Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2^R1441G mice

Mitochondrial dysfunction causes energy deficiency and nigrostriatal neurodegeneration which is integral to the pathogenesis of Parkinson disease (PD). Clearance of defective mitochondria involves fission and ubiquitin-dependent degradation via mitophagy to maintain energy homeostasis. We hypothesize that LRRK2 (leucine-rich repeat kinase 2) mutation disrupts mitochondrial turnover causing accumulation of defective mitochondria in aging brain. We found more ubiquitinated mitochondria with aberrant morphology associated with impaired function in aged (but not young) LRRK2^R1441G knockin mutant mouse striatum compared to wild-type (WT) controls. LRRK2^R1441G mutant mouse embryonic fibroblasts (MEFs) exhibited reduced MAP1LC3/LC3 activation indicating impaired macroautophagy/autophagy. Mutant MEFs under FCCP-induced (mitochondrial uncoupler) stress showed increased LC3-aggregates demonstrating impaired mitophagy. Using a novel flow cytometry assay to quantify mitophagic rates in MEFs expressing photoactivatable <i>mito</i>-PAmCherry, we found significantly slower mitochondria clearance in mutant cells. Specific LRRK2 kinase inhibition using GNE-7915 did not alleviate impaired mitochondrial clearance suggesting a lack of direct relationship to increased kinase activity alone. DNM1L/Drp1 knockdown in MEFs slowed mitochondrial clearance indicating that DNM1L is a prerequisite for mitophagy. DNM1L knockdown in slowing mitochondrial clearance was less pronounced in mutant MEFs, indicating preexisting impaired DNM1L activation. DNM1L knockdown disrupted mitochondrial network which was more evident in mutant MEFs. DNM1L-Ser616 and MAPK/ERK phosphorylation which mediate mitochondrial fission and downstream mitophagic processes was apparent in WT using FCCP-induced stress but not mutant MEFs, despite similar total MAPK/ERK and DNM1L levels. In conclusion, aberrant mitochondria morphology and dysfunction associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in mutant LRRK2 MEFs and mouse brain. <b>Abbreviations:</b> ATP: adenosine triphosphate; BAX: BCL2-associated X protein; CDK1: cyclin-dependent kinase 1; CDK5: cyclin-dependent kinase 5; CQ: chloroquine; CSF: cerebrospinal fluid; DNM1L/DRP1: dynamin 1-like; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LAMP2A: lysosomal-associated membrane protein 2A; LRRK2: leucine-rich repeat kinase 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MMP: mitochondrial membrane potential; PAmCherry: photoactivatable-mCherry; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB10: RAB10, member RAS oncogene family; RAF: v-raf-leukemia oncogene; SNCA: synuclein, alpha; TEM: transmission electron microscopy; VDAC: voltage-dependent anion channel; WT: wild type; SQSTM1/p62: sequestosome 1.线粒体功能障碍可引发能量缺乏与黑质纹状体神经退行性变，这与帕金森病（Parkinson disease, PD）的发病机制密不可分。缺陷线粒体的清除涉及分裂过程及经由线粒体自噬（mitophagy）的泛素依赖性降解，以此维持能量稳态。我们提出假说：富亮氨酸重复激酶2（leucine-rich repeat kinase 2, LRRK2）突变会破坏线粒体周转，导致衰老大脑中缺陷线粒体异常积累。相较于野生型（wild-type, WT）对照小鼠，衰老（而非年轻）的LRRK2^R1441G敲入突变小鼠纹状体中，可观察到更多形态异常的泛素化线粒体，且其功能受损。LRRK2^R1441G突变小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）表现出MAP1LC3/LC3（微管相关蛋白1轻链3，microtubule-associated protein 1 light chain 3）激活水平降低，提示巨自噬/自噬功能受损。在羰基氰化物-4-(三氟甲氧基)苯腙（carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP，线粒体解偶联剂）诱导的应激条件下，突变型MEFs的LC3聚集量增加，表明线粒体自噬功能受损。我们采用一种新型流式细胞术分析法，对表达光激活线粒体靶向-PAmCherry（photoactivatable mito-PAmCherry）的MEFs的线粒体自噬速率进行定量，结果发现突变细胞的线粒体清除速率显著减慢。使用GNE-7915进行特异性LRRK2激酶抑制，并未改善受损的线粒体清除能力，这表明该表型与单纯的激酶活性升高缺乏直接关联。敲低DNM1L/DRP1（动力蛋白1样，dynamin 1-like）可减慢MEFs的线粒体清除速率，提示DNM1L是线粒体自噬的必要前提条件。在突变型MEFs中，敲低DNM1L对线粒体清除速率的减缓作用更为微弱，表明DNM1L的激活已预先存在缺陷。敲低DNM1L会破坏线粒体网络，这一现象在突变型MEFs中更为显著。在FCCP诱导的应激下，野生型细胞中可检测到介导线粒体分裂及下游线粒体自噬过程的DNM1L-Ser616磷酸化与MAPK/ERK（丝裂原活化蛋白激酶/细胞外调节蛋白激酶，mitogen-activated protein kinase/extracellular regulated protein kinases）磷酸化，但突变型MEFs中未出现该现象，尽管两者的总MAPK/ERK和DNM1L水平并无差异。综上，在突变型LRRK2的MEFs和小鼠大脑中，均存在异常的线粒体形态与功能障碍，同时伴随线粒体自噬受损及DNM1L-MAPK/ERK信号通路异常。**缩写：** ATP: 三磷酸腺苷（adenosine triphosphate）；BAX: BCL2相关X蛋白（BCL2-associated X protein）；CDK1: 细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1）；CDK5: 细胞周期蛋白依赖性激酶5（cyclin-dependent kinase 5）；CQ: 氯喹（chloroquine）；CSF: 脑脊液（cerebrospinal fluid）；DNM1L/DRP1: 动力蛋白1样（dynamin 1-like）；ELISA: 酶联免疫吸附测定（enzyme-linked immunosorbent assay）；FACS: 荧光激活细胞分选（fluorescence-activated cell sorting）；FCCP: 羰基氰化物-4-(三氟甲氧基)苯腙（carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone）；GAPDH: 甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；LAMP2A: 溶酶体相关膜蛋白2A（lysosomal-associated membrane protein 2A）；LRRK2: 富亮氨酸重复激酶2（leucine-rich repeat kinase 2）；MAP1LC3/LC3: 微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3）；MAPK1/ERK2: 丝裂原活化蛋白激酶1（mitogen-activated protein kinase 1）；MEF: 小鼠胚胎成纤维细胞（mouse embryonic fibroblast）；MFN1: 线粒体融合蛋白1（mitofusin 1）；MMP: 线粒体膜电位（mitochondrial membrane potential）；PAmCherry: 光激活mCherry（photoactivatable-mCherry）；PD: 帕金森病（Parkinson disease）；PINK1: PTEN诱导的推定激酶1（PTEN induced putative kinase 1）；PRKN/PARKIN: 帕金RBR E3泛素蛋白连接酶（parkin RBR E3 ubiquitin protein ligase）；RAB10: RAS癌基因家族成员RAB10（RAB10, member RAS oncogene family）；RAF: v-raf白血病致癌基因（v-raf-leukemia oncogene）；SNCA: α-突触核蛋白（synuclein, alpha）；TEM: 透射电子显微镜（transmission electron microscopy）；VDAC: 电压依赖性阴离子通道（voltage-dependent anion channel）；WT: 野生型（wild type）；SQSTM1/p62: 隔离体1（sequestosome 1）。