Healthy mitochondria are required for maintaining neuronal integrity and function. Mitochondria undergo dynamic fission-fusion and damaged mitochondria are cleared by autophagy (termed as mitophagy). Recently, there have been debates on whether mitophagy occurs locally in axons or whether damaged mitochondria need to be transported to the neuronal soma for lysosome-mediated turnover (Cai et al., 2012; Ashrafi et al., 2014; Maday and Holzbaur, 2014). To address this question, we have developed a Drosophila wing nerve model, which allows us to label axons and mitochondria by different fluorescent proteins to study their changes during aging in vivo.
We found mitochondria in axons were significantly shorter in aged flies, suggesting that mitochondria become fragmented during aging. Alternatively, it may be due to reduced mitophagy in aged axons. We hence studied the consequence of manipulating key mitophagy genes Pink1 and Parkin in axons. To our great surprise, unlike in muscles where the Pink1B9 null mutation caused severe mitochondria abnormality and muscle degeneration (Clark et al., 2006; Park et al., 2006), Pink1B9 flies showed normal mitochondria morphology, size and density in neurons, and did not cause spontaneous axon degeneration. This result suggests that local axonal mitophagy might not be an essential mechanism in maintaining neural integrity during aging. Consistently, we found that co-localization of mitochondria and autophagosomes almost exclusively occurred in the neuronal soma and was essentially not observed in axons, even in aged flies. The co-localization occurrence in soma did not decrease with age, suggesting a constant mitophagy level in neurons during aging. Co-localization of axonal mitochondria and autophagosomes in vivo were observed only after axotomy in the injured wing nerve. This is consistent with previous in vitro studies where extreme insults such as H2O2 or CCCP were used to induce mitophagy in cultured neurites.
Next, we overexpressed Pink1 or Parkin to see if promoting mitophagy mitigated age-associated accumulation of damaged mitochondria in neurons. Unexpectedly, however, both flies showed increase in fragmented mitochondria and severe spontaneous axon degeneration. Upregulation of mitochondria fusion genes could fully rescue the axonal phenotypes of Parkin OE. Together, our results indicate that mitophagy is dispensable for axonal integrity and we have additional evidence suggesting that fission-fusion plays a major role in keeping healthy and functional mitochondria in axons during normal aging.