Protein synthesis in slowed aging: insights into shared characteristics of long-lived mouse models

2014 Spring.The following dissertation describes a series of experiments with the overall aim to understand the role that changes in protein synthesis have in slowed aging. The specific aims of the three sets of experiments were 1) to determine if chronic administration of the mTORC1 inhibitor rapamycin to mice increases proteostatic mechanisms in skeletal muscle, heart, and liver; 2) to determine if an underdeveloped anterior pituitary, caused by deletion of the Pit-1 gene in mice, increases proteostatic mechanisms in skeletal muscle, heart, and liver of long-lived Snell dwarf mice; 3) and to determine if transient nutrient restriction during the suckling period in mice (i.e. crowded litter), increases proteostatic mechanisms in skeletal muscle, heart, and liver later in life. In Experiment #1 we found that mitochondrial proteins were preferentially synthesized in skeletal muscle and that global protein synthesis in the heart was maintained despite reduced cellular proliferation and mTORC1 activity in mice fed rapamycin compared to normal diet controls. Originally we determined that these data were indicative of an improved somatic maintenance of skeletal muscle mitochondria and the heart proteome. Since we could not account for changes to other energetic processes (e.g. metabolism), we reasoned that our data was more consistent with proteostasis, a component of somatic maintenance. In Experiment #2 we developed a novel method for assessing proteostasis and determined that Snell dwarf mice had an increase in proteostatic mechanisms across sub-cellular fractions within skeletal muscle and heart compared control mice, despite differential rates of protein synthesis in the face of decreased mTORC1. Together with our previous investigations into rapamycin fed and caloric restriction models of long-life we concluded that increased proteostatic mechanisms may be a shared characteristic of models of slowed aging. In Experiment #3 we demonstrate that the crowded litter mouse transitions from growth to maintenance as it ages. Furthermore, in the crowded litter mouse, we demonstrate that proteostasis is not dependent upon decreased mTORC1. Our results indicate that decreased mTORC1 does not necessarily correlate to decreases in protein synthesis across all sub-cellular fractions. Discerning which proteins and the mechanism(s) of how specific proteins can be preferentially synthesized despite decreases in protein synthesis in other fractions and decreased mTORC1, may give further insight into characteristics of slowed aging. Further, we demonstrate that increases in proteostasic mechanisms are a shared characteristic of multiple unique models of slowed aging and therefore, provides a basis for future work aimed at slowing the aging process

AGE-RELATED DIFFERENCES IN MITOPHAGY RESPONSE TO ACUTE EXERCISE IN SKELETAL MUSCLE OF MICE

BACKGROUND: mitophagy, a process that is responsible for eliminating damaged mitochondria, gradually declines during aging. This decline, particularly prominent in aging skeletal muscle, correlates with compromised mitochondrial function, impacting mobility and muscle strength in older individuals. Previous research demonstrates that after 6 hours of acute exercise, young mice exhibit heightened mitophagy compared to their sedentary counterparts. However, many studies revealed that the accumulation of damaged mitochondria due to oxidative stress, DNA mutations and dysfunctional lysosome can overwhelm the mitophagy process, reducing its efficiency in removing dysfunctional mitochondria in aged skeletal muscle. Therefore, our hypothesis is exercise-induced mitophagy in skeletal muscle is impaired with aging. METHODS: We transfected pMito-timer into Flexor digitorum brevis (FDB) muscle of 3 (young) and 25 (old) month old mice. After 10 days recovery, mice were familiarized to treadmill running for three days (10 min at 10m/min). On the fourth day, mice performed 90 minutes treadmill running exercise. Young mice ran 10 min at 13m/min, 10 minutes at speed 16m/min, 50 minutes at 19m/min, and finally 20 minutes at 21m/min. Based on the NMR data, the old mice have higher body fat mass and lower muscle mass in compare with young group. Therefore, old mice cannot have same performance as young group. In this regard, we normalized and adjusted the running protocol for workload equality. We calculated the work performance based on body weight by using the equation (Body weight (kg)*distance (m)*time (m)* 0.05 incline). Also, we monitored blood lactate before and after exercise to monitor relative exercise intensity. After 6 hours, we harvested FDB to observe mitophagy by confocal microscopy.RESULTS: Young mice had significantly higher mitophagy following exercise, as seen previously. However, mitophagy was significantly elevated in sedentary old mice compared to either young group. Furthermore, there was no effect of exercise in old mice to further elevate mitophagy above sedentary, age-matched counterparts.CONCLUSION: Our study shows age-related differences in skeletal muscle mitophagy and the mitophagy response to acute exercise. To better comprehend our findings, we need to investigate how metabolic preferences during acute exercise differ between young and old mice and how these variances impact mitophagy