https://orcid.org/0000-0003-4755-9357
ABSTRACT
Mitophagy is the sole mechanism for neurons to eliminate superfluous or damaged mitochondria. Although the critical implications of mitophagy have been emphasized in a variety of neurological disorders, it remains ambiguous how neurons control the quality of axonal mitochondria. By employing an oxygen-glucose-deprivation and reperfusion (OGD-Rep) model in cultured neurons, our recent results clearly documented the prompt recovery of retrograde transport of axonal mitochondria to neuronal soma. Moreover, by selectively labeling axonal mitochondria, we found that these axonal mitochondria appear in neuronal soma and are eliminated via autophagosomes in priority. This mitochondrial movement from axon to soma has a critical contribution to overall neuronal mitophagy under ischemia. Because forced expression of an anchoring protein, SNPH (Syntaphilin), significantly blocks mitophagy, and aggravates mitochondrial dysfunction and neuronal injury. Conversely, promoted retrograde mitochondrial movement facilitates neuronal mitophagy and attenuates ischemic neuronal demise. In conclusion, we propose stimulating the somatic autophagy of axonal mitochondria after ischemic insults. These findings may provide further insight into how neurons control the mitochondrial quality in pathological conditions and offer novel strategies to cure neurological disorders.
The critical roles of mitophagy in neuronal homeostasis have been established during the last decade. Most of the current knowledge on mitophagy regulation, however, has been gained by studying immortal cells. Distinct from these cells, neurons are highly polarized and characterized by long axons, in which almost half of the total neuronal mitochondrial mass is located. These discrepancies raise an open question as to whether and how these distal mitochondria in axons are eliminated. An acceptable proposal suggests the involvement of mitochondrial mobility in axonal mitochondrial elimination. This paradigm is primarily based on the observations that mitochondria escape from axonal anchoring and move in a retrograde direction (back to the soma) upon stress. Nevertheless, the ultimate destiny of these retrograde-moving mitochondria remains unclear. Alternatively, some other lines of evidence support the immobility of axonal mitochondria in mature neurons and emphasize that it is the arrest of axonal mitochondria that facilitates their subsequent elimination through sequestration within local autophagosomes. Nevertheless, the association between axonal mitochondria mobility and mitophagy remains ambiguous.
To gain further insight into how axonal mitochondria are removed, we subjected primary cultured mouse cortical neurons to oxygen-glucose deprivation and reperfusion (OGD-Rep), which is a common model mimicking cerebral ischemia, and has been proven to trigger mitochondria damage and mitophagy. We documented mitochondria loss both in neuronal soma and axons after OGD-Rep. Unexpectedly, direct mitophagy was not observed in axons, and axonal mitochondrial loss cannot be reversed by Atg7 or Prkn/Park2 gene deletion [Citation1]. These results urged us to track axonal mitochondria in our model. By labelling axonal mitochondria selectively, we clearly showed the appearance of axonal mitochondria in neuronal soma after OGD-Rep. It is likely that axonal mitochondria move to neuronal soma after ischemic stress. To clarify this, we visualized and recorded the axonal mitochondria movement during the OGD-Rep procedures. Axonal mitochondria are arrested immediately after OGD, possibly due to the low intracellular energy status. Interestingly, we recorded the prompt recovery of retrograde movement of axonal mitochondria after reperfusion onset. It is noteworthy that the prompt recovery occurred selectively on retrograde-moving mitochondria while the anterograde-moving mitochondria quantity did not alter significantly. In addition, the recovery of retrograde mitochondrial movement declines along with prolonged OGD duration. Surprisingly enough, the transported mitochondria seem to have priority in being engulfed by phagophores in neuronal soma. This phenomenon implies a novel mechanism by which neurons might recognize, sort and deliver damaged axonal mitochondria to the soma, where lysosomes are more enriched, for degradation.
We next asked about the causative role of retrograde transport in axonal mitochondrial elimination. For clarification, the axonal mitochondria movement was arrested by transfecting Syntaphilin, an anchoring protein, to cultured neurons. It showed that axonal mitochondrial immobility significantly compromised OGD-Rep-induced neuronal mitophagy. To regulate axonal mitochondrial movement more accurately, we employed a rapalog-induced protein interaction assay. This approach allowed us to manipulate axonal mitochondrial traffic by conjugating mitochondria to motor proteins, namely KIF5B and BICD2, in culture neurons. The results indicated that forced retrograde mitochondrial transport (by conjugating BICD2) promoted neuronal mitophagy while force anterograde transport (by conjugating KIF5B) did the reverse. These data provided the first evidence indicating the requirement of retrograde transport for the autophagy of axonal mitochondria, at least in ischemic scenario. In line with the previous findings, we next proved that axonal mitochondria arrest aggravated, while promoted retrograde transport attenuated, the impairment of mitochondrial respiration and neuronal demise caused by ischemia. Interestingly, we finally observed the recovery of anterograde mobility of axonal mitochondria in a late phase (12 h) after reperfusion. It is likely that neurons may deliver healthy mitochondria to these distal compartments for functional recovery, if they can overcome ischemia.
Taken together, we identified the somatic autophagy of axonal mitochondria in ischemic neurons. Axonal mitochondrial have longer half-life and higher vulnerability to stress compare with the neuronal mitochondria in other compartments. Hence, the ‘Come-and-Eat’ pattern may accelerate axonal mitochondrial turnover before they further contaminate the overall mitochondrial pool. In the perspective of therapy, our finding provided the rationale to cure neurological disorders by correcting axonal mitochondrial mobility.
Disclosure statement
No potential conflict of interest was reported by the authors.