Assessing Apoptosis Following Knockout of BH3-Only Proteins Bim, Puma, Hrk, and Bmf

Apoptosis is a form of programmed cell death that plays a crucial role in neuronal development and disease. Key regulators of apoptosis include members of the BCL-2 family of proteins. In particular, the BH3-only proteins Bim, Puma, Hrk, and Bmf play critical roles in facilitating the intrinsic mitochondrial pathway of apoptosis.&nbsp; Despite their known involvement in the apoptotic pathway, the extent of functional redundancy among these proteins remains unclear. Using <em>in vitro </em>CRISPR-Cas9-mediated gene knockout, this study aimed to assess the individual contributions of BH3-only protein family members, Bim, Puma, Hrk, and Bmf on neuronal survival in primary sympathetic neurons. Primary neurons were dissected from the superior cervical ganglion of postnatal mice, cultured in the presence of nerve growth factor (NGF), and individual BH3-only proteins were targeted for knockout using CRISPR-Cas9 technology. On postnatal day 5, neurons were deprived of NGF to induce apoptosis and neuronal survival was assessed using time-lapse microscopy. Quantitative analysis revealed that the knockout of each individual BH3-only protein slowed the rate of apoptosis, however, neuronal death still progressed, suggesting partial redundancy in function. Notably, Hrk knockout resulted in the greatest delay in apoptosis onset, whereas Bim knockout resulted in the shortest onset delay. These findings provide insight into the complex interplay of BH3-only proteins during neuronal apoptosis, with potential implications for neurodegenerative diseases and therapeutic interventions targeting apoptotic pathways.