The Role of Autophagy in Maintaining Mitochondrial Morphology in C.

elegans Muscle
Karen Lin

ABSTRACT The process of autophagy has been shown to be required by almost all pathways that prolong lifespan in Caenorhabditis elegans worms. Similarly, the mitochondria are also credited with the regulation of aging. Not surprisingly, research has shown that both autophagy and mitochondrial metabolism are involved in each others regulation to some extent. By investigating how autophagy plays a role in mitochondria morphology, it could yield more information about how both autophagy and the mitochondria interact with each other to regulate aging. Here it is shown that RNAi knockdown of autophagy (atg) genes bec-1, atg-14, atg-18, and lgg-1 results in severe changes in the organization and morphology of the mitochondria in the muscle. As bec-1 has been also shown to function in the retrograde transport from early endosome to Golgi, it was next investigated whether other genes involved in retrograde transport also had an effect on mitochondria morphology. However, RNAi knockdown against unc-51 and vps-35 had no effect on the mitochondria. This suggests a specific role for autophagy in maintaining mitochondria morphology that is independent of vesicle trafficking. The discovery of this regulation brings research one step closer in understanding the role of autophagy in the health of an individual as well as a possible mechanism for how autophagy can help prolong lifespan and slow the aging process.

INTRODUCTION Macroautophagy, which will be referred to as autophagy for the rest of the article, is a type of cellular degradation using lysosomal machinery in which the cellular contents are recycled (Toth et al., 2008). The process of autophagy is depicted in Figure 1. In many cases, induction of autophagy has been shown to increase under stressful conditions and is required for the long lifespan phenotype (Melendez et al., 2003). Autophagy preserves cellular resources by eliminating inefficient organelles and recycling these materials to the rest of the body. Additionally, autophagy has been shown to induce permanent death in cells that are damaged beyond repair. In both cases, autophagy acts to preserve cellular homeostasis by removing cells that threaten this equilibrium. Not surprisingly, autophagy has been found to be deficient in many cancer and disease stricken patients, suggesting a critical role for autophagy in preventing tumorigenesis and maintaining individual health (Mizushima et al., 2008; Levine and Kroemer, 2008). Therefore, determining the means by which autophagy is regulated would allow for not only better medical treatments and means of prevention but also methods to increase individual life span and improve health.
Diagram 1: Autophagy machinery. The diagram below (from Mizushima et al., 2008) shows how macroautophagy begins and ends. It begins with a double membrane bound structure of unknown origin, the phagomore, and encircles cytosolic proteins and organelles. When the membrane structure completely encloses the organelles, it is called the autophagosome. This structure will go on and merge with a lysosome, forming what is known as an autolysosome, ready to degrade other cellular components. It differs from microautophagy, which is the degradation of materials directly through the lysosome.

Autophagy and aging in C. elegans Under unfavorable conditions, the nematode Caenorhabditis elegans goes through a developmental arrest stage referred to as dauer, which is an alternate third larval stage that specializes in helping them survive harsh environmental conditions, including dietary restriction. Interestingly, dauer development requires autophagy. This was determined when daf-2 mutants treated with bec-1 RNAi formed abnormal dauers and resulted in shorter lifespan (Melendez et al., 2003). Mutants in the daf-2 gene that normally encodes an insulin-like growth receptor exhibit extended organism lifespan as well as increased autophagy levels. The bec-1 gene is required for autophagy. Autophagy is required for extended lifespan in daf-2 mutants. This was supported by the speculation that autophagic influence on aging is regulated through the insulin/IGF-1 signaling pathway (Yen and Klionsky, 2008). For example, eat-2 mutant animals are diet restricted and exhibit long life span. The reduction of lifespan of eat-2 mutants after treatment with bec-1 RNAi confirmed the role of autophagy in lifespan extension, in this case by dietary restriction (Hansen et al., 2008). Clearly, autophagy plays an important role in aging in C.elegans. However, recent studies have implicated another role for autophagy, its role in mitochondrial maintenance. Relationship between autophagy and the mitochondria According to the mitochondrial lysosomal axis theory of aging, aging is the result of the accumulation of damaged mitochondria possibly as a result of imperfect autophagy (Brunk and Terman, 2002). It is suggested that mitochondrial age-related damage is the result of reactive oxygen species (ROS), an inevitable product of oxygen metabolism (de Grey, 1997). Mitochondria suffering from ROS damage become less efficient. They produce not only less energy for the cell but also less ROS as well. This decrease of ROS production makes them less

