Alzheimer’s disease (AD) is characterized by the accumulation of ß-amyloid (Aß), which impairs neural stem cell function and contributes to cognitive decline. ß-hydroxybutyrate (BHB), a ketone body elevated during fasting or ketogenic diets, has been suggested to exert neuroprotective effects. This study aimed to investigate the effects of BHB and Aß on the stemness and mitochondrial integrity of human neural stem cells.

Human neural progenitor cells were treated with increasing concentrations of BHB (0.5–5 mM) and Aß (0.1–10 µM) to assess their individual effects on stemness and mitochondrial structure. Stemness was evaluated by analyzing the expression of markers such as PAX6, SOX2 and Nestin through qPCR and immunocytochemistry. Cell viability and morphology were assessed using phase-contrast microscopy and viability assays. To examine mitochondrial integrity, immunocytochemical staining and qPCR for TOMM20, a mitochondrial outer membrane protein, was performed to assess mitochondrial morphology and gene expression. In a subsequent experiment, hNSCs were pretreated with BHB for 24 hours before Aß exposure to evaluate potential protective effects on both stemness and mitochondrial structure.

The results demonstrated that BHB treatment preserved the expression of PAX6, SOX2 and Nestin in a concentration-dependent manner and maintained normal cell morphology and viability. Aß exposure led to a reduction in stemness marker expression, disrupted mitochondrial morphology, and decreased gene expression. TOMM20 staining revealed that Aß-treated cells exhibited abnormal, punctate mitochondrial patterns, indicating mitochondrial stress. Notably, BHB pretreatment before Aß exposure significantly preserved stemness marker expression and restored mitochondrial morphology toward a more filamentous, as revealed by TOMM20 staining and gene expression.

In conclusion, BHB supports neural stemness and preserves mitochondrial integrity in hNSCs under Aß-induced stress. The protective effects of BHB may involve modulation of mitochondrial dynamics, as evidenced by TOMM20 analysis. These findings suggest a potential therapeutic role for BHB in neurodegenerative diseases through the preservation of endogenous regenerative capacity and mitochondrial condition.