Prasanth Potluri

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.

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.

Development and Characterization of a Conditional Mitochondrial Complex I Assembly System

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE·HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another ∼24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1–6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as “accessory.” It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res−) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res− cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res− cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res− cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.

Cyanide Detoxification by the Cobalamin Precursor Cobinamide

Cyanide is a highly toxic agent that inhibits mitochondrial cytochrome-c oxidase, thereby depleting cellular ATP. it contributes to smoke inhalation deaths in fires and could be used as a weapon of mass destruction. Cobalamin (vitamin B12) binds cyanide with a relatively high affinity and is used in Europe to treat smoke inhalation victims. Cobinamide, the penultimate compound in cobalamin biosynthesis, binds cyanide with about 1010 greater affinity than cobalamin, and we found It was several-fold more effective than cobalamin in (i) reversing cyanide inhibition of oxidative phosphorylation in mammalian cells; (ii) rescuing mammalian cells and Drosophila melanogaster from cyanide toxicity; and (iii) reducing cyanide inhibition of Drosophila Malpighian tubule secretion. Cobinamide could be delivered by oral ingestion, inhalation, or injection to Drosophila, and it was as effective when administered up to 5 mins post-cyanide exposure as when given preexposure. We conclude that cobinamide is an effective cyanide detoxifying agent that has potential use as a cyanide antidote, both in smoke inhalation victims and in persons exposed to cyanide used as a weapon of mass destruction.

Mitochondrial dysfunction in CA1 hippocampal neurons of the UBE3A deficient mouse model for Angelman syndrome

Angelman syndrome (AS) is a severe neurological disorder caused by a deficiency of ubiquitin protein ligase E3A (UBE3A), but the pathophysiology of the disease remains unknown. We now report that in the brains of AS mice in which the maternal UBE3A allele is mutated (m-) and the paternal allele is potentially inactivated by imprinting (p+) (UBE3A m-\p+), the mitochondria are abnormal and exhibit a partial oxidative phosphorylation (OXPHOS) defect. Electron microscopy of the hippocampal region of the UBE3A m-\p+ mice (n=6) reveals small, dense mitochondria with altered cristae, relative to wild-type littermates (n=6) and reduced synaptic vesicle density. The specific activity of OXPHOS complex III is reduced in whole brain mitochondria in UBE3A m-\p+ (n=5) mice versus wild-type littermates (n=5). Therefore, mitochondrial dysfunction may contribute to the pathophysiology of Angelman syndrome.

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.

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination.

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination.

The Cobalamin Precursor Cobinamide Detoxifies Nitroprusside-Generated Cyanide

Sodium nitroprusside is used to treat hypertensive emergencies and acute heart failure. It acts by releasing nitric oxide (NO), a highly potent vasodilator, but unfortunately, for each NO molecule released, five cyanide ions are released. Thus, nitroprusside therapy is limited by cyanide toxicity. Therefore, a cyanide scavenger could be beneficial when administering nitroprusside. Hydroxocobalamin, which has a relatively high binding affinity for cyanide, has been shown to reduce cyanide levels in nitroprusside-treated patients. Cobinamide, the penultimate precursor in hydroxocobalamin biosynthesis, has a much greater affinity for cyanide than cobalamin, and binds two cyanide ions. We now show that cobinamide is highly effective in neutralizing cyanide ions released by nitroprusside in cultured mammalian cells, Drosophila melanogaster, and mice. Cobinamide also binds NO, but at molar concentrations 2.5–5 times that of nitroprusside, it did not decrease NO concentrations or the physiological effectiveness of nitroprusside. We conclude that cobinamide could be a valuable adjunct to nitroprusside therapy.

A neonatal polyvisceral failure linked to a de novo homoplasmic mutation in the mitochondrially encoded cytochrome b gene.

Mutations within the mitochondrially encoded cytochrome b (MTCYB) gene are heteroplasmic and lead to severe exercise intolerance. We describe an unusual clinical presentation secondary to a novel homoplasmic mutation within MTCYB. The m.15635T>C transition (S297P) was carried by a newborn who presented with a polyvisceral failure. This mutation was responsible for a complex III deficiency. It was homoplasmic in all tissues tested and was undetectable in patients mother. Functional analyses, including studies on patients cybrid cell lines, demonstrate the pathogenicity of this variant. Our data show that mutations within MTCYB can be responsible for severe phenotype at birth.