Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H₂O₂ treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H₂O₂ treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Telomerase Reverse Transcriptase (TERT), in addition to its well-known role in telomere lengthening, also has non-canonical functions, including gene regulation and protection against apoptosis. Beyond its nuclear functions, it is now recognized for its presence inside mitochondria. However, the biological role of TERT in mitochondrial physiological activity, with its specific mechanism of action, still needs to be clarified. This work clearly demonstrates the presence of TERT inside the mitochondrion under physiological conditions, in different cellular contexts, both with endogenous and ectopic TERT expression, and regardless of the presence of telomerase RNA counterpart TERC. TERT was shown to bind mitochondrial DNA, influencing mitochondrial replication and transcription. Furthermore, electron microscopy analysis of morphology revealed TERT-induced fragmentation of the mitochondrial network. Collectively, our findings suggest that TERT may play a role in regulating mitochondrial biogenesis and dynamics, and influencing processes such as fission and mitophagy, essential for maintaining mitochondrial homeostasis and closely connected to cellular states.

Previous studies have demonstrated that Telomerase Reverse Transcriptase (TERT) is localized inside mitochondria, where it associates with mitochondrial DNA (mtDNA), thereby influencing replication and transcription. Using a combination of quantitative fluorescence microscopy and biochemistry, we demonstrate that TERT binds indirectly to mtDNA through the interaction with mitochondrial transcription factor A (TFAM) to induce mitochondrial fragmentation. These findings reveal a novel mitochondrial interaction involving TERT and highlight a role for TERT and TFAM in the regulation of auto/mitophagy. This interaction is an important factor for mitochondrial function and contributes to the remodelling of mitochondrial respiratory capacity. Highlights: TERT predominantly binds TFAM inside mitochondria. TERT induces mitochondrial fission. TERT/TFAM cooperates to enhance auto/mitophagy. TERT-overexpression promotes remodelling of the electron transport chain.

Ionizing radiation (IR) induces DNA damage that activates innate immunity through the cyclic GMP–AMP synthase (cGAS) pathway, yet the mechanisms determining why only a subset of radiation-induced micronuclei (MNi) become cGAS-positive remain unclear. Here, we investigated the dose- and time-dependent dynamics of cGAS-positive MNi and the molecular events regulating their formation in HaCaT human keratinocytes. cGAS recruitment occurred with delayed kinetics relative to total MNi formation and was associated with micronuclear envelope destabilization and lamin B1 loss. Structural observations support a sequential model in which early recruitment of the autophagy receptor p62 precedes micronuclear envelope destabilization and subsequent cGAS recruitment. Reactive oxygen species (ROS) generated following irradiation emerged as key upstream regulators by promoting p62 recruitment and micronuclear destabilization. Accordingly, ROS scavenging with N-acetylcysteine markedly reduced both p62-positive and cGAS-positive MNi. Conditioned-medium experiments further demonstrated that diffusible ROS-dependent signals generated by irradiated cells promote the formation of cGAS-positive MNi in non-irradiated bystander cells. Finally, cGAS activation was accompanied by delayed induction of type I interferons and ISG15, which was attenuated by ROS scavenging. Together, these findings identify oxidative stress as a critical regulator linking radiation-induced genomic instability to innate immune activation through p62-dependent micronuclear sensing.