Subcellular Localization of Telomerase Reverse Transcriptase Regulates Gene-Specific Modulation of Mitochondrial DNA...

Telomerase reverse transcriptase (TERT) exhibits non-canonical neuroprotective functions; its overexpression protects against hypoxic-ischemic brain injury and preserves mitochondrial function. Here, we identified a novel phenomenon in an in vitro model in which oxygen-glucose deprivation/reoxygenation induced a phase-dependent redistribution of endogenous TERT in HT22 neuronal cells, characterized by early mitochondrial accumulation followed by predominant nuclear localization. We aimed to determine whether subcellular localization of TERT dictates its functional specificity in regulating the copy number of mitochondrial DNA-encoded genes in neurons. HT22 cells expressing TERT, targeted to either the mitochondria (mito-TERT) or nucleus (nuc-TERT), were generated using adenoviral transfection. Subcellular localization was confirmed using western blotting. Mitochondrial DNA copy number was assessed using quantitative polymerase chain reaction (qPCR), and direct mito-TERT binding was assessed using chromatin immunoprecipitation (ChIP)-qPCR; ND1 and ND2 mRNA levels were measured using qRT-PCR. Both nuc-TERT and mito-TERT models were successfully established. qPCR analysis showed that mito-TERT specifically upregulated the copy number of mitochondrial complex I genes (nicotinamide adenine dinucleotide-hydrogen dehydrogenase subunit [ND]1/2), whereas nuc-TERT broadly upregulated genes across complexes I, III, IV, and V. A regulation specificity index showed that mito-TERT selectively enhanced ND1 copy number. ChIP-qPCR confirmed mito-TERT enrichment at the ND1 and ND2 promoters. Together, these findings demonstrate that TERT regulates mitochondrial DNA copy number through distinct, compartment-dependent mechanisms. Mito-TERT exhibited gene-specific regulation via direct promoter binding, whereas nuc-TERT displayed broader effects. The stress-induced redistribution of TERT and these mechanistic insights reveal a novel paradigm for multiphasic TERT-mediated neuroprotection in brain injury.