Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)–encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)–binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation

Regulation of mitochondrial permeability transition pore by PINK1

Background: Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson’s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains. Results: In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1−/− cells recapitulate the respiratory defect in isolated mitochondria from PINK1−/− mouse brains, suggesting that these PINK1−/− cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1−/− cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1−/− cells, and this genotypic difference between PINK1−/− and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1−/− cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1−/− and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1−/− compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1. Conclusions: Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1−/− cells

Disruption of fusion results in mitochondrial heterogeneity and dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzo yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated

Striatal neuroinflammation promotes parkinsonism in rats

The specific role of neuroinflammation in the pathogenesis of Parkinson's disease remains to be fully elucidated. By infusing lipopolysaccharide (LPS) into the striatum, we investigated the effect of neuroinflammation on the dopamine nigrostriatal pathway. Here, we report that LPS-induced neuroinflammation in the striatum causes progressive degeneration of the dopamine nigrostriatal system, which is accompanied by motor impairments resembling parkinsonism. Our results indicate that neurodegeneration is associated with defects in the mitochondrial respiratory chain related to extensive S-nitrosylation/nitration of mitochondrial proteins. Mitochondrial injury was prevented by treatment of L-N^6^-(l-iminoethyl)-lysine, an inducible nitric oxide synthase (iNOS) inhibitor, suggesting that iNOS-derived NO is responsible for mitochondrial dysfunction. Furthermore, the nigral dopamine neurons exhibited intracytoplasmic [alpha]-synuclein and ubiquitin accumulation. These results demonstrate that degeneration of nigral dopamine neurons by neuroinflammation is associated with mitochondrial malfunction induced by NO-mediated S-nitrosylation/nitration of mitochondrial proteins

Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency

Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients

Emerging functions of mammalian mitochondrial fusion and fission

Mitochondria provide a myriad of services to the cell, including energy production, calcium buffering and regulation of apoptosis. How these diverse functions are coordinated among the hundreds of mitochondria in a given cell is largely unknown, but is probably dependent on the dynamic nature of mitochondria. In this review, we explore the latest developments in mitochondrial dynamics in mammals. These studies indicate that mitofusins and OPA1 are essential for mitochondrial fusion, whereas Fis1 and Drp1 are essential for mitochondrial fission. The overall morphology of the mitochondrial population depends on the relative activities of these two sets of proteins. In addition to the regulation of mitochondrial shape, these molecules also play important roles in cell and tissue physiology. Perturbation of mitochondrial fusion results in defects in mitochondrial membrane potential and respiration, poor cell growth and increased susceptibility to cell death. These cellular observations may explain why mitochondrial fusion is essential for embryonic development. Two inherited neuropathies, Charcot–Marie–Tooth type 2A and autosomal dominant optic atrophy, are caused by mutations in mitofusin 2 and OPA1, suggesting that proper regulation of mitochondrial dynamics is particularly vital to neurons. Mitochondrial fission accompanies several types of apoptotic cell death and appears important for progression of the apoptotic pathway. These studies provide insight into how mitochondria communicate with one another to coordinate mitochondrial function and morphology

Overproduction of PDR3 Suppresses Mitochondrial Import Defects Associated with a TOM70 Null Mutation by Increasing the Expression of TOM72 in Saccharomyces cerevisiae

Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol. We previously reported that the presequence of the F1-ATPase beta subunit precursor (pre-F1beta ) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P. Hajek, J. Y. Koh, L. Jones, and D. M. Bedwell, Mol. Cell. Biol. 17:7169-7177, 1997). We also found that a mutant form of pre-F1beta with a minimal targeting signal (Delta 1,2 pre-F1beta) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation. We have now analyzed the consequences of reducing the pre-F1beta targeting signal to a minimal unit in more detail. We found that Delta 1,2 pre-F1beta is more dependent upon the Tom70p receptor for import than WT pre-F1beta is, resulting in a growth defect on a nonfermentable carbon source at 15°C. Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Delta 1,2 pre-F1beta import signal in an import-competent conformation. We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Delta 1,2 pre-F1beta in a tom70Delta strain. The overproduction of PDR3 mediated this effect by increasing the import of Delta 1,2 pre-F1beta into mitochondria. This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions. Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue. These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis

Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson's, Alzheimer's and Huntington's disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy