线粒体融合蛋白2在心血管疾病中的研究现状

线粒体是一个处于不断地融合与分裂过程中的动态细胞器。线粒体融合蛋白2(Mfn2)作为广泛分布于线粒体外膜和线粒体结合内质网膜上具有多重功能的蛋白,参与维持正常细胞功能。除了参与线粒体融合外,Mfn2还能够调节线粒体代谢、促进损伤线粒体的自噬、增强线粒体与内质网交流、维持内质网功能及通过调控线粒体外膜通透性和渗透性钙转运孔道的启闭参与细胞死亡过程等。另外,Mfn2基因还可通过调控Ras-Raf-ERK/MAPK和Ras-PI3K-Akt信号通路分别参与调控血管平滑肌细胞的增殖和凋亡过程。Mfn2的这一系列重要的生物学功能有助于其参与高血压、肺动脉高压、动脉粥样硬化、急性缺血/再灌损伤、扩张性心肌病、心肌肥大、心衰和肥胖糖尿病等多种心血管疾病的发生发展过程。研究Mfn2与心血管疾病的相关性也许能为临床提供一个心血管疾病潜在治疗的靶点。因此,本文将综述Mfn2在心血管疾病相关研究中的现状。     

线粒体与心房颤动

心肌是能量代谢非常活跃的组织,线粒体的含量非常丰富。心房颤动(房颤)患者心房细胞中除了线粒体形态发生改变外,线粒体相关的蛋白、线粒体DNA(mtDNA)、能量代谢等方面均发生了改变,这些改变导致线粒体功能不全同时也破坏了线粒体内部的稳态。房颤心房肌细胞线粒体的改变可能在房颤的发生与维持机制中起着重要的作用。     

血管内皮细胞线粒体动力学相关功能与心血管疾病关系的研究进展

细胞线粒体动力学相关功能是指线粒体通过不断地融合与分裂、线粒体自噬及线粒体-内质网结构偶联来维持细胞正常生理功能的过程。其异常与神经退行性病变、肿瘤、视神经萎缩及糖尿病等疾病的发生发展关系密切。近年来,血管内皮细胞（vascular endothelial cell,VEC）线粒体相关功能在心血管疾病中的研究受到广泛关注,研究发现VEC线粒体相关功能异常在心肌缺血/再灌注（I/R）损伤、冠状动脉粥样硬化、肺动脉高压及扩张型心肌病等疾病的发生发展中发挥重要作用。本文就VEC线粒体动力学相关功能及与心血管疾病的关系进行简要阐述。     

线粒体内质网结构偶联的蛋白组成和功能

线粒体内质网结构偶联(MAM)是线粒体与内质网二者之间形成的一个动态膜偶联结构,并且MAM可以参与这两个细胞器之间信息交流。研究证实MAM参与调控钙信号、脂质平衡、线粒体动态变化、线粒体自噬和内质网应激反应等。MAM与心血管疾病、神经退行性疾病和代谢性疾病等密切相关。本文综述了MAM的蛋白组成、功能以及与疾病的关系。     

AKAP1调控线粒体稳态及其在心血管疾病中的研究进展

线粒体是细胞的能量工厂,同时也是细胞内信号传导的枢纽,可调节细胞增殖、分化和存活。A型激酶锚定蛋白（A-kinase anchoring protein,AKAP）1是一种支架蛋白,因其可与蛋白激酶（protein kinase,PK） A结合而得名。近年研究发现AKAP1可将多种信号蛋白以及mRNA招募至线粒体外膜,调节线粒体功能及一系列相关生理病理过程。研究报道AKAP1可参与调控心肌肥厚、心肌细胞凋亡、血管舒张等活动,提示AKAP1与心血管疾病密切相关。本文将对AKAP1及其信号复合物调控线粒体功能的分子机制进行综述,并阐述AKAP1在心血管疾病中的研究进展。     

线粒体相关内质网膜:心血管疾病治疗的新靶点

线粒体相关内质网膜是指内质网和线粒体之间高度动态的紧密连接部分,参与维持内质网和线粒体的正常功能,与细胞脂质代谢、钙稳态、线粒体动力学、自噬和凋亡、内质网应激和炎症等密切相关。研究显示线粒体相关内质网膜功能异常或者数量和结构改变参与心血管疾病的发生发展。本文总结了线粒体相关内质网膜的功能,阐述了其在心血管疾病中的作用及可能机制,为线粒体相关内质网膜成为心血管疾病治疗的新靶点提供理论参考。     

