Mitochondrial protein import regulates cytosolic protein homeostasis and neuronal integrity

Neurodegeneration is characterized by protein aggregate deposits and mitochondrial malfunction. Reduction in Tom40 (translocase of outer membrane 40) expression, a key subunit of the translocase of the outer mitochondrial membrane complex, led to accumulation of ubiquitin (Ub)-positive protein aggregates engulfed by Atg8a-positive membranes. Other macroautophagy markers were also abnormally accumulated. Autophagy was induced but the majority of autophagosomes failed to fuse with lysosomes when <i>Tom40</i> was downregulated. In <i>Tom40</i> RNAi tissues, autophagosome-like (AL) structures, often not sealed, were 10 times larger than starvation induced autophagosomes. <i>Atg5</i> downregulation abolished <i>Tom40</i> RNAi induced AL structure formation, but the Ub-positive aggregates remained, whereas knock down of <i>Syx17</i>, a gene required for autophagosome-lysosome fusion, led to the disappearance of giant AL structures and accumulation of small autophagosomes and phagophores near the Ub-positive aggregates. The protein aggregates contained many mitochondrial preproteins, cytosolic proteins, and proteasome subunits. Proteasome activity and ATP levels were reduced and the ROS levels was increased in <i>Tom40</i> RNAi tissues. The simultaneous inhibition of proteasome activity, reduction in ATP production, and increase in ROS, but none of these conditions alone, can mimic the imbalanced proteostasis phenotypes observed in <i>Tom40</i> RNAi cells. Knockdown of <i>ref(2)P</i> or ectopic expression of Pink1 and park greatly reduced aggregate formation in <i>Tom40</i> RNAi tissues. In nerve tissues, reduction in Tom40 activity leads to aggregate formation and neurodegeneration. Rather than diminishing the neurodegenerative phenotypes, overexpression of Pink1 enhanced them. We proposed that defects in mitochondrial protein import may be the key to linking imbalanced proteostasis and mitochondrial defects. <b>Abbreviations:</b> AL: autophagosome-like; Atg12: Autophagy-related 12; Atg14: Autophagy-related 14; Atg16: Autophagy-related 16; Atg5: Autophagy-related 5; Atg6: Autophagy-related 6; Atg8a: Autophagy-related 8a; Atg9: Autophagy-related 9; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; cDNA: complementary DNA; COX4: Cytochrome c oxidase subunit 4; CRISPR: clustered regularly interspaced short palindromic repeats; Cyt-c1: Cytochrome c1; DAPI: 4,6-diamidino-2-phenylindole dihydrochloride; Dcr-2: Dicer-2; FLP: Flippase recombination enzyme; FRT: FLP recombination target; GFP: green fluorescent protein; GO: gene ontology; gRNA: guide RNA; Hsp60: Heat shock protein 60A; HDAC6: Histone deacetylase 6; htt: huntingtin; Idh: Isocitrate dehydrogenase; IFA: immunofluorescence assay; Irp-1A: Iron regulatory protein 1A; kdn: knockdown; Marf: Mitochondrial assembly regulatory factor; MitoGFP: Mitochondrial-GFP; MS: mass spectrometry; MTPAP: mitochondrial poly(A) polymerase; Nmnat: Nicotinamide mononucleotide adenylyltransferase; OE: overexpression; Pink1/PINK1: PTEN-induced putative kinase 1; polyQ: polyglutamine; PRKN: parkin RBR E3 ubiquitin protein ligase; Prosα4: proteasome α4 subunit; Prosβ1: proteasome β1 subunit; Prosβ5: proteasome β5 subunit; Prosβ7: proteasome β7 subunit; ref(2)P: refractory to sigma P; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Rpn11: Regulatory particle non-ATPase 11; Rpt2: Regulatory particle triple-A ATPase 2; scu: scully; sicily: severe impairment of CI with lengthened youth; sesB: stress-sensitive B; Syx17: Syntaxin17; TEM: transmission electron microscopy; ttm50: tiny tim 50; Tom: translocase of the outer membrane; Tom20: translocase of outer membrane 20; Tom40: translocase of outer membrane 40; Tom70: translocase of outer membrane 70; UAS: upstream active sequence; Ub: ubiquitin; VNC: ventral nerve cord; ZFYVE1: zinc finger FYVE-type containing 1

