an increase in neuronal cell proliferation as evidenced by elevated cell counts, and of the proliferation marker Ki-67 and PCNA gene expression. Conversely, neuronal cell differentiation was impaired, as indicated by reduced neurite outgrowth. Notably, differentiated neuronal cells show a significantly increased WDFY3 expression compared to undifferentiated cells, suggesting involvement of ALFY in neuronal differentiation. Lastly, we found that treatment with lithium chloride was associated with an increased expression of WDFY3, which may point to a potential therapeutic option for patients with impaired ALFY function.