Hypoxia Triggers BNIP3/BNIP3L-Associated Mitophagy in HT22 Immortalized Hippocampal Neurons: A Temporal Descriptive Study

Hypoxia poses a significant threat to hippocampal neurons, but the temporal dynamics of mitophagy pathways in neuronal models remain incompletely characterized. This study aims to delineate time-dependent mitochondrial structural damage, fragmentation, and mitophagic responses in hippocampal neuronal cells under sustained hypoxia and to explore the key mitophagy receptors involved. Murine HT22 immortalized hippocampal neuronal cells were exposed to 1% O2 for 6–72 h. Mitochondrial integrity was assessed using Western blot and transmission electron microscopy. Mitochondrial fragmentation was quantified via HSP60 intensity, mitochondrial length, and number of fragments per cell using immunofluorescence and MitoTracker live-cell labeling. Mitophagic activity was evaluated using LC3B puncta quantification, LC3B–Tom20 colocalization, and mitochondria–lysosome proximity via LysoTracker co-labeling. The transcriptional responses of five mitophagy-related genes (Bnip3, Bnip3l, Pink1, Fundc1, and Atg5) were screened using RT-qPCR, with protein validation by Western blot and immunofluorescence. Prolonged hypoxia progressively reduced Tim23 and Tom20, with ultrastructural damage and mitochondrial fragmentation seen. Mitophagy-associated phenotypes were evident, as shown by increased LC3B puncta, LC3B–Tom20 colocalization, and mitochondria–lysosome proximity. Bnip3 and Bnip3l showed the earliest and most pronounced transcriptional upregulation, with BNIP3 and BNIP3L protein levels elevated from 12 to 48 h. BNIP3 and BNIP3L intensities increased after 48 h, exhibiting granular and filamentous morphologies, respectively. This study provides a temporal framework for hypoxia-induced mitophagy in hippocampal neurons, identifying BNIP3/BNIP3L as early and prominent responders, which may inform therapeutic targeting in hypoxic brain injury.