Understanding Coenzyme Q.

Coenzyme Q (CoQ), also known as ubiquinone, comprises a benzoquinone head group and a long isoprenoid sidechain. It is thus extremely hydrophobic and resides in membranes. It is best known for its complex function as an electron transporter in the mitochondrial electron transport chain (ETC) and in several other cellular processes. In fact, CoQ appears to be central to the redox balance of the cell. Remarkably, its structure and properties have not changed from bacteria to vertebrates. In metazoans, it is synthesized in all cells and is found in most, and maybe all, biological membranes. CoQ is also known as a nutritional supplement, mostly because of its involvement with antioxidant defenses. However, whether there is any health benefit from oral consumption of CoQ is not well established. Here we review the function of CoQ as a redox active molecule in the ETC and other enzymatic systems, its role as a pro-oxidant in reactive oxygen species generation, and its separate involvement in antioxidant mechanisms. We also review CoQ biosynthesis, which is particularly complex because of its extreme hydrophobicity, as well as the biological consequences of primary and secondary CoQ deficiency, including in human patients. Primary CoQ deficiency is a rare inborn condition due to mutation in CoQ biosynthetic genes. Secondary CoQ deficiency is much more common as it accompanies a variety of pathological conditions, including mitochondrial disorders as well as aging. In this context, we discuss the importance, but also the great difficulty, of alleviating CoQ deficiency by CoQ supplementation.

The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans.

The increased longevity of the C. elegans electron transport chain mutants isp-1 and nuo-6 is mediated by mitochondrial ROS (mtROS) signaling. Here we show that the mtROS signal is relayed by the conserved, mitochondria-associated, intrinsic apoptosis signaling pathway (CED-9/Bcl2, CED-4/Apaf1, and CED-3/Casp9) triggered by CED-13, an alternative BH3-only protein. Activation of the pathway by an elevation of mtROS does not affect apoptosis but protects from the consequences of mitochondrial dysfunction by triggering a unique pattern of gene expression that modulates stress sensitivity and promotes survival. In vertebrates, mtROS induce apoptosis through the intrinsic pathway to protect from severely damaged cells. Our observations in nematodes demonstrate that sensing of mtROS by the apoptotic pathway can, independently of apoptosis, elicit protective mechanisms that keep the organism alive under stressful conditions. This results in extended longevity when mtROS generation is inappropriately elevated. These findings clarify the relationships between mitochondria, ROS, apoptosis, and aging.

Accessory subunit NDUFB4 participates in mitochondrial complex I supercomplex formation.

Mitochondrial electron transport chain complexes organize into supramolecular structures called respiratory supercomplexes (SCs). The role of respiratory SCs remains largely unconfirmed despite evidence supporting their necessity for mitochondrial respiratory function. The mechanisms underlying the formation of the I1III2IV1 "respirasome" SC are also not fully understood, further limiting insights into these processes in physiology and diseases, including neurodegeneration and metabolic syndromes. NDUFB4 is a complex I accessory subunit that contains residues that interact with the subunit UQCRC1 from complex III, suggesting that NDUFB4 is integral for I1III2IV1 respirasome integrity. Here, we introduced specific point mutations to Asn24 (N24) and Arg30 (R30) residues on NDUFB4 to decipher the role of I1III2-containing respiratory SCs in cellular metabolism while minimizing the functional consequences to complex I assembly. Our results demonstrate that NDUFB4 point mutations N24A and R30A impair I1III2IV1 respirasome assembly and reduce mitochondrial respiratory flux. Steady-state metabolomics also revealed a global decrease in citric acid cycle metabolites, affecting NADH-generating substrates. Taken together, our findings highlight an integral role of NDUFB4 in respirasome assembly and demonstrate the functional significance of SCs in regulating mammalian cell bioenergetics.

Biallelic variants in COQ7 cause distal hereditary motor neuropathy with upper motor neuron signs.

