Abstract
Intrinsic apoptosis relies on the ability of the BCL-2 family to induce the formation of pores on the outer mitochondrial membrane. Previous studies have shown that both BAX and BAK are essential during murine embryogenesis, and reports in human cancer cell lines identified non-canonical roles for BAX and BAK in mitochondrial fission during apoptosis. BAX and BAK function in human brain development remains elusive due to the lack of appropriate model systems. Here, we generated BAX/BAK double knockout human-induced pluripotent stem cells (hiPSCs), hiPSC-derived neural progenitor cells (hNPCs), neural rosettes, and cerebral organoids to uncover the effects of BAX and BAK deletion in an in vitro model of early human brain development. We found that BAX and BAK-deficient cells have abnormal mitochondrial morphology and give rise to aberrant cortical structures. We suggest crucial functions for BAX and BAK during human development, including maintenance of homeostatic mitochondrial morphology, which is crucial for proper development of progenitors and neurons of the cortex. Human pluripotent stem cell-derived systems can be useful platforms to reveal novel functions of the apoptotic machinery in neural development.
Highlights
The intrinsic cell death pathway can be initiated by various stimuli including metabolic stress and exposure to cytotoxic agents
The deletion of Bcl-2associated X protein (BAX)/Bcl-2 homologous antagonist/killer (BAK) in human-induced pluripotent stem cells (hiPSCs) was validated by immunoblot (Fig. 1a) and DNA sequencing
To confirm that BAX and BAK deletion did not result in the induction of basal necrosis, hiPSCs were stained for 7-aminoactinomycin D (7-AAD)
Summary
The intrinsic cell death pathway can be initiated by various stimuli including metabolic stress and exposure to cytotoxic agents. The response to these stimuli is mediated by the B-cell lymphoma 2 (BCL-2) family, including proapoptotic and antiapoptotic members that are evolutionarily conserved[1]. Antiapoptotic members, which include BCL-2, B-cell lymphoma-extralarge (BCL-XL), and myeloid cell leukemia 1 (MCL-1) preserve the integrity of the outer mitochondrial membrane by keeping the proapoptotic effectors Bcl-2associated X protein (BAX) and Bcl-2 homologous antagonist/killer (BAK) in an inactive state[2,3]. BAK-deficient mice show normal development, suggesting BAK has redundant functions with other proapoptotic BCL-2 family members[13]. 10% of mice lacking both BAX and BAK survive to adulthood.
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