BACKGROUND: The mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10) is implicated in neurodegenerative disorders, particularly Alzheimer’s disease (AD), through its interplay with the amyloid-β peptide (Aβ). However, its independent pathological role in AD remains unclear. METHODS: To explore the individual effects of HSD10 and amyloid precursor protein (APP) overexpression (including the Aβ42-generating APPSwe/Ind variant), monoclonal HEK293 cell lines were developed. Cellular fitness was evaluated by measuring ATP levels, cell viability, and cytotoxicity measurements under glucose and galactose culture conditions. Mitochondrial metabolic changes were analysed using mitochondrial electron flow measurements in response to various metabolic substrates. HSD10 enzymatic activity was monitored using a fluorogenic probe, and two HSD10 inhibitors were tested for their ability to reduce cytotoxic effects. Statistical significance was determined using appropriate tests as detailed in the methods section. RESULTS: The overexpression of HSD10 or APPSwe/Ind led to mitochondrial dysfunction and reduced viability, particularly under glucose-deprived conditions. HSD10-driven cytotoxicity was linked to its enzymatic activity and associated with impaired TCA cycle function, reduced β-oxidation, and increased oxidative stress. In contrast, APPSwe/Ind overexpression induced Aβ42 production, glucose hypermetabolism, and enhanced β-oxidation. Aβ42 also affected HSD10 activity and further amplified its cytotoxic effects. The benzothiazole-based HSD10 inhibitor 34 restored cell viability under both HSD10 overexpression and Aβ42-rich conditions. CONCLUSIONS: HSD10 and Aβ42 each contribute to mitochondrial impairment via distinct metabolic pathways. These findings established HSD10 as an independent pathological factor in AD and support the potential of HSD10 inhibitors, particularly inhibitor 34, as therapeutic agents targeting mitochondrial dysfunction in AD. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13195-025-01821-8.

Biomedical Research Centre University Hospital Hradec Kralove Sokolska 581 Hradec Kralove 500 03 Czech Republic

Faculty of Science Department of Chemistry University of Hradec Kralove Rokitanskeho 62 Hradec Kralove 500 03 Czech Republic

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