Our results provide direct evidence that mitophagy selectively removes and degrades damaged mitochondria. In hepatocytes incubated in nutritionally replete growth medium, only photoirradiated mitochondria that depolarized were sequestered into autophagosomes. When mitochondria were photoirradiated at lower laser power, mitochondria initially depolarized but then recovered polarization as indicated by reuptake of TMRM. Such mitochondria did not undergo mitophagy. Thus, photodamage leading to sustained mitochondrial depolarization was required to initiate sequestration into autophagosomes.
Mitochondrial depolarization occurred in a dose-dependent manner after photoirradiation with 488-nm light (). The lowest light exposure, namely that used to image hepatocytes, did not cause mitochondrial depolarization or induce mitophagy. Initially, after a light exposure about 2.5×103
times greater than that used for imaging, mitochondria depolarized, but they then recovered their membrane potential within about 3
min. At greater photoirradiation, mitochondria depolarized in a sustained fashion. At highest illumination, a bystander effect occurred in which depolarization not only occurred in mitochondria under the light beam but also in adjacent mitochondria outside the illuminated region (). Bystander photoirradiation suggests that a toxic agent formed in illuminated regions diffused into immediately adjacent areas. This toxic agent is most likely ROS, such as singlet oxygen, which is produced during exposure to strong light. Photoxicity-dependent mitophagy occurred in the absence of TMRM or other added fluorophore and, thus, did not require photosensitization of an exogenous absorber (). In addition, mitophagy did not occur with 543-nm light (). Previous work shows that photoirradiation of 400- to 500-nm light causes oxygen-dependent inactivation of flavoproteins and succinate dehydrogenase that is mediated by production of ROS (1
). Thus, photodamage to mitochondria in our experiments is likely via
photoexcitation of succinate dehydrogenase and other mitochondrial flavoproteins.
ROS induce the MPT in mitochondria, leading to depolarization, uncoupling, and more ROS formation (34
). After photodamage, sustained mitochondrial depolarization appeared to be a prerequisite for subsequent mitophagy. Mitochondria that depolarized transiently after light exposure did not undergo subsequent mitophagy, whereas photodamaged mitochondria that underwent sustained depolarization were reproducibly sequestered into autophagosomes (). Moreover, this latter group of mitochondria became permeable to calcein, indicative of the inner membrane permeabilization of the MPT (). Thus, sustained mitochondrial depolarization and associated inner membrane permeabilization seemed to be required for autophagy signaling. These results are consistent with involvement of the MPT in photodamage-induced mitophagy, as previously proposed for autophagy stimulated by nutritional deprivation (10
). Moreover, when cells are treated with a mitochondrial uncoupler such as carbonylcyanide m-chlorophenylhydrazone, depolarized mitochondria are removed (33
Photodamage-induced mitophagy, however, differed from nutrient deprivation-induced mitophagy in several ways. In nutrient deprivation-induced mitophagy, small (0.2–0.3
μm) pre-autophagosomal structures associate with polarized mitochondria and grow into crescent-shaped phagophores that envelope and enclose individual polarized mitochondria into mitophagosomes (19
). Mitochondrial depolarization only occurs at or after formation of mitophagosomes. Subsequently, as the mitophagosomal vesicles acidify and fuse with lysosomes, GFP-LC3 is released and/or degraded (20
). However, when the MPT is inhibited, autophagy also becomes inhibited, indicating that the MPT likely plays a role in the nutrient deprivation-induced mitophagy, possibly in coordination with autophagosomal sequestration.
By contrast, in mitophagy induced by photodamage with 488-nm light, only depolarized mitochondria were targeted for autophagic degradation. Moreover, instead of being enveloped by a crescent-shaped phagophore, the periphery of photodamaged mitochondria became decorated with small speckled aggregates of GFP-LC3 that later coalesced into rings enveloping the entire mitochondrion ( and ). Additionally, mild acidification of photodamaged mitochondria occurred before assembly of continuous GFP-LC3-decorated rings. Subsequently, after completion of the GFP-LC3 ring, the mitophagosomal vesicle became more intensely acidic. Future studies will be needed to determine whether depolarized mitochondria themselves undergo mild acidification or whether a sequestration membrane encloses photodamaged mitochondria before recruitment of GFP-LC3 ().
Mitophagy stimulated by 488-nm light also occurred in the absence of TMRM, indicating that TMRM was not acting as a photosensitizer (). Photoirradiation with 543-nm did not induce mitophagy (). Unexpectedly, photoirradiation with 543-nm light did lead to loss of mitochondrial TMRM fluorescence (). This may reflect direct photobleaching of TMRM rather than photodamage and depolarization of mitochondria. However, if mitochondria remained polarized, reaccumulation of TMRM from the cytosol and extracellular medium into mitochondria would be expected to occur over time, and such reuptake did not occur. Alternatively, mitophagy after photoirradiation may depend on the nature of the mitochondrial injury, and damage to flavoproteins by 488-nm irradiation may be the more potent mitophagy inducer. Additional studies will be needed to characterize what specific mitochondrial injuries induce mitophagy the most.
A particularly noteworthy difference between nutrient deprivation-induced mitophagy and photodamage-induced mitophagy is that the latter was not blocked by PI3K inhibition with 3MA (10
) or wortmannin (100
) () (18
). Class III PI3K/p150 interacts with Beclin 1, a mammalian homologue of Atg6 that is required for an early stage of autophagosome formation during nutrient deprivation (47
). Rather, in our study, PI3K inhibition appeared to augment GFP-LC3 association with photodamaged mitochondria (). These findings indicate that activation of mitophagy after photodamage occurs independently of PI3K signaling, which is in striking contrast to autophagy after other stimuli, such as nutritent deprivation, that is blocked by PI3K inhibitors (see ). Indeed, photodamage-induced GFP-LC3 ring formation was more robust after PI3K inhibition, which suggests that subsequent processing of mitophagosomes may require PI3K, as shown for the processing of mitophagosomes in nutrient deprivation-induced autophagy (19
Mitochondria of nonproliferating tissues such as heart, brain, liver, and kidney have a half-life of 10 to 25 days (29
). In this normal turnover, old and presumably dysfunctional mitochondria are removed by mitophagy and replaced by biogenesis of new mitochondria. Such mitophagy serves the physiological function of segregating and degrading dysfunctional mitochondria that might otherwise release ROS, pro-apoptotic proteins, and other toxic mediators. Since mitochondria are a primary site of ROS generation, mitochondrial DNA (mtDNA) is prone to oxidative damage. Due to limited mtDNA repair mechanisms, damaged mtDNA likely accumulates with time. Since mtDNA is nearly 100% active in transcription (compared to 2% or 3% for nuclear DNA), damage to mtDNA will lead quickly to mitochondrial dysfunction. Decreased mitophagy may promote accumulation of mtDNA mutations in aging rodents (6
), whereas caloric restriction and rapamycin, inducers of autophagy, increase longevity in rodents (7
). Moreover, mitochondria with mutant mtDNA are selectively removed in heteroplasmic cells (44
In conclusion, photoirradiation by 488-nm light caused mitochondrial depolarization, inner membrane permeabilization, and subsequent selective mitophagy, consistent with previous reports of photodynamic induction of the MPT and involvement of the MPT in mitophagy (10
). However, upstream signaling for photodamage-induced mitophagy bypassed the classical PI3K signaling pathway of nutrient deprivation-induced autophagy, although PI3K still appeared to be needed for downstream processing of newly formed mitophagosomes. Thus, mitophagy is an important mechanism to sequester and degrade damaged mitochondria in otherwise viable and healthy cells.