COQ7 encodes a hydroxylase responsible for the penultimate step of coenzyme Q10 (CoQ10) biosynthesis in mitochondria. CoQ10 is essential for multiple cellular functions, including mitochondrial oxidative phosphorylation, lipid metabolism, and reactive oxygen species homeostasis. Mutations in COQ7 have been previously associated with primary CoQ10 deficiency, a clinically heterogeneous multisystemic mitochondrial disorder. We identified COQ7 biallelic variants in nine families diagnosed with distal hereditary motor neuropathy with upper neuron involvement, expending the clinical phenotype associated with defects in this gene. A recurrent p.Met1? change was identified in five families from Brazil with evidence of a founder effect. Fibroblasts isolated from patients revealed a substantial depletion of COQ7 protein levels, indicating protein instability leading to loss of enzyme function. High-performance liquid chromatography assay showed that fibroblasts from patients had reduced levels of CoQ10, and abnormal accumulation of the biosynthetic precursor DMQ10. Accordingly, fibroblasts from patients displayed significantly decreased oxygen consumption rates in patients, suggesting mitochondrial respiration deficiency. Induced pluripotent stem cell-derived motor neurons from patient fibroblasts showed significantly increased levels of extracellular neurofilament light protein, indicating axonal degeneration. Our findings indicate a molecular pathway involving CoQ10 biosynthesis deficiency and mitochondrial dysfunction in patients with distal hereditary motor neuropathy. Further studies will be important to evaluate the potential benefits of CoQ10 supplementation in the clinical outcome of the disease.

ROS regulation of RAS and vulva development in Caenorhabditis elegans.

Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H202, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H202 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.

The efficacy of coenzyme Q10 treatment in alleviating the symptoms of primary coenzyme Q10 deficiency: A systematic review.

Coenzyme Q10 (CoQ10 ) is necessary for mitochondrial electron transport. Mutations in CoQ10 biosynthetic genes cause primary CoQ10 deficiency (PCoQD) and manifest as mitochondrial disorders. It is often stated that PCoQD patients can be treated by oral CoQ10 supplementation. To test this, we compiled all studies describing PCoQD patients up to May 2022. We excluded studies with no data on CoQ10 treatment, or with insufficient description of effectiveness. Out of 303 PCoQD patients identified, we retained 89 cases, of which 24 reported improvements after CoQ10 treatment (27.0%). In five cases, the patient's condition was reported to deteriorate after halting of CoQ10 treatment. 12 cases reported improvement in the severity of ataxia and 5 cases in the severity of proteinuria. Only a subjective description of improvement was reported for 4 patients described as responding. All reported responses were partial improvements of only some symptoms. For PCoQD patients, CoQ10 supplementation is replacement therapy. Yet, there is only very weak evidence for the efficacy of the treatment. Our findings, thus, suggest a need for caution when seeking to justify the widespread use of CoQ10 for the treatment of any disease or as dietary supplement.

Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans.

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development.

Mitochondrial and cytoplasmic ROS have opposing effects on lifespan.

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan - elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.

Pathogenicity of two COQ7 mutations and responses to 2,4-dihydroxybenzoate bypass treatment.

Primary ubiquinone (co-enzyme Q) deficiency results in a wide range of clinical features due to mitochondrial dysfunction. Here, we analyse and characterize two mutations in the ubiquinone biosynthetic gene COQ7. One mutation from the only previously identified patient (V141E), and one (L111P) from a 6-year-old girl who presents with spasticity and bilateral sensorineural hearing loss. We used patient fibroblast cell lines and a heterologous expression system to show that both mutations lead to loss of protein stability and decreased levels of ubiquinone that correlate with the severity of mitochondrial dysfunction. The severity of L111P is enhanced by the particular COQ7 polymorphism (T103M) that the patient carries, but not by a mitochondrial DNA mutation (A1555G) that is also present in the patient and that has been linked to aminoglycoside-dependent hearing loss. We analysed treatment with the unnatural biosynthesis precursor 2,4-dihydroxybenzoate (DHB), which can restore ubiquinone synthesis in cells completely lacking the enzymatic activity of COQ7. We find that the treatment is not beneficial for every COQ7 mutation and its outcome depends on the extent of enzyme activity loss.

CEP-1, the Caenorhabditis elegans p53 homolog, mediates opposing longevity outcomes in mitochondrial electron transport chain mutants.

Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the "aging" Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses.

Molecular genetics of ubiquinone biosynthesis in animals.

Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and Saccharomyces cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including Caenorhabditis elegans, rodents, and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general.

