Mitophagy, the elimination of damaged mitochondria through autophagy, promotes neuronal survival in
cerebral ischemia. Previous studies found deficient mitophagy in ischemic neurons, but the mechanisms are still largely unknown. We determined that BNIP3L/NIX, a mitophagy receptor, was degraded by proteasomes, which led to mitophagy deficiency in both ischemic neurons and brains. BNIP3L exists as a monomer and homodimer in mammalian cells, but the effects of homodimer and monomer on mitophagy are unclear. Site-specific mutations in the transmembrane domain of BNIP3L (S195A and G203A) only formed the BNIP3L monomer and failed to induce mitophagy. Moreover, overexpression of wild-type BNIP3L, in contrast to the monomeric BNIP3L, rescued the mitophagy deficiency and protected against cerebral ischemic injury. The macroautophagy/autophagy inhibitor 3-MA and the
proteasome inhibitor MG132 were used in cerebral ischemic brains to identify how BNIP3L was reduced. We found that
MG132 blocked the loss of BNIP3L and subsequently promoted mitophagy in ischemic brains. In addition, the dimeric form of BNIP3L was more prone to be degraded than its monomeric form.
Carfilzomib, a drug for
multiple myeloma therapy that inhibits proteasomes, reversed the BNIP3L degradation and restored mitophagy in ischemic brains. This treatment protected against either acute or chronic ischemic
brain injury. Remarkably, these effects of
carfilzomib were abolished in bnip3l-/- mice. Taken together, the present study linked BNIP3L degradation by proteasomes with mitophagy deficiency in
cerebral ischemia. We propose
carfilzomib as a novel
therapy to rescue ischemic
brain injury by preventing BNIP3L degradation.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ATG7: autophagy related 7; BCL2L13: BCL2-like 13 (apoptosis facilitator); BNIP3L/NIX: BCL2/adenovirus E1B interacting
protein 3-like;
CCCP:
carbonyl cyanide 3-chlorophenylhydrazone; CFZ:
carfilzomib; COX4I1:
cytochrome c oxidase subunit 4I1; CQ:
chloroquine; GAPDH:
glyceraldehyde-3-phosphate dehydrogenase; GFP:
green fluorescent protein; I-R:
ischemia-reperfusion; MAP1LC3A/LC3A: microtube-associated
protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtube-associated
protein 1 light chain 3 beta; O-R:
oxygen and
glucose deprivation-reperfusion; OGD:
oxygen and
glucose deprivation; PHB2:
prohibitin 2; pMCAO: permanent
middle cerebral artery occlusion; PRKN/PARK2: parkin RBR E3
ubiquitin protein ligase; PT: photothrombosis; SQSTM1: sequestosome 1; tMCAO: transient
middle cerebral artery occlusion; TOMM20: translocase of outer mitochondrial membrane 20; TTC: 2,3,5-triphenyltetrazolium hydrochloride.