Mitochondrial Retrograde Transport Promoted-mitophagy And Its Protective Role In Ischemic Neuronal Injury

Cerebral ischemia results from the sudden shortage of blood supply to the brain,which has become one of the leading causes of mortality worldwide.Although in the face of increasing clinical demand,effective therapies for cerebral ischemia are still lacking.Autophagy is an intracellular degradation process by delivering substrates to lysosomes through double-membrane vesicles termed autophagosomes.Autophagic elimination of damaged or superfluous mitochondria is defined as mitophagy.We have previously proved that ischemia-reperfusion activates neuronal mitophagy which further protects against cerebral ischemic injury by inhibiting damaged mitochondria-induced apoptosis.These results highlight mitophagy as a potential target for stroke therapy.However,the cellular process of neuronal mitophagy is still unclear.Highly polarized morphology distinguishes neurons from other cells.Their long and thin axons are not only unique in morphology but pivotal to neuronal functions.Mitochondria are distributed evenly within neurons including distal axons where they support the functions of synapses by suppling energy and buffering Ca2+.However,lysosomes are predominantly located in neuronal soma,raising a question as to whether and how axonal mitochondria are cleared.Investigations into the process of axonal mitochondrial clearance will provide more insight into the mechanisms underlying neuronal mitophagy and offer novel strategies to resuce cerebral ischemia.In the present study,we aimed to clarify the process of axonal mitochondrial clearance in ischemic neurons.Part 1Somatic autophagy of axonal mitochondria in ischemic neuronsPrimary cultured cortical neurons from embryonic mice brains were subjected to oxygen-glucose deprivation plus reperfusion(OGD-Rep).OGD-Rep reduced the mitochondrial proteins COX ? and Tim23,which was reversed by the lysosome inhibitor chloroquine.These results suggested an autophagy-dependent mitochondrial elimination.Mitochondrial matrix protein Hsp60 was immunostained to visualize mitochondrial loss in greater details.Compared with control,OGD-Rep induced significant and equal mitochondrial loss in neuronal somas and axons.Intriguingly,chloroquine reversed mitochondrial loss only in soma but not in axons after OGD-Rep,indicating mitophagy may only occur in neuronal soma after ischemic stress.To further investigate the neuronal compartments where mitophagy occurred after OGD-Rep,the co-localization of mitochondria and autophagosomes was monitored in neurons by transfecting mitoGFP and mCherry-LC3,respectively.After OGD-Rep,autophagosomes increased in both neuronal soma and axons,while the number of mitochondria engulfed by autophagosomes increased significantly only in soma but not in axons.To clarify axonal mitophagy,Atg7 knockout neurons from Atg7fl/fl crossed with nestin-Cre mice were used(Atg7fl/fl;nes-Cre).Atg7 knockout abolished mitochondrial loss in neuronal soma but not in axons after OGD-Rep,which supported the absence of direct axonal mitophagy.To track the axonal mitochondria that were lost during OGD-Rep,the axonal and somatic mitochondria were separately labeled by taking advantage of microfluidic chambers.Axon-derived mitochondria increased in neuronal soma after OGD-Rep and engulfed by autophagosomes,indicating somatic autophagy of axonal mitochondria after OGD-Rep.More intriguingly,axonal mitochondria,compared to somatic and dendritic mitochondria,made up the majority of mitochondria that underwent mitophagy after OGD-Rep.In the present investigation,we found that axonal mitochondria were eliminated by somatic rather than local mitophagy during OGD-Rep.Since neuronal mitochondrial distribution is regulated by mitochondrial transport,we would further investigate the roles of mitochondrial transport in neuronal mitophagy during ischemia-reperfusion.Part 2Mitochondrial retrograde transport protects ischemic neurons through mitophagyMitochondria undergo dynamic movements along with microtubules.Mitochondrial anterograde transport is responsible for transporting mitochondria to terminus whilst axonal mitochondria return to soma through retrograde transport.Time-lapse confocal microscopy was employed to monitor axonal mitochondrial movements in ischemic neurons.OGD arrested mitochondrial mobility in axons and mitochondrial anterograde transport was more vulnerable to the OGD insult.However,reperfusion induced a prompt recovery of retrograde,but not anterograde,movements of axonal mitochondria.To identify the association of mitochondrial retrograde trafficking with somatic mitophagy,the axonal mitochondrial mobility was arrested by overexpression of axonal mitochondrial anchor protein syntaphilin(SNPH).SNPH significantly abolished axonal mitochondrial loss after OGD-Rep.Remarkably,the overall mitophagy induced by OGD-Rep was counteracted by SNPH as reflected by accumulation of mitochondrial proteins,revealing that axonal mitochondrial movement was required for ischemic neuronal mitophagy.Furthermore,to manipulate the direction of mitochondrial movements,a protein dimerization tool was employed to facilitate the interaction of mitochondria with motor proteins for anterograde and retrograde transport.Reinforced interaction of mitochondria with motor protein for anterograde transport compromised OGD-Rep-induced mitophagy.Conversely,enhanced retrograde movement of axonal mitochondria was sufficient to reinforce the OGD-Rep-induced mitophagy.Importantly,reinforced mitochondrial retrograde transport prevented mitochondrial dysfunction and protected ischemic neurons whilst the inhibition of mitochondrial retrograde transport aggravated ischemic injury.Atg7 deletion abolished the neuroprotection of mitochondrial retrograde transport,indicating mitochondrial retrograde transport protects against ischemic injury through mitophagy.Futhermore,OGD-Rep induced mitochondrial retrograde transport independent of mitochondrial fragmentation and membrane potential loss.Mitochondria with more ROS in matrix tend to move retrogradely after OGD-Rep,indicating the involvement of mitochondrial oxidative stress in mitochondrial retrograde transport.In the present investigation,we establish a direct link of mitochondrial retrograde movement with mitophagy which further conferred neuroprotective effects against ischemia.In addition,mitochondrial oxidative stress seems to trigger mitochondrial retrograde transport after ischemic insult.Our findings provide a new concept that to reduce ischemic neuronal inj ury by correcting mitochondrial motility.