Mitochondrial respiration without ubiquinone biosynthesis.

Ubiquinone (UQ), a.k.a. coenzyme Q, is a redox-active lipid that participates in several cellular processes, in particular mitochondrial electron transport. Primary UQ deficiency is a rare but severely debilitating condition. Mclk1 (a.k.a. Coq7) encodes a conserved mitochondrial enzyme that is necessary for UQ biosynthesis. We engineered conditional Mclk1 knockout models to study pathogenic effects of UQ deficiency and to assess potential therapeutic agents for the treatment of UQ deficiencies. We found that Mclk1 knockout cells are viable in the total absence of UQ. The UQ biosynthetic precursor DMQ9 accumulates in these cells and can sustain mitochondrial respiration, albeit inefficiently. We demonstrated that efficient rescue of the respiratory deficiency in UQ-deficient cells by UQ analogues is side chain length dependent, and that classical UQ analogues with alkyl side chains such as idebenone and decylUQ are inefficient in comparison with analogues with isoprenoid side chains. Furthermore, Vitamin K2, which has an isoprenoid side chain, and has been proposed to be a mitochondrial electron carrier, had no efficacy on UQ-deficient mouse cells. In our model with liver-specific loss of Mclk1, a large depletion of UQ in hepatocytes caused only a mild impairment of respiratory chain function and no gross abnormalities. In conjunction with previous findings, this surprisingly small effect of UQ depletion indicates a nonlinear dependence of mitochondrial respiratory capacity on UQ content. With this model, we also showed that diet-derived UQ10 is able to functionally rescue the electron transport deficit due to severe endogenous UQ deficiency in the liver, an organ capable of absorbing exogenous UQ.

A microfluidic device for efficient chemical testing using Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has been employed as a popular model organism in many fields of biological research. In this paper, we present a microfluidic device for facilitating chemical testing using C. elegans. For testing chemicals on chip, the device houses single nematodes in microfluidic chambers and precisely adjusts the chamber's chemical environment during experiments. Eight nematodes can be readily loaded into the chambers through separate loading channels in a quick and gentle manner. In addition, a custom-made software with a graphic user interface is also created for quantitative analysis of locomotion parameters (swimming frequency and bend amplitude) of the nematodes in response to chemical stimuli, thus greatly enhancing the efficiency of data collection. We perform proof-of-concept experiments using two chemicals, zinc ion (Zn(2+)) and glucose, to demonstrate the effectiveness of the microfluidic device.

Superoxide dismutase is dispensable for normal animal lifespan.

Reactive oxygen species (ROS) are toxic oxygen-containing molecules that can damage multiple components of the cell and have been proposed to be the primary cause of aging. The antioxidant enzyme superoxide dismutase (SOD) is the only eukaryotic enzyme capable of detoxifying superoxide, one type of ROS. The fact that SOD is present in all aerobic organisms raises the question as to whether SOD is absolutely required for animal life and whether the loss of SOD activity will result in decreased lifespan. Here we use the genetic model organism Caenorhabditis elegans to generate an animal that completely lacks SOD activity (sod-12345 worms). We show that sod-12345 worms are viable and exhibit a normal lifespan, despite markedly increased sensitivity to multiple stresses. This is in stark contrast to what is observed in other genetic model organisms where the loss of a single sod gene can result in severely decreased survival. Investigating the mechanism underlying the normal lifespan of sod-12345 worms reveals that their longevity results from a balance between the prosurvival signaling and the toxicity of superoxide. Overall, our results demonstrate that SOD activity is dispensable for normal animal lifespan but is required to survive acute stresses. Moreover, our findings indicate that maintaining normal stress resistance is not crucial to the rate of aging.

Mitochondrial oxidative stress alters a pathway in Caenorhabditis elegans strongly resembling that of bile acid biosynthesis and secretion in vertebrates.

Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA-biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.

How genetic analysis tests theories of animal aging.

