BNIP3L/NIX degradation leads to mitophagy deficiency in ischemic brains

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 <i>bnip3l</i>^-/- 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. <b>Abbreviations:</b> 3-MA: 3-methyladenine; AAV: adeno-associated virus; <i>ATG7</i>: 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.

线粒体自噬（mitophagy）是通过自噬途径清除受损线粒体的过程，在脑缺血中可促进神经元存活。既往研究发现缺血神经元中线粒体自噬功能缺陷，但具体机制尚未完全阐明。本研究证实，线粒体自噬受体BCL2/腺病毒E1B相互作用蛋白3样（BNIP3L/NIX）可被蛋白酶体降解，进而导致缺血神经元及脑组织中的线粒体自噬功能缺陷。BNIP3L/NIX在哺乳动物细胞中以单体和同源二聚体两种形式存在，但二者对线粒体自噬的调控作用尚不明确。对BNIP3L/NIX跨膜结构域进行位点特异性突变（S195A和G203A）后，其仅能以单体形式存在，且无法诱导线粒体自噬。此外，与单体形式的BNIP3L/NIX不同，过表达野生型BNIP3L/NIX可逆转线粒体自噬功能缺陷，并对脑缺血损伤起到保护作用。本研究通过在缺血脑组织中使用巨自噬/自噬抑制剂3-甲基腺嘌呤（3-MA）和蛋白酶体抑制剂MG132，探究BNIP3L/NIX的降解机制。结果显示，MG132可抑制BNIP3L/NIX的降解，并进而促进缺血脑组织中的线粒体自噬。此外，BNIP3L/NIX的二聚体形式较单体形式更易被降解。卡非佐米（Carfilzomib，CFZ）是一种用于治疗多发性骨髓瘤的蛋白酶体抑制剂，可逆转缺血脑组织中BNIP3L/NIX的降解并恢复线粒体自噬功能。该治疗手段可对急性或慢性缺血性脑损伤起到保护作用。值得注意的是，在<i>bnip3l</i>^-/-小鼠中，卡非佐米的上述保护作用完全消失。综上，本研究证实了蛋白酶体介导的BNIP3L/NIX降解与脑缺血中线粒体自噬功能缺陷之间的关联。本研究提出，卡非佐米可通过抑制BNIP3L/NIX的降解，成为治疗缺血性脑损伤的新型手段。<b>缩略词表：</b>3-MA：3-甲基腺嘌呤（3-methyladenine）；AAV：腺相关病毒（adeno-associated virus）；ATG7：自噬相关7（autophagy related 7）；BCL2L13：BCL2样蛋白13（凋亡促进因子，BCL2-like 13 (apoptosis facilitator)）；BNIP3L/NIX：BCL2/腺病毒E1B相互作用蛋白3样（BCL2/adenovirus E1B interacting protein 3-like）；CCCP：羰基氰化物间氯苯腙（carbonyl cyanide 3-chlorophenylhydrazone）；CFZ：卡非佐米（carfilzomib）；COX4I1：细胞色素c氧化酶亚基4I1（cytochrome c oxidase subunit 4I1）；CQ：氯喹（chloroquine）；GAPDH：甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；GFP：绿色荧光蛋白（green fluorescent protein）；I-R：缺血再灌注（ischemia-reperfusion）；MAP1LC3A/LC3A：微管相关蛋白1轻链3α（microtube-associated protein 1 light chain 3 alpha）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtube-associated protein 1 light chain 3 beta）；O-R：氧糖剥夺再灌注（oxygen and glucose deprivation-reperfusion）；OGD：氧糖剥夺（oxygen and glucose deprivation）；PHB2：抑制素2（prohibitin 2）；pMCAO：永久性大脑中动脉阻塞（permanent middle cerebral artery occlusion）；PRKN/PARK2：帕金RBR E3泛素蛋白连接酶（parkin RBR E3 ubiquitin protein ligase）；PT：光血栓模型（photothrombosis）；SQSTM1：隔离体1（sequestosome 1）；tMCAO：暂时性大脑中动脉阻塞（transient middle cerebral artery occlusion）；TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）；TTC：氯化三苯基四氮唑（2,3,5-triphenyltetrazolium hydrochloride）