(The) effects of CLCN4 variants on human neurodevelopment

Abstract

The gene CLCN4, which is highly expressed in the brain, exhibits variants that have been linked to neurodevelopmental disorders, such as intellectual disabilities, epileptic seizures, and autistic features. The expression patterns of CLCN4 and the observed phenotypes resulting from CLCN4 variants strongly suggest a significant role of CLCN4 in brain development. However, there is limited knowledge regarding the biological roles for CLCN4 in neurodevelopment and the specific effects of CLCN4 variants on neurodevelopmental processes. To address this issues, genome editing technique was applied to generate the human embryonic stem cells (hESCs) harboring CLCN4 variant, which were subsequently differentiated into brain organoids and neurons. The brain organoids derived from hESCs carrying CLCN4 variants exhibited a decreased population of excitatory neurons. Notably, when neural progenitor cells (NPCs) were differentiated into excitatory neurons, the experienced early-stage neuronal cell death and abnormal neurite development were observed in CLCN4-variant neurons. Also, it was verified that an accumulation of vesicles in the endo-lysosomal pathway and a decrease in autophagy within CLCN4-variant neurons. RNA sequencing analysis of CLCN4-variant neurons revealed altered expression of genes involved in neurodevelopmental processes. Through the gene expression profile analysis, MEG3, was found to be the most significantly changed. Manipulation of MEG3 expression could rescue phenotypes observed in CLCN4-variant neurons, including cell death. This study highlights the impact of CLCN4 variants on neuro-developmental phenotypes, particularly cell death, and emphasizes the potential of MEG3 activation in restoring these phenotypes. The findings not only underscore the pivotal role of CLCN4 in brain development but also offer potential therapeutic implications for the treatment of CLCN4-related neurodevelopmental disorders.