内质网–线粒体交互在心血管疾病中的作用研究进展

线粒体和内质网的稳态在维持心血管正常功能中发挥重要作用,线粒体或内质网的结构功能异常参与了众多心血管疾病的发生发展。近年来研究发现线粒体与内质网存在物理和功能的交互,其交互作用调控线粒体、内质网功能,进而影响心肌细胞和平滑肌细胞的线粒体动力学平衡、钙转运及磷脂合成和转运。内质网–线粒体交互异常被认为是冠心病、心力衰竭、肺动脉高压和动脉粥样硬化等心血管疾病的关键机制。因此,理解内质网-线粒体交互机制可为预防和改善心血管疾病提供崭新靶点。     

线粒体突变和心血管疾病等的关系研究进展

线粒体是使细胞能量生成的场所,线粒体基因组(mitochondrial DNA,mtDNA)编码参与线粒体呼吸链的13个蛋白亚基,2个rRNA和22个tRNA。mtDNA突变是引起多因素疾病和部分遗传疾病的重要原因之一,本文介绍线粒体基因组学、mtDNA疾病模型,mtDNA突变导致心血管疾病等的临床特征及其治疗和预防的研究进展。     

冠状动脉内皮细胞线粒体损伤在心肌梗死中的研究进展

心血管疾病严重影响人们健康。心血管疾病中的心肌梗死严重危及生命。研究表明,冠状动脉内皮细胞的损伤会加重心肌梗死,而内皮细胞线粒体在内皮细胞损伤中扮演了重要角色,因此内皮细胞线粒体的研究也越来越受到人们关注。现就冠状动脉内皮细胞线粒体的结构及其损伤在氧化应激、炎症、动脉粥样硬化和心肌梗死后缺血再灌注损伤方面的研究进展和治疗进行综述,以期为心肌梗死的相关研究及治疗提供参考。     

虎杖苷改善氧化应激介导的肺泡上皮细胞线粒体损伤的机制

目的探讨虎杖苷对氧化应激介导的肺泡上皮细胞线粒体损伤的影响及其可能机制。方法A549细胞分为4组:对照组接受0.1%浓度的DMSO处理60 min;模型组给予同等浓度DMSO预处理30 min后以H 2O 2250μmol/L刺激30 min;治疗组接受虎杖苷50μmol/L预处理30 min后予以H 2O 2250μmol/L刺激30 min;抑制剂组接受线粒体自噬抑制剂mdivi-110μmol/L及虎杖苷50μmol/L预处理30 min后以H 2O 2250μmol/L刺激30 min。Western blotting法检测线粒体膜蛋白[线粒体外膜转位酶(TOM20)和线粒体内膜转位酶(TIM23)],及线粒体生成调节因子[线粒体转录因子(mTFA)和过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)],同时Keima法检测细胞红色/绿色荧光面积以反映线粒体自噬水平。JC-1探针检测线粒体膜电位(MMP)。DCFH-DA荧光探针检测细胞活性氧(ROS)水平。荧光素-荧光素酶法检测细胞ATP水平。细胞计数试剂盒(CCK-8)检测细胞活性。应用SPSS 20.0软件进行统计分析。结果(1)线粒体自噬水平。与对照组比较,模型组细胞TOM20及TIM23表达显著下降;与模型组比较,治疗组TOM20及TIM23表达显著下降;与治疗组比较,抑制剂组TOM20及TIM23显著增加,差异均有统计学意义(P<0.01)。但4组细胞mTFA及PGC-1α水平比较差异均无统计学意义(P>0.05)。Keima法显示,4组细胞红色/绿色荧光面积比值变化趋势与TOM20及TIM23水平变化相反。(2)MMP水平。对照组、模型组、治疗组与抑制剂组MMP分别为(100.0±5.9)%、(54.2±4.8)%、(70.8±3.6)%和(56.0±6.1)%。与对照组比较,模型组细胞MMP显著下降,治疗组MMP较模型组显著增加,而抑制剂组MMP较治疗组显著下降,差异有统计学意义(P<0.01)。(3)ROS水平、细胞活性及ATP水平。与对照组比较,模型组细胞ROS水平显著上升,细胞活性、ATP水平显著下降;与模型组比较,治疗组ROS水平显著下降,细胞活性、ATP水平显著增加;与治疗组比较,抑制剂组细胞ROS水平显著上升,细胞活性、ATP水平显著下降,差异有统计学意义(P<0.01)。结论虎杖苷可以显著改善氧化应激介导的肺泡上皮细胞线粒体损伤,其机制可能与上调线粒体自噬有关。     

In vivo protein-interaction mapping of a mitochondrial translocator protein Tom22 at work

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.     

Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient

The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.     

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.     

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.     