likely to be targeted for autophagic degradation. The result is an accumulation of deficient mitochondria in the cell, which will contribute to aging, as suggested by the mitochondrial lysosomal axis theory of aging (Brunk and Terman, 2002). Consequently, it appears that autophagic activity regulates aging not only through nutritional pathways but also through mitochondrial maintenance. Therefore, determining what the correlation between autophagy and the mitochondria and their influence on aging has been the topic of hot debate. Functioning as the site of cellular respiration, mitochondria are heavily influenced by autophagy. For example, deletion of atg genes atg1, atg6, atg8, and atg12 leads to mitochondrial related defects in yeast (Zhang et al, 2007). Additionally, in the process of mitophagy, the removal of mitochondria by autophagic means, research shows that the mitochondrial permeability transition pore (mtPTP) releases ROS to signal autophagy to target mitochondria for degradation (Elmore et al, 2001). Furthermore, mitophagy specificity has been shown to be heavily influenced by atg genes atg11 and atg32 (Gottlieb et al, 2010). These findings all suggest a critical role for autophagy in mitochondrial maintenance. In regards to aging, further elucidation of this relationship between autophagy and mitochondria could yield a better understanding of longevity pathways in C. elegans. The research presented here aims to expand that information. It investigates the role of several atg genes and their effect on mitochondria morphology in the C. elegans muscle tissue. Based on the results of Zhang et al., (2007), it is expected that the mitochondria will be detrimentally affected. The results presented here may help better elucidate the relationship between autophagy and mitochondria. This may bring research one step further in understanding autophagy which may not just improve medical treatments for age-related diseases but may also yield insight on how to maintain a healthier, longer life.

MATERIALS AND METHODS Strain N2 double label(dl) Ex[myo3pro::OuterMembraneINV-YFP + myo3pro::MatrixCFP + rol-6] Line 5 strain was a kind gift from Dr. Alex van der Bliek, UCLA Biological Chemistry. This strain was maintained on Nematode Growth Medium (NGM) agar medium with E. coli strain OP50 at 15C as described (Brenner, 1974). RNAi experiments Bacteria expressing specific dsRNA were obtained from the Ahringer (Kamath et al., 2003) and the Vidal libraries (Rual et al., 2004). Clones that target the following genes were used: bec-1, atg-14, atg-18, lgg-1, unc-51, vps-35, and W09C5.8 (cytochrome C oxidase IV). As a negative control, bacteria carrying the empty vector L4440 were used. Single colonies were grown on bacteria RNAi growth plates that contained tetracycline and carbenicillin. Fresh clones were picked and grown in Lysogeny Broth containing the antibiotic ampicillin (LB/Amp) overnight at 250 cycles per minute at 37C. Next, cultures were plated on NGM plates containing carbenicillin with 2 mM IPTG at room temperature over night. L4 animals carrying the reporter transgene were placed onto plates seeded with HT115 E. coli bacteria expressing dsRNA for the corresponding target gene. These animals were then transferred the next day onto fresh plates expressing the dsRNA for the particular gene that corresponded with the original plates that were discarded. The now adult animals were removed the next day but the eggs remained intact. Once progeny reached L4 stage, rollers carrying the transgene were examined on a Zeiss fluorescence microscope.

RESULTS To investigate the role of autophagy in mitochondrial morphology, RNAi was used to knock down the expression of several atg genes in wild type N2 worms that carried the mitochondria marker transgene Ex[myo3pro::OuterMembraneINV-YFP + myo3pro::MatrixCFP + rol-6] Line 5. In animals fed with the bacteria that expressed the empty vector (negative control), their mitochondria were found to have elongated tubular structures organized in parallel rows (Fig1a). However, this level of structural organization was impaired in animals fed RNAi bacteria against W09C5.8 (cytochrome C oxidase IV). As shown by Lee et al., (2003), knockdown of cytochrome C oxidase IV, a key component of the mitochondrial electron transport chain, results in lack of organization in the mitochondria of these worms. Instead of long, neatly arranged mitochondria, the mitochondria found in these worms were severely fragmented. These animals served as a positive control in this study (Fig1b). Similarly, animals fed RNAi bacteria against bec-1 yielded mitochondria with disrupted organization. Although these mitochondria retained their tubular shape, they took on a disoriented disrupted pattern (Fig1c). Furthermore, atg-14 RNAi treatment affected the morphology of mitochondria more severely. Not only were the mitochondria disorganized, but the narrowing of the muscle fiber demonstrated that the muscle itself had become disfigured (Fig1d). The results were similar for the atg-18 RNAi treatment. These mitochondria had a smudged disoriented appearance (Fig1e). On another note, lgg-1 RNAi treatment resulted in very dotted mitochondrial structures (Fig1f). In contrast, unc51 RNAi and vps-35 RNAi treatments yielded mitochondria that remained more similar to the morphology seen in the empty vector (negative control). The mitochondria of unc-51 or vps-35 RNAi animals were straighter and appeared to follow in parallel to each other, more organized than any of the other mitochondria observed under any other RNAi treatment (Fig1g and Fig1h).

