The effects of single amino acid alteration on CLCN4 in human neurogenesis

Abstract

Diverse mutations on different positions along the CLCN4 have been identified in patients with intellectual disability, autistic traits, and epileptic seizures. CLCN4 is a gene encoding voltage gated chloride channel 4, CLC-4. While the expression profile of the CLCN4 shows the highest expression level in the brain among the other tissues, the biological roles of CLCN4 in brain development and diseases remain largely unknown. In this study, to investigate the effects of CLCN4 mutations in neuronal development, we generated human embryonic stem cells (hESCs) carrying mutant CLCN4 by applying the genome-editing technique. At first, we differentiated the hESCs carrying the CLCN4 mutation to neural progenitor cells (NPCs) and obtained adequate NPCs similar to the control. Next, NPCs were differentiated into neurons. The neurons with mutant CLCN4 displayed abnormal morphology at the early stage of neurogenesis compared to the control neurons and eventually died before maturation. We also observed a reduced number of neurons in the forebrain organoids carrying CLCN4 mutations. Our data show the requirement of CLCN4 for the survival of developing neurons. Currently, we are working on investigating molecular mechanisms of how CLCN4 variants cause neuronal cell death.