Telomerase reverse transcriptase in motion - dynamics of subcellular localization and functional consequences in aging and senescence

• Applicants: Professor Dr. Joachim Altschmied; Professorin Dr. Judith Haendeler
• Subject Area: Biogerontology and Geriatric Medicine ; Biophysics
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 429746873

Telomerase plays a central role in aging processes by counteracting telomere shortening. Its catalytic subunit Telomerase Reverse Transcriptase (TERT) has, besides its task in telomere-preservation, also non-telomeric functions. It has been demonstrated that TERT is present in mitochondria, where it has a protective role. Of note, post-mitotic cells of the cardiovascular system possess TERT in both compartments. Under certain conditions, it is exported from the nucleus with a concomitant increase in the mitochondria. Up to now it is unclear, if the same TERT molecules that leave the nucleus enter the mitochondria. Combining the unique expertise and methods of the partners in this proposal will allow for the first time to follow existing TERT molecules in single cells, also under conditions relevant for cellular senescence, aging and age-related diseases. One aim of this project is to track already existing TERT molecules fused to the photoconvertible Dendra2 protein, which after irradiation changes its fluorescence properties and can thereby be distinguished from newly synthesized molecules, in living cells. This will be done in several cell types of different proliferation capacity stably expressing TERT-Dendra2 and, additionally, by microinjection of the recombinant protein. To study the remote possibility of a transfer of TERT from the nucleus to the mitochondria in a non-canonical way, we will destroy the mitochondrial targeting sequence in TERT to prevent mitochondrial import via the translocases of the mitochondrial membranes. The above-described increase in mitochondrial TERT leads to the hypothesis that this protein affects intra-mitochondrial processes at various levels. Therefore, we will investigate by Förster Resonance Energy Transfer, Single Molecule Localization Microscopy and immunoprecipitation, whether TERT interacts with Mitochondrial Transcription Factor A and Manganese Superoxide Dismutase, as all three proteins have been demonstrated to be associated with mitochondrial DNA. Furthermore, the impact of TERT on the mitochondrial transcriptome will be explored by comprehensive profiling of mitochondrial RNAs from cells completely devoid of TERT, containing TERT in the nucleus and the mitochondria or exclusively in the latter. In addition, changes in the respiratory chain will be examined by O2-consumption and at the level of respiratory chain supercomplex assembly. Finally, we will treat cardiomyocytes and cardiac fibroblasts with stimuli relevant for cellular senescence, aging and age-related diseases and follow TERT travel by life cell imaging; these tracking experiments will be mirrored biochemically. On the functional side, we will measure respiration, apoptosis, hypertrophy, senescence and myofibroblast differentiation potential. These studies will for the first time shed light on the dynamic distribution of existing TERT molecules and its cellular consequences in senescence, aging and age-related diseases.

• DFG Programme: Research Grants
• International Connection: Russia
• Partner Organisation: Russian Foundation for Basic Research, until 3/2022
• Cooperation Partner: Valentin I. Borshchevskiy, Ph.D., until 3/2022