ILLUSTRATIONS
Figure 1: The effect of RNAi of atg genes on mitochondrial morphology in N2 double label(dl) Ex[myo3pro::OuterMembraneINV-YFP + myo3pro::MatrixCFP + rol-6] Line 5 worms. The morphologies of the worm were observed in the muscle after L4440 RNAi ( a), W09C5.8 (cyt. C oxidase IV) RNAi (b), bec-1 RNAi (c), atg-14 RNAi (d), and atg-18 RNAi (e) were conducted on this strain. lgg-1 RNAi (f), unc-51 RNAi (g), and vps-35 RNAi (h) were conducted on different occasions.

Graph 1: A total of twenty N2 double label(dl) Ex[myo3pro::OuterMembraneINV-YFP + myo3pro::MatrixCFP + rol-6] Line 5 worms were looked at for each RNAi. The percentage of worms that corresponded to the phenotype pictured in Figure 1a-h is represented below for each RNAi for three trials.

100

Percentage of Worms

90 trial 1 trial 2 80 trial 3

70 L4440 W09C5.8 bec-1 atg-14 atg-18 lgg-1 unc-51 vps-35 RNAi

DISCUSSION The disorganized mitochondria seen in worms with RNAi against autophagy genes were similar to the phenotype of worms with RNAi against W09C5.8 (cytochrome C oxidase IV). This suggests that the mitochondria in autophagy deficient animals were impaired to some extent. For example, there was evidence of mitochondria fragmentation in atg-14, atg-18, or lgg-1 RNAi. In contrast, RNAi against the retrograde transport genes unc-51 or vps-35 had little to no effect on the morphology of the mitochondria. This suggests a specific role for autophagy in mitochondrial morphology. However, it is unclear whether the presence of small dot-like mitochondria seen in the lgg-1 RNAi animals is the result of increased fission of mitochondria or an aggregation of mitochondria with decreased fusion. What is even more puzzling is that these results seem to contradict the conclusion Lee et al., (2003) arrived at with their experiments which showed that worms with 9

increased mitochondrial damage live longer. The worms presented here definitely exhibited significantly abnormal mitochondria morphology but this is due to the lack of autophagy genes. If what Lee et al., (2003) proposes is true, it would mean that lack of atg genes lead to longevity in worms. This reasoning seems highly unlikely because previous research has shown the importance of autophagy in extension of lifespan in C. elegans (Melendez et al., 2003; Toth et al., 2008; Hansen et al., 2008; Lapierre et al, 2011). The only plausible explanation is that the lifespan extension phenotype observed in worms by Lee et al., (2003) was caused by the induction of autophagy. It is possible that the mitochondrial defects of their worms triggered autophagy and as a result, extended the worms lifespan. Interestingly, in regards to worm phenotype, the defects that may have stimulated autophagy have the same appearance as the defects that are the result of impaired autophagy. Regardless of whether or not autophagy expands lifespan in worms with impaired mitochondria function, it is clearly demonstrated here that autophagy has a role in maintaining mitochondrial morphology. Surprisingly, previous studies have indicated a mitochondrial role in supplying membranes for autophagosome biogenesis (Hailey et al., 2010). This relationship may help explain the presence of disrupted organization and fragmented phenotype found in the mitochondrial membranes when autophagy was impaired. What has yet to be determined is whether the change in mitochondria morphology has any affect on mitochondria efficiency, or whether it was a result of a deficiency that caused the change in morphology in the first place. Therefore, measuring ATP content and oxygen consumption rates as a follow up procedure would be important. Additionally, a lifespan assay would be useful to determine the consistency of the results of Lee et al., (2003). If the assay showed the worms had shortened lifespan, however, the next question would be to investigate

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why. This could give insight on the mechanisms by which autophagy, if it did, extended lifespan in the Lee et al., (2003) experiments. A potential problem in this is that the shortened lifespan may be due to natural illness as a result of mitochondria deficiencies, not a phenotype of lack of autophagy genes. This complication makes it difficult to determine whether autophagy is necessary for longevity in worms with compromised mitochondria. Future work would also include whether the change in mitochondria morphology is a developmental defect. Determining this would lead to greater understanding on how autophagy and the mitochondria influence the aging process. Questions that remain unanswered include what the mechanisms by which autophagy maintains mitochondria are and how this influences aging. Since the mitochondria are known for their functions in aerobic cellular respiration, it is likely that nutritional pathways are involved. It would be especially interesting if the target of rapamycin (TOR) nutritional sensing pathway was involved. It would emphasize autophagys influence on mitochondria maintenance because TOR is known to act as a negative regulator of autophagy. This and other means by which autophagy are regulated are still unclear. However, ongoing and future research will help reduce this gap in information. It will bring new advancements in medical treatments and breakthroughs in understanding the mechanisms of aging.

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ACKNOWLEDGEMENTS I would like to take the opportunity to thank all those who have helped me come this far. I thank my mentor Alicia Melendez for her expertise and her continuous support throughout the progression of my experiment. I also thank my teacher Marisa Wagner for her patience and endless dedication to helping me with my project. Most of all, I have a lot to thank for my mom for her continuous encouragement and motivation for me. Without their help, I would not have accomplished as much as I did and I would not be the scientist I am today.

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