I. A genetic approach for mitochondrial degradation. Marcomolecules in mitochondria are prone to damage because of their close vicinity to the free radicals, the by products of mitochondrial electron transferring. However there is no significant accumulation of dysfunctional mitochondrial in normally aged tissues. It is conceivable that post mitotic cells ought to constantly monitor mitochondrial abundance and fitness and are able to cleanse the defective mitochondria and replenish with new organelles. Indeed, mitochondria undergo constant turnover in post mitotic cells. On the other hand, accumulation of defective mitochondria has been associated with many types of human neurodegenerative diseases, further indicating a crucial involvement of this mitochondrial surveillance system in maintaining cell wellness and tissue integrity. But it remains to be determined on first ground whether defective mitochondria are selectively removed. Little is known regarding the mechanism of mitochondrial degradation, which is essential for maintaining overall fitness of mitochondria population, and likely play a role in mitochondrial inheritance. We have developed a genetic approach to quantitatively assay mitochondrial degradation, and test in principle whether cells can selectively eliminate damaged mitochondria. To delineate the cellular responses against defective mitochondria, we are applying this assay for a genome wide RNAi screening in S2 cells. We expressed a bacterial toxin, PorB at low level in culture cells and tissues. PorB localized to and damaged a subset of cellular mitochondria. As a reference, an endogenous mitochondrial protein, Tom20 was also expressed. Mitochondrion is believed to be turned over by autophagic engulfment of whole organelle. Thus the behavior of reporter proteins will reflect the fate of the particular mitochondriaon that they reside in. We found that PorB was turned over much faster than Tom20 after a pulse of induction. The fast decline of PorB coincided with the appearance of vesicles containing abnormal mitochondria. Also most of PorB harboring mitochondria were associated with lysosomes, suggesting that defective mitochondria were degraded through a lysosome dependent process. Genetic analysis revealed that proteasome and autophagy were required for the removal of damaged mitochondria, and demonstrated Parkin dependent degradation of damaged mitochondria in muscle. This study present a direct evidence of selective degradation of defective mitochondria in otherwise healthy cells, also provide a genetic handle dissect the molecular players and cellular pathways participating in the process. A genome wide screening is currently underway. Accumulation of defective mitochondria ahs been linked to the pathogenesis of many neurodegenerative diseases. Most mitochondrial DNA diseases are caused by heteroplasmic mtDNA mutations. Uncovering the mechanism of mitochondrial turnover not only will facilitate our understanding of basic biology regarding mitochondrial homeostasis, it may also provide new angle for managing age-related neurological and mtDNA diseases. II. Mitochondrial DNA mutations on stem cell aging Mitochondria are prone to damage. Besides converting energy to ATP and carrying out biosynthesis, mitochondria also generate much of free radials that can damage proteins, lipids and DNA. To make matters worse, mitochondrial DNA has no histone protection and lacks recombination or other significant repair mechanisms. Mutations on the electron transport chains complexes could result in the generation of more free radicals and exacerbate the mitochondrial damage in a feed-forward cycle. Accumulation of mutations on mtDNA during lifetime has been postulated to cause age related decline of biogenetics and tissue homeostasis. Recent studies that engineered mice with elevated rate of mtDNA mutagenesis display premature aging, attest the mtDNA mutation in aging process in principle. However the levels of mtDNA mutations from various tissue of normally aged animals including liver, heart, brain and others are rather too low to possibly elicit any pathological consequences, which argues against a causative role of mtDNA mutations in physiological aging, particularly in the post mitotic tissues. DNA replication is the source of mutations. In the adulthood, most of tissues are consist of post mitotic cells that have no or very limited replication, which might explain the lack of mtDNA mutations in most somatic tissues (please refer to the next segment for more detail on this issue). On the other hand, mtDNA mutations in these actively dividing cells in the adulthood, i.e., stem cells could reach high level along lifespan and compromise stem cells activities. Considering the essential roles of stem cells in tissue homeostasis, age dependent decline of stem cells functions could contribute to the aging of whole organism. In the past year, we have been utilizing the drosophila female germline stem cells, one of the best-characterized and genetically tractable models to study the aging of stem cells. Normal aged female flies completely cease to produce any egg about six-week old, while most of germline stem cells remain in the niches, indicating that some type of cellular abnormality occurred and impede stem cell differentiation. We used mtDNA mutation selection as an indication of mtDNA mutation load in female germline stem cells. We found that the frequency of escapers increased dramatically in old female comparing to young flies, reflecting a higher level of mtDNA mutations in aged germline stem cells. We also found old ovary demonstrated deficiency in mtDNA encoded respiratory complex, indicating that mtDNA mutations level is high enough to compromise the functionality of aged stem cells. In addition, virgin female that have reduced number of stem cell division cycles has higher mitochondrial activity, fecundity comparing to these crossed flies. Our results suggested that continues division of stem cells would lead to the accumulation of mtDNA mutations, which in turn might compromise stem cell activity and eventually trigger the shutdown of oogenesis. We are now trying to genetically alter mtDNA mutations level and test whether mtDNA mutations play a causal role in stem cell aging.