神经退行性疾病以蛋白质聚集沉积与线粒体功能异常为典型特征。线粒体外膜转位酶复合物的关键亚基Tom40（translocase of outer membrane 40）表达量下调，会导致泛素（Ubiquitin, Ub）阳性的蛋白聚集物被Atg8a阳性膜包裹并发生蓄积。其他巨自噬标志物亦出现异常蓄积现象。当Tom40表达被抑制时，自噬虽被诱导激活，但绝大多数自噬体无法与溶酶体完成融合。
在Tom40 RNA干扰（RNA interference, RNAi）的组织样本中，自噬体样（autophagosome-like, AL）结构（通常未完全密封）的尺寸是饥饿诱导型自噬体的10倍。敲低Atg5（Autophagy-related 5）可阻断Tom40 RNAi诱导的自噬体样结构形成，但泛素阳性聚集物仍持续存在；而敲低自噬体-溶酶体融合必需基因Syx17（Syntaxin17），则会导致巨型自噬体样结构消失，且在泛素阳性聚集物附近出现小型自噬体与前自噬泡的蓄积。
该蛋白聚集物包含大量线粒体前体蛋白、胞质蛋白以及蛋白酶体亚基。在Tom40 RNAi组织中，蛋白酶体活性与ATP（adenosine triphosphate）水平显著降低，而活性氧（reactive oxygen species, ROS）水平则升高。同时抑制蛋白酶体活性、降低ATP生成并升高ROS，而非单独施加上述任一条件，可精准模拟Tom40 RNAi细胞中观察到的蛋白质稳态失衡表型。
敲低ref(2)P（refractory to sigma P）或异位过表达Pink1（PTEN-induced putative kinase 1）与parkin（parkin RBR E3 ubiquitin protein ligase, PRKN），可大幅减少Tom40 RNAi组织中的聚集物形成。在神经组织中，Tom40活性降低会引发聚集物形成与神经退行性变。与预期相悖的是，过表达Pink1非但未减轻神经退行性表型，反而加剧了该症状。本研究提出假说：线粒体蛋白导入缺陷可能是连接蛋白质稳态失衡与线粒体功能异常的关键分子环节。
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AL：自噬体样（autophagosome-like）；Atg12：自噬相关蛋白12（Autophagy-related 12）；Atg14：自噬相关蛋白14（Autophagy-related 14）；Atg16：自噬相关蛋白16（Autophagy-related 16）；Atg5：自噬相关蛋白5（Autophagy-related 5）；Atg6：自噬相关蛋白6（Autophagy-related 6）；Atg8a：自噬相关蛋白8a（Autophagy-related 8a）；Atg9：自噬相关蛋白9（Autophagy-related 9）；ATP：三磷酸腺苷（adenosine triphosphate）；Cas9：CRISPR相关蛋白9（CRISPR associated protein 9）；cDNA：互补DNA（complementary DNA）；COX4：细胞色素c氧化酶亚基4（Cytochrome c oxidase subunit 4）；CRISPR：成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats）；Cyt-c1：细胞色素c1（Cytochrome c1）；DAPI：4',6-二脒基-2-苯基吲哚二盐酸盐（4,6-diamidino-2-phenylindole dihydrochloride）；Dcr-2：Dicer-2；FLP：翻转酶重组酶（Flippase recombination enzyme）；FRT：FLP重组靶点（FLP recombination target）；GFP：绿色荧光蛋白（green fluorescent protein）；GO：基因本体论（gene ontology）；gRNA：向导RNA（guide RNA）；Hsp60：热休克蛋白60A（Heat shock protein 60A）；HDAC6：组蛋白脱乙酰酶6（Histone deacetylase 6）；htt：亨廷顿蛋白（huntingtin）；Idh：异柠檬酸脱氢酶（Isocitrate dehydrogenase）；IFA：免疫荧光测定（immunofluorescence assay）；Irp-1A：铁调节蛋白1A（Iron regulatory protein 1A）；kdn：敲低；Marf：线粒体组装调控因子（Mitochondrial assembly regulatory factor）；MitoGFP：线粒体靶向绿色荧光蛋白（Mitochondrial-GFP）；MS：质谱分析法（mass spectrometry）；MTPAP：线粒体多聚(A)聚合酶（mitochondrial poly(A) polymerase）；Nmnat：烟酰胺单核苷酸腺苷酰转移酶（Nicotinamide mononucleotide adenylyltransferase）；OE：过表达（overexpression）；Pink1/PINK1：PTEN诱导的假定激酶1（PTEN-induced putative kinase 1）；polyQ：多聚谷氨酰胺（polyglutamine）；PRKN：帕金RBR E3泛素连接酶（parkin RBR E3 ubiquitin protein ligase）；Prosα4：蛋白酶体α4亚基（proteasome α4 subunit）；Prosβ1：蛋白酶体β1亚基（proteasome β1 subunit）；Prosβ5：蛋白酶体β5亚基（proteasome β5 subunit）；Prosβ7：蛋白酶体β7亚基（proteasome β7 subunit）；ref(2)P：对σP具有抗性（refractory to sigma P）；RFP：红色荧光蛋白（red fluorescent protein）；RNAi：RNA干扰（RNA interference）；ROS：活性氧（reactive oxygen species）；Rpn11：调节颗粒非ATP酶11（Regulatory particle non-ATPase 11）；Rpt2：调节颗粒三磷酸腺苷酶2（Regulatory particle triple-A ATPase 2）；scu：scully；sicily：复合物I严重损伤伴青年期延长（severe impairment of CI with lengthened youth）；sesB：应激敏感蛋白B（stress-sensitive B）；Syx17：突触融合蛋白17（Syntaxin17）；TEM：透射电子显微镜（transmission electron microscopy）；ttm50：tiny tim 50；Tom：线粒体外膜转位酶（translocase of the outer membrane）；Tom20：线粒体外膜转位酶20（translocase of outer membrane 20）；Tom40：线粒体外膜转位酶40（translocase of outer membrane 40）；Tom70：线粒体外膜转位酶70（translocase of outer membrane 70）；UAS：上游激活序列（upstream active sequence）；Ub：泛素（Ubiquitin）；VNC：腹神经索（ventral nerve cord）；ZFYVE1：含锌指FYVE结构域蛋白1（zinc finger FYVE-type containing 1）