Each animal species displays a specific life span, rate of aging and pattern of development of age-dependent diseases. The genetic bases of these related features are being studied experimentally in invertebrate and vertebrate model systems as well as in humans through medical records. Three types of mutants are being analyzed: (i) short-lived mutants that are prone to age-dependent diseases and might be models of accelerated aging; (ii) mutants that show overt molecular defects but that do not live shorter lives than controls, and can be used to test specific theories about the molecular causes of aging and age-dependent diseases; and (iii) long-lived mutants that might advance the understanding of the molecular physiology of slow-aging animals and aid the discovery of molecular targets that could be used to manipulate rates of aging to benefit human health. Here, I analyze some of what we know today and discuss what we should try to find out in the future to understand the aging phenomenon.

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans.

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O(⋅) (-)), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.

Novel Homozygous Variant in COQ7 in Siblings With Hereditary Motor Neuropathy.

Background and Objectives: Coenzyme Q10 (CoQ10) is an important electron carrier and antioxidant. The COQ7 enzyme catalyzes the hydroxylation of 5-demethoxyubiquinone-10 (DMQ10), the second-to-last step in the CoQ10 biosynthesis pathway. We report a consanguineous family presenting with a hereditary motor neuropathy associated with a homozygous c.1A > G p.? variant of COQ7 with abnormal CoQ10 biosynthesis. Methods: Affected family members underwent clinical assessments that included nerve conduction testing, histologic analysis, and MRI. Pathogenicity of the COQ7 variant was assessed in cultured fibroblasts and skeletal muscle using a combination of immunoblots, respirometry, and quinone analysis. Results: Three affected siblings, ranging from 12 to 24 years of age, presented with a severe length-dependent motor neuropathy with marked symmetric distal weakness and atrophy with normal sensation. Muscle biopsy of the quadriceps revealed chronic denervation pattern. An MRI examination identified moderate to severe fat infiltration in distal muscles. Exome sequencing demonstrated the homozygous COQ7 c.1A > G p.? variant that is expected to bypass the first 38 amino acid residues at the n-terminus, initiating instead with methionine at position 39. This is predicted to cause the loss of the cleavable mitochondrial targeting sequence and 2 additional amino acids, thereby preventing the incorporation and subsequent folding of COQ7 into the inner mitochondrial membrane. Pathogenicity of the COQ7 variant was demonstrated by diminished COQ7 and CoQ10 levels in muscle and fibroblast samples of affected siblings but not in the father, unaffected sibling, or unrelated controls. In addition, fibroblasts from affected siblings had substantial accumulation of DMQ10, and maximal mitochondrial respiration was impaired in both fibroblasts and muscle. Discussion: This report describes a new neurologic phenotype of COQ7-related primary CoQ10 deficiency. Novel aspects of the phenotype presented by this family include pure distal motor neuropathy involvement, as well as the lack of upper motor neuron features, cognitive delay, or sensory involvement in comparison with cases of COQ7-related CoQ10 deficiency previously reported in the literature.

Elevated mitochondrial reactive oxygen species generation affects the immune response via hypoxia-inducible factor-1alpha in long-lived Mclk1+/- mouse mutants.

Mitochondrial reactive oxygen species (ROS) are believed to stabilize hypoxia-inducible factor (HIF)-1alpha, a transcriptional regulator of the immune response. Mclk1 encodes a mitochondrial protein that is necessary for ubiquinone biosynthesis. Heterozygote Mclk1(+/-) mutant mice are long-lived despite increased mitochondrial ROS and decreased energy metabolism. In this study, Mclk1(+/-) mutant mice in the C57BL/6J background displayed increased basal and induced expression of HIF-1alpha in liver and macrophages in association with elevated expression of inflammatory cytokines, in particular TNF-alpha. Mutant macrophages showed increased classical and decreased alternative activation, and mutant mice were hypersensitive to LPS. Consistent with these observations in vivo, knock-down of Mclk1 in murine RAW264.7 macrophage-like cells induced increased mitochondrial ROS as well as elevated expression of HIF-1alpha and secretion of TNF-alpha. We used an antioxidant peptide targeted to mitochondria to show that altered ROS metabolism is necessary for the enhanced expression of HIF-1alpha, which, in turn, is necessary for increased TNF-alpha secretion. These findings provide in vivo evidence for the action of mitochondrial ROS on HIF-1alpha activity and demonstrate that changes in mitochondrial function within physiologically tolerable limits modulate the immune response. Our results further suggest that altered immune function through a limited increase in HIF-1alpha expression can positively impact animal longevity.