Antonio Davila

Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming

Significance Mitochondria generate signals that regulate nuclear gene expression via retrograde signaling, but this phenomenon is rendered more complex by the quantitative differences in the percentage of mutant and normal mtDNAs that can exist within patient cells. This study demonstrates that depending upon its relative cytoplasmic levels, a single mtDNA point mutation can cause a discrete set of cellular transcriptional responses within cells of the same nuclear background. This qualitative regulation of nuclear gene expression by quantitative changes in mtDNA mutant levels challenges the traditional “single mutation–single disease” concept and provides an alternative perspective on the molecular basis of complex metabolic and degenerative diseases, cancer, and aging. Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNALeu(UUR) 3243A>G mutation and harbor ∼10–30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50–90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90–100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20–30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50–90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a “complex” disease.

A Mutation of β-Actin That Alters Depolymerization Dynamics Is Associated with Autosomal Dominant Developmental Malformations, Deafness, and Dystonia

Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer-sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia.

A novel NDUFA1 mutation leads to a progressive mitochondrial complex I-specific neurodegenerative disease

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patients mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA encoded MWFE polypeptide has been shown to interact with various mtDNA encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.

Role of SUV3 Helicase in Maintaining Mitochondrial Homeostasis in Human Cells

In yeast mitochondria, RNA degradation takes place through the coordinated activities of ySuv3 helicase and yDss1 exoribonuclease (mtEXO), whereas in bacteria, RNA is degraded via RNaseE, RhlB, PNPase, and enolase. Yeast lacking the Suv3 component of the mtEXO form petits and undergo a toxic accumulation of omega intron RNAs. Mammalian mitochondria resemble their prokaryotic origins by harboring a polyadenylation-dependent RNA degradation mechanism, but whether SUV3 participates in regulating RNA turnover in mammalian mitochondria is unclear. We found that lack of hSUV3 in mammalian cells subsequently yielded an accumulation of shortened polyadenylated mtRNA species and impaired mitochondrial protein synthesis. This suggests that SUV3 may serve in part as a component of an RNA degradosome, resembling its yeast ancestor. Reduction in the expression levels of oxidative phosphorylation components correlated with an increase in reactive oxygen species generation, whereas membrane potential and ATP production were decreased. These cumulative defects led to pleiotropic effects in mitochondria such as decreased mtDNA copy number and a shift in mitochondrial morphology from tubular to granular, which eventually manifests in cellular senescence or cell death. Thus, our results suggest that SUV3 is essential for maintaining proper mitochondrial function, likely through a conserved role in mitochondrial RNA regulation.

Testing the Oxidative Stress Hypothesis of Aging in Primate Fibroblasts: Is There a Correlation Between Species Longevity and Cellular ROS Production?

The present study was conducted to test predictions of the oxidative stress theory of aging assessing reactive oxygen species production and oxidative stress resistance in cultured fibroblasts from 13 primate species ranging in body size from 0.25 to 120 kg and in longevity from 20 to 90 years. We assessed both basal and stress-induced reactive oxygen species production in fibroblasts from five great apes (human, chimpanzee, bonobo, gorilla, and orangutan), four Old World monkeys (baboon, rhesus and crested black macaques, and patas monkey), three New World monkeys (common marmoset, red-bellied tamarin, and woolly monkey), and one lemur (ring-tailed lemur). Measurements of cellular MitoSox fluorescence, an indicator of mitochondrial superoxide (O2(·-)) generation, showed an inverse correlation between longevity and steady state or metabolic stress-induced mitochondrial O2(·-) production, but this correlation was lost when the effects of body mass were removed, and the data were analyzed using phylogenetically independent contrasts. Fibroblasts from longer-lived primate species also exhibited superior resistance to H(2)O(2)-induced apoptotic cell death than cells from shorter-living primates. After correction for body mass and lack of phylogenetic independence, this correlation, although still discernible, fell short of significance by regression analysis. Thus, increased longevity in this sample of primates is not causally associated with low cellular reactive oxygen species generation, but further studies are warranted to test the association between increased cellular resistance to oxidative stressor and primate longevity.

Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

Background: Elevating NAD throughout the body improves oxidative metabolism in skeletal muscle and counteracts effects of high fat feeding. Results: Boosting NAD synthesis specifically in muscle does not recapitulate these effects. Conclusion: NAD content does not limit oxidative metabolism in young, healthy muscles. Significance: Contrary to the presumption of direct, tissue-autonomous effects, the primary sites of action for NAD precursors remain unknown. The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.

Assessment of mitochondrial membrane potential using an on-chip microelectrode in a microfluidic device.

The mitochondrial membrane potential is used to generate and regulate energy in living systems, driving the conversion of ADP to ATP, regulating ion homeostasis, and controlling apoptosis, all central to human health and disease. Therefore, there is a need for tools to study its regulation in a controlled environment for potential clinical and scientific applications. For this aim, an on-chip tetraphenylphosphonium (TPP(+)) selective microelectrode sensor was constructed in a microfluidic environment. The concentration of isolated mitochondria (Heb7A) used in a membrane potential measurement was 0.3 ng microL(-1), four orders of magnitude smaller than the concentration used in conventional assays (3 microg microL(-1)). In addition, the volume of the chamber (85 microL) is 2 orders of magnitude smaller than traditional experiments. As a demonstration, changes in the membrane potential are clearly measured in response to a barrage of well-known substrates and inhibitors of the electron transport chain. This general approach, which to date has not been demonstrated for study of mitochondrial function and bio-energetics in generally, can be instrumental in advancing the field of mitochondrial research and clinical applications by allowing high throughput studies of the regulation, dynamics, and statistical properties of the mitochondrial membrane potential in response to inhibitors and inducers of apoptosis in a controlled (microfluidic) chemical environment.