Inaccurate Translation of the Mitochondrial Genetic Code as a Mechanism Contributing to Aging

Abstract

I. SUMMARY During the last decades, incidence of age-related metabolic and degenerative diseases increased steadily in all industrialized countries turning aging issues into the focus of many socioeconomic discussions. Aging is characterized by a progressive loss of physiological integrity due to accumulation of changes, malfunctions and congregating damage to organs and tissue systems. It is challenging to dissect the interconnectedness between the elicitors, their relative contribution to aging, and the influence between different type of damages and compensatory responses provoked by them. Several studies have shown that impaired mitochondrial function is involved in the pathogenesis of aging and age-related neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease. Several theories exist on how aging and mitochondria depend on and influence each other. Most of them focus on reactive oxygen species (ROS) production, reduced respiration, mitochondrial DNA (mtDNA) mutations and morphological alterations of mitochondria. Next to these aspects, mitochondrial translation came into focus as experiments in yeast have shown that increasing the accuracy of mitochondrial ribosomes extended cellular lifespan. So, could it be that reduced accuracy shortens lifespan? And could this be a potential source for age-related mitochondrial dysfunction and a trigger for premature aging? In this thesis we are focusing on consequences of mitoribosomal mistranslation on the behavioral phenotype of aging mice. Mutations which reduce the translational fidelity of the ribosome have been found to naturally occur in bacteria as well as in yeast. Our collaboration partners in the Böttger lab identified the candidate position in mammalian mitoribosomal proteins homologue to two of these mutations as amino acid substitutions V338Y and G315R in the nuclear encoded mitoribosomal protein MRPS5 (uS5m). This protein is an important building block of the fully assembled mitoribosome and plays a crucial part in fine-tuning the mitoribosomal translational machinery. We studied these two mutations separately in aging female mice to evaluate the effect of mistranslation on aging. Because neurons and muscles are particularly dependent on mitochondrial function and as long-lived cells particularly sensitive to the accumulation of damage, a strong focus was placed on the behavioral screening of the knock-in lines at three ages (9, 14 and 19 months). Behavioral phenotyping included standard behavioral tests of sensorimotor function, emotional behavior, as well as learning and memory. In addition, new approaches based on fully automated behavioral profiling of group-housed mice in their home cage were used. This was complemented by the monitoring of clinical signs of aging, the characterization of mitochondrial function, as well as functional genomic, proteomic and metabolomic analyses in cooperation with the Böttger lab and others. Overall the results from the V338Y mouse line suggest that the mutation increases anxiety and reduces explorative behavior in 9 months old mice, making their behavior similar to that of 19 months old wild-type mice of the same genetic background. Performance in hippocampus-dependent learning tasks strongly depended on the paradigm. In aversive environments e.g. the water maze place navigation task, the performance of V338Y mutants was reduced while in T-maze or IntelliCage experiments with less aversive settings, no decline was observed. By studying the G315R mouse line, no evidence for a phenotypic difference between mutants and wild-types could be found. The only difference was found in the water maze place navigation task where a mild deficit appeared in the probe trial. Later, it was realized that the G315R mutation is not producing a sterical hindrance at the interface of MRPS5 (uS5m) and bS18-2 (mS40) in the mitoribosome as it can be observed in the underlying mutation G104R in E. coli. Therefore, this line was further considered as additional control line for discussing results from the V338Y mouse line. Unfortunately male animals for this study were already too old for regrouping when we started planning the experimental process. As all animals should experience the same tests to avoid possible confounding effects due to prior learnings, we decided to work with only females in the whole study. With a retrospective study on variability in male and female mouse behavior during the water maze place navigation task and the open field test, we could show that including female mice in experiments does not cause a relevant increase of data variability – contrary to the view of many. Therefore, we assume that the results from the V338Y and G315R line are as variable as if only male mice would have been tested. Questions regarding a sex-dependent mutation effect though cannot be answered with our study. The observed cognitive impairment under stress, the anxiety-related behavioral deterioration, and the enhanced metabolic aging in the V338Y line let us conclude that the data presented in this study indicates an association of translational fidelity with aging processes during lifetime. Then again, despite the pivotal role of mitochondria in cellular physiology, the observed behavioral alterations due to mistranslation of mtDNA-encoded proteins can be considered rather little at the organismal level. Further investigations on the behavioral as well as on the cellular level are needed to understand if and how the mitochondrion can adapt and reach for functional complementation to face the consequences of such a fundamental mutation.