膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A (TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1 (Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶（TOM）复合物和线粒体内膜转位酶（TIM）复合物之间的桥接因子而介导PTEN诱导激酶（PINK1）输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。     

Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.     

Expression of mutant SOD1 in astrocytes induces functional deficits in motoneuron mitochondria

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration resulting in paralysis and eventual death. ALS is regarded as a motoneuron-specific disorder but increasing evidence indicates non-neuronal cells play a significant role in disease pathogenesis. Although the precise aetiology of ALS remains unclear, mutations in the superoxide dismutase (SOD1) gene are known to account for approximately 20% of familial ALS. We examined the influence of SOD1(G93A) expression in astrocytes on mitochondrial homeostasis in motoneurons in a primary astrocyte : motoneuron co-culture model. SOD1(G93A) expression in astrocytes induced changes in mitochondrial function of both SOD1(G93A) and wild-type motoneurons. In the presence of SOD1(G93A) astrocytes, mitochondrial redox state of both wild-type and SOD1(G93A) motoneurons was more reduced and mitochondrial membrane potential decreased. While intra-mitochondrial calcium levels [Ca(2+)](m) were elevated in SOD1(G93A) motoneurons, changes in mitochondrial function did not correlate with [Ca(2+)](m). Thus, expression of SOD1(G93A) in astrocytes directly alters mitochondrial function even in embryonic motoneurons, irrespective of genotype. These early deficits in mitochondrial function induced by surrounding astrocytes may increase the vulnerability of motoneurons to other neurotoxic mechanisms involved in ALS pathogenesis.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Defective mitochondrial mRNA maturation is associated with spastic ataxia

In human mitochondria, polyadenylation of mRNA, undertaken by the nuclear-encoded mitochondrial poly(A) RNA polymerase, is essential for maintaining mitochondrial gene expression. Our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the Old Order Amish, identified a mutation of MTPAP associated with the disease phenotype. When subjected to poly(A) tail-length assays, mitochondrial mRNAs from affected individuals were shown to have severely truncated poly(A) tails. Although defective mitochondrial DNA maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mRNA maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders.     

Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer's disease: implications to mitochondria-targeted antioxidant therapeutics

Synaptic pathology and mitochondrial oxidative damage are early events in Alzheimer's disease (AD) progression. Loss of synapses and synaptic damage are the best correlates of cognitive deficits found in AD patients. Recent research on amyloid beta (Aβ) and mitochondria in AD revealed that Aβ accumulates in synapses and synaptic mitochondria, leading to abnormal mitochondrial dynamics and synaptic degeneration in AD neurons. Further, recent studies using live-cell imaging and primary neurons from amyloid beta precursor protein (AβPP) transgenic mice revealed reduced mitochondrial mass, defective axonal transport of mitochondria and synaptic degeneration, indicating that Aβ is responsible for mitochondrial and synaptic deficiencies. Tremendous progress has been made in studying antioxidant approaches in mouse models of AD and clinical trials of AD patients. This article highlights the recent developments made in Aβ-induced abnormal mitochondrial dynamics, defective mitochondrial biogenesis, impaired axonal transport and synaptic deficiencies in AD. This article also focuses on mitochondrial approaches in treating AD, and also discusses latest research on mitochondria-targeted antioxidants in AD. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.     

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.     

Mitochondrial polyadenylic acid-containing RNA: localization and characterization

The RNA from the mitochondrial fraction of animal cells contains a polyadenylic acid sequence, approximately 55 nucleotides in length, which migrates at about 4 S in gel electrophoresis and which is attached to high molecular weight RNA. The experiments reported here indicate that: (a) the 4 S poly(A) sequence is found only in the mitochondrial fraction; (b) the RNA containing 4 S poly(A) is located within structures (presumably mitochondria) which protect it from pancreatic ribonuclease; (c) no RNA containing the longer poly(A) of nuclear origin appears to be located in mitochondria; (d) the 4 S poly(A), but not the longer poly(A), is attached to RNA which hybridizes to mitochondrial DNA; and (e) this poly(A) sequence is located at the 3′ end of the RNA molecule.The poly(A)-containing RNA can be isolated by affinity to oligodeoxyribothymidylic acid cellulose and resolved into approximately eight distinct species by acrylamide gel electrophoresis. These may correspond to individual mitochondrial messenger RNA molecules.     

Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae.

A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a [rho0] mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA. This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S. cerevisiae. A corresponding decrease in the level of mitochondrial DNA was not observed.     

Protective Effects of Hesperidin Against Amyloid-β (Aβ) Induced Neurotoxicity Through the Voltage Dependent Anion Channel 1 (VDAC1)-Mediated Mitochondrial Apoptotic Pathway in PC12 Cells

Amyloid-β (Aβ) is known to exert cytotoxic effects by inducing mitochondrial dysfunction. Additionally, the mitochondrial voltage-dependent anion channel 1 (VDAC1), which is involved in the release of apoptotic proteins with possible relevance in Alzheimer’s disease (AD) neuropathology, plays an important role in maintaining mitochondrial function and integrity. However, the application of therapeutic drugs, especially natural products in (AD) therapy via VDAC1-regulated mitochondrial apoptotic pathway has not aroused extensive attention. In the present study, we investigated neuroprotective effects of hesperidin, a bioactive flavonoid compound, on Aβ_25–35-induced neurotoxicity in PC12 cells and also examined the potential cellular signalling mechanism. Our results showed that treatment with hesperidin significantly inhibited Aβ_25–35-induced apoptosis by reversing Aβ-induced mitochondrial dysfunction, including the mitochondrial permeability transition pore opening, intracellular free calcium increase and reactive oxygen species production. Further study indicated that hesperidin can increase the level of VDAC1 phosphorylation through enhancing the activity of the glycogen synthasekinase-3β and decrease the level of hexokinaseI in mitochondrial, resulting in mitochondrial release of cytochrome c. Furthermore, hesperidin inhibited mitochondria-dependent downstream caspase-mediated apoptotic pathway, such as that involving caspase-9 and caspase-3. These results demonstrate that hesperidin can protect Aβ-induced neurotoxicity via VDAC1-regulated mitochondrial apoptotic pathway, and they raise the possibility that hesperidin could be developed into a clinically valuable treatment for AD and other neuronal degenerative diseases associated with mitochondrial dysfunction.     

Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons

Mitochondrial dysfunction is an underpinning event in many neurodegenerative disorders. Less clear, however, is how mitochondria become injured during neuronal demise. Nitric oxide (NO) evokes rapid mitochondrial fission in cortical neurons. Interestingly, proapoptotic Bax relocates from the cytoplasm into large foci on mitochondrial scission sites in response to nitrosative stress. Antiapoptotic Bcl-xL does not prevent mitochondrial fission despite its ability to block Bax puncta formation on mitochondria and to mitigate neuronal cell death. Mitofusin 1 (Mfn1) or dominant-negative dynamin-related protein 1(K38A) (Drp1(k38A)) inhibits mitochondrial fission and Bax accumulation on mitochondria induced by exposure to an NO donor. Although NO is known to cause a bioenergetic crisis, lowering ATP by glycolytic or mitochondrial inhibitors neither induces mitochondrial fission nor Bax foci formation on mitochondria. Taken together, these data indicate that the mitochondrial fission machinery acts upstream of the Bcl-2 family of proteins in neurons challenged with nitrosative stress.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

Isolation,crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.     

Mitochondrial permeability transition pore in Alzheimer's disease: Cyclophilin D and amyloid beta

Amyloid beta (Aβ) plays a critical role in the pathophysiology of Alzheimer's disease. Increasing evidence indicates mitochondria as an important target of Aβ toxicity; however, the effects of Aβ toxicity on mitochondria have not yet been fully elucidated. Recent biochemical studies in vivo and in vitro implicate mitochondrial permeability transition pore (mPTP) formation involvement in Aβ-mediated mitochondrial dysfunction. mPTP formation results in severe mitochondrial dysfunction such as reactive oxygen species (ROS) generation, mitochondrial membrane potential dissipation, intracellular calcium perturbation, decrease in mitochondrial respiration, release of pro-apoptotic factors and eventually cell death. Cyclophilin D (CypD) is one of the more well-known mPTP components and recent findings reveal that Aβ has significant impact on CypD-mediated mPTP formation. In this review, the role of Aβ in the formation of mPTP and the potential of mPTP inhibition as a therapeutic strategy in AD treatment are examined.