Prevention of the ischemia-induced decrease in mitochondrial Tom20 content by ischemic preconditioning

Preserved mitochondrial function (respiration, calcium handling) and integrity (cytochrome c release) is central for cell survival following ischemia/reperfusion. Mitochondrial function also requires import of proteins from the cytosol via the translocase of the outer and inner membrane (TOM and TIM complexes). Since mitochondrial function following ischemia/reperfusion is better preserved by ischemic preconditioning (IP), we now investigated whether expression of parts of the import machinery is affected by ischemia/reperfusion without or with IP in vivo. We analyzed the mitochondrial content of the presequence receptor Tom20, the pore forming unit Tom40 and Tim23. Goettinger minipigs were subjected to 90 min of low-flow ischemia without or with preconditioning by 10 min ischemia and 15 min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall of the left ventricle and from the control posterior wall. Infarct size was significantly reduced by IP (20.1 +/- 1.6% of area at risk (non-preconditioned) vs. 6.5 +/- 2.5% of area at risk (IP)). Using Western blot analysis, the ratio of Tom20 (normalized to Ponceau S) between mitochondria isolated from the anterior ischemic and posterior control wall was reduced (0.72 +/- 0.11, a.u., n = 8), whereas the mitochondrial Tom20 content was preserved by IP (1.17 +/- 0.16 a.u., n = 7, P < 0.05). The mitochondrial Tom40, Tim23 and adenine nucleotide transporter (ANT) contents were not significantly different between non-preconditioned and preconditioned myocardium. The preservation of the mitochondrial Tom20 protein level may contribute to the improved mitochondrial function after IP.     

Determinism and contingencies shaped the evolution of mitochondrial protein import

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei. The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Intracellular endosymbionts lack protein import systems, whereas such systems are a defining feature of mitochondria and plastids, both of which evolved from bacterial endosymbionts (1–3). Today, more than 95% of all mitochondrial proteins are imported from the cytosol, which makes mitochondrial protein import a key process required for mitochondrial biogenesis (4–6). The question of how mitochondrial protein import evolved is therefore central to understand how the endosymbiotic bacterial ancestor of mitochondria converted into an organelle that is genetically integrated into the host cell (7–9).Proteins are targeted to mitochondria by internal or external import signals, the most frequent one of which is the N-terminal presequence found in 60 to 70% of all imported proteins (10, 11). Interestingly, the various mitochondrial import signals are conserved even between highly diverged eukaryotes (6). The import signals are decoded by receptors, which are integral mitochondrial outer membrane (OM) proteins that are associated with the heterooligomeric protein translocase of the OM (TOM complex) (6, 12). Contrary to the core components of the TOM complex (Tom40, Tom22, and Tom7), which are highly conserved in essentially all eukaryotes, these receptors evolved independently in different eukaryotic lineages, even though they recognize the same conserved import signals (6).The best studied prototypical import receptors are Tom20 and Tom70 of yeast, orthologs of which are found in all members of the eukaryotic supergroup of the opisthokonts (13). Tom20 is an N-terminally anchored OM membrane protein, and its cytosolic domain contains a single tetratricopeptide repeat (TPR). Tom20 preferentially recognizes precursor proteins that have N-terminal presequences. It binds to the hydrophobic surface of the presequence and transfers the precursors to the highly conserved Tom22 that functions as a secondary receptor (14–17). Tom70 is the primary receptor for proteins that have multiple membrane spanning domains, such as mitochondrial carrier proteins, but also binds to hydrophobic precursor proteins that have presequences (18–20). Moreover, it has been shown that binding of Tom70 to the mitochondrial presequence-like stretches that are present in the mature part of many precursor proteins increases the import efficiency (21). Tom70 is N-terminally anchored in the membrane. Its large cytosolic domain consists of 11 TPR motifs. The three TPR motifs proximal to the membrane interact with cytosolic Hsp70 or Hsp90, from which Tom70 can receive precursor proteins (22, 23). The remaining eight TPR motifs directly recognize substrate proteins (24, 25). In yeast, Tom20 and Tom70 have partially redundant functions. Tom70 is not essential for growth and respiration. Loss of Tom20 causes a stronger phenotype; it abolishes respiration but is not lethal. Finally, even the deletion of Tom70 and Tom20 does not kill the cells, provided that the secondary receptor Tom22 is still present (15, 26–29).A single import receptor, termed Tom20, is associated with the TOM complex of plant mitochondria. Yeast and plant Tom20 (30) are superficially similar: both have a single transmembrane domain (TMD) and a soluble domain containing one (in yeast) and two TPR motifs (in plants). Furthermore, both proteins have the same domain organization provided that they are aligned in an antiparallel way. Thus, whereas yeast Tom20 is N-terminally anchored, plant Tom20 is a C-terminally anchored protein. This strongly suggests that yeast and plant Tom20, while both being import receptors, have different evolutionary origins (31, 32). Moreover, plants have another TPR domain-containing OM protein, termed OM64, that is not associated with the TOM complex, but implicated in protein import (31, 33).ATOM46 and ATOM69 are the two receptor subunits of the atypical translocase of the OM (ATOM) of trypanosomatids (34). ATOM69 is superficially similar to yeast Tom70. Both have the same molecular mass and multiple TPR-like motifs. ATOM69, in addition, has an N-terminal CS/Hsp20-like domain, which potentially can bind to cytosolic chaperones. Analogous to plant Tom20, ATOM69 is C-terminally membrane-anchored, whereas yeast Tom70 has an N-terminal TMD. ATOM46 also has an N-terminal membrane anchor and a cytosolic armadillo (ARM) repeat domain, a protein–protein interaction module specific for eukaryotes. The cytosolic domains of ATOM69 and ATOM46 were shown to bind a number of different precursor proteins and are essential for normal growth (34). ATOM69 and ATOM46 have been found in all kinetoplastids as well as in euglenoids (35). Except for the TPR domain in ATOM69, the two import receptors of trypanosomes do not resemble the TOM subunits of other species, indicating that they evolved independently from both the yeast and the plant receptors.Recently, an analysis of the TOM complex in Trichomonas vaginalis hydrogenosomes, which are mitochondria-derived hydrogen-producing organelles that lack their own genome (36), identified Tom36 and Tom46 (37). The two proteins are paralogues and consist of an N-terminal CS/Hsp20-like domain, three TPR-like sequences, and a C-terminal membrane anchor, which is reminiscent of trypanosomal ATOM69, although the mass of both hydrogenosomal proteins is much lower than that of ATOM69. Moreover, HHpred analysis, using Tom36 as a query, retrieved ATOM69 as the first hit (37). The cytosolic domains of Tom36 and Tom46 were able to bind hydrogenosomal precursor proteins, suggesting they may function as protein import receptors. However, despite the similarities between ATOM69 and Trichomonas Tom36/Tom46, phylogenetic analysis suggests that they evolved independently of each other, and therefore reflect yet another example of convergent evolution, although a diversification of a common ancestor cannot be ruled out (37).Here, we have investigated the substrate specificity of the trypanosomal import receptors ATOM46 and ATOM69 using inducible RNA interference (RNAi) cell lines and biochemical methods. We could correlate the observed receptor preference with specific features of the recognized substrate proteins, such as the presence of a predicted presequence, average hydrophobicity, and presence of TMDs. Moreover, we devised a method that allows for identification of which trypanosomal precursor proteins can be recognized by heterologous import receptors. Using this method, the mitochondrial proteomes are quantitatively compared between Trypanosoma brucei cell lines lacking either ATOM46 or ATOM69 and with T. brucei cell lines in which ATOM46 or ATOM69 were replaced by either Tom20 from yeast or Tom36 from Trichomonas.     

Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection

We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.     

The mitochondrial organelle and the heart

The heart is highly dependent for its function on oxidative energy generated in mitochondria, primarily by fatty acid beta-oxidation, respiratory electron chain and oxidative phosphorylation. Defects in mitochondrial structure and function have been found in association with cardiovascular diseases such as dilated and hypertrophy cardiomyopathy, cardiac conduction defects and sudden death, ischemic and alcoholic cardiomyopathy, as well as myocarditis. While a subset of these mitochondrial abnormalities have a defined genetic basis (e.g. mitochondrial DNA changes leading to oxidative phosphorylation dysfunction,fatty acid beta-oxidation defects due to specific nuclear DNA mutations), other abnormalities appear to be due to a more sporadic or environmental cardiotoxic insult or have not yet been characterized.This review focuses on abnormalities in mitochondrial bioenergetic function and mitochondrial DNA defects associated with cardiovascular diseases, their significance in cardiac pathogenesis as well as on the available diagnostic and therapeutic options. A concise background concerning mitochondrial biogenesis and bioenergetic pathways during cardiac growth,development and aging will also be provided.     

ALS-linked mutant superoxide dismutase 1 (SOD1) alters mitochondrial protein composition and decreases protein import

Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. Mutant SOD1 preferentially associates with the cytoplasmic face of mitochondria from spinal cords of rats and mice expressing SOD1 mutations. Two-dimensional gels and multidimensional liquid chromatography, in combination with tandem mass spectrometry, revealed 33 proteins that were increased and 21 proteins that were decreased in SOD1(G93A) rat spinal cord mitochondria compared with SOD1(WT) spinal cord mitochondria. Analysis of this group of proteins revealed a higher-than-expected proportion involved in complex I and protein import pathways. Direct import assays revealed a 30% decrease in protein import only in spinal cord mitochondria, despite an increase in the mitochondrial import components TOM20, TOM22, and TOM40. Recombinant SOD1(G93A) or SOD1(G85R), but not SOD1(WT) or a Parkinson's disease-causing, misfolded α-synuclein(E46K) mutant, decreased protein import by >50% in nontransgenic mitochondria from spinal cord, but not from liver. Thus, altered mitochondrial protein content accompanied by selective decreases in protein import into spinal cord mitochondria comprises part of the mitochondrial damage arising from mutant SOD1.