LOSS OF NEUROD2 EXACERBATES NEURONAL VULNERABILITY TO OXYGEN–GLUCOSE DEPRIVATION AND REPERFUSION

Hypoxia remains a critical global health challenge, characterized by high rates of mortality and long-term disability, which suggests research of alternative therapeutic strategies to enhance neuronal resilience and functional recovery. NeuroD2 (ND2), a neuron-specific transcription factor essential for neuronal differentiation, neuroprotection, and plasticity, represents a promising candidate for such interventions.
In this study, we investigated the neuroprotective role of ND2 using an in vitro hypoxia model. ND2 expression was either upregulated or suppressed via lentiviral-mediated vectors, respectively. Ischemic injury was induced by 8 hours of oxygen–glucose deprivation (OGD), followed by 16 hours of reperfusion under normoxic conditions. Cellular survival was assessed using cell survival assay, while DNA fragmentation was quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). To elucidate the molecular mechanisms underlying ND2-mediated effects, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was conducted to identify differentially expressed proteins and signaling pathways regulated by ND2 modulation.
Under OGD conditions, ND2 overexpression significantly enhanced cell survival and reduced DNA fragmentation, while ND2 inhibition increased apoptosis and cell death. Proteomic analysis revealed that ND2 modulation altered key mitochondrial respiratory proteins (Ndufa12, Ndufb9), apoptosis-related factors (Aifm1), and intracellular signaling and chromatin remodeling regulators (Mapk3, Smarcc2). These findings suggest that ND2 overexpression may be neuroprotective and necessary for neuronal cells, as inhibition of ND2 increases DNA damage and cellular stress.
Collectively, our results identify ND2 as a critical regulator of neuronal survival under ischemia-reperfusion conditions and highlight its potential as a therapeutic target for hypoxia- and ischemia-related neurological disorders.