Sofia Garcia

Cytochrome c Oxidase Is Required for the Assembly/Stability of Respiratory Complex I in Mouse Fibroblasts

ABSTRACT Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.

Cytochrome c oxidase deficiency in neurons decreases both oxidative stress and amyloid formation in a mouse model of Alzheimer's disease

Defects in the mitochondrial cytochrome c oxidase (COX) have been associated with Alzheimers Disease, in which the age-dependent accumulation of β-amyloid plays an important role in synaptic dysfunction and neurodegeneration. To test the possibility that age-dependent decline in the mitochondrial respiratory function, especially COX activity, may participate in the formation and accumulation of β-amyloid, we generated mice expressing mutant amyloid precursor protein and mutant presenilin 1 in a neuron-specific COX-deficient background. A neuron-specific COX-deficient mouse was generated by the Cre-loxP system, in which the COX10 gene was deleted by a CamKIIα promoter-driven Cre-recombinase. COX10 is a farnesyltransferase involved in the biosynthesis of heme a, required for COX assembly and function. These KO mice showed an age-dependent COX deficiency in the cerebral cortex and hippocampus. Surprisingly, COX10 KO mice exhibited significantly fewer amyloid plaques in their brains compared with the COX-competent transgenic mice. This reduction in amyloid plaques in the KO mouse was accompanied by a reduction in Aβ42 level, β-secretase activity, and oxidative damage. Likewise, production of reactive oxygen species from cells with partial COX activity was not elevated. Collectively, our results suggest that, contrary to previous models, a defect in neuronal COX does not increase oxidative damage nor predispose for the formation of amyloidgenic amyloid precursor protein fragments.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

A defect in the mitochondrial Complex III, but not Complex IV, triggers early ROS-dependent damage in defined brain regions

We have created two neuron-specific mouse models of mitochondrial electron transport chain deficiencies involving defects in complex III (CIII) or complex IV (CIV). These conditional knockouts (cKOs) were created by ablation of the genes coding for the Rieske iron-sulfur protein (RISP) and COX10, respectively. RISP is one of the catalytic subunits of CIII and COX10 is an assembly factor indispensable for the maturation of Cox1, one of the catalytic subunits of CIV. Although the rates of gene deletion, protein loss and complex dysfunction were similar, the RISP cKO survived 3.5 months of age, whereas the COX10 cKO survived for 10-12 months. The RISP cKO had a sudden death, with minimal behavioral changes. In contrast, the COX10 cKO showed a distinctive behavioral phenotype with onset at 4 months of age followed by a slower but progressive neurodegeneration. Curiously, the piriform and somatosensory cortices were more vulnerable to the CIII defect whereas cingulate cortex and to a less extent piriform cortex were affected preferentially by the CIV defect. In addition, the CIII model showed severe and early reactive oxygen species damage, a feature not observed until very late in the pathology of the CIV model. These findings illustrate how specific respiratory chain defects have distinct molecular mechanisms, leading to distinct pathologies, akin to the clinical heterogeneity observed in patients with mitochondrial diseases.

Pathophysiology and fate of hepatocytes in a mouse model of mitochondrial hepatopathies

Background: Although oxidative phosphorylation defects can affect the liver, these conditions are poorly understood, partially because of the lack of animal models. Aims: To create and characterise the pathophysiology of mitochondrial hepatopathies in a mouse model. Methods: A mouse model of mitochondrial hepatopathies was created by the conditional liver knockout (KO) of the COX10 gene, which is required for cytochrome c oxidase (COX) function. The onset and progression of biochemical, molecular and clinical phenotypes were analysed in several groups of animals, mostly at postnatal days 23, 56, 78 and 155. Results: Biochemical and histochemical analysis of liver samples from 23–56-day-old KO mice showed liver dysfunction, a severe COX deficiency, marked mitochondrial proliferation and lipid accumulation. Despite these defects, the COX-deficient hepatocytes were not immediately eliminated, and apoptosis followed by liver regeneration could be observed only at age 78 days. Hepatocytes from 56–78-day-old KO mice survived despite very low COX activity but showed a progressive depletion of glycogen stores. In most animals, hepatocytes that escaped COX10 ablation were able to proliferate and completely regenerate the liver between days 78 and 155. Conclusions: The results showed that when faced with a severe oxidative phosphorylation defect, hepatocytes in vivo can rely on glycolysis/glycogenolysis for their bioenergetic needs for relatively long periods. Ultimately, defective hepatocytes undergo apoptosis and are replaced by COX-positive cells first observed in the perivascular regions.

Partial complex I deficiency due to the CNS conditional ablation of Ndufa5 results in a mild chronic encephalopathy but no increase in oxidative damage

Deficiencies in the complex I (CI; NADH-ubiquinone oxidoreductase) of the respiratory chain are frequent causes of mitochondrial diseases and have been associated with other neurodegenerative disorders, such as Parkinsons disease. The NADH-ubiquinone oxidoreductase 1 alpha subcomplex subunit 5 (NDUFA5) is a nuclear-encoded structural subunit of CI, located in the peripheral arm. We inactivated Ndufa5 in mice by the gene-trap methodology and found that this protein is required for embryonic survival. Therefore, we have created a conditional Ndufa5 knockout (KO) allele by introducing a rescuing Ndufa5 cDNA transgene flanked by loxP sites, which was selectively ablated in neurons by the CaMKIIα-Cre. At the age of 11 months, mice with a central nervous system knockout of Ndufa5 (Ndufa5 CNS-KO) showed lethargy and loss of motor skills. In these mice cortices, the levels of NDUFA5 protein were reduced to 25% of controls. Fully assembled CI levels were also greatly reduced in cortex and CI activity in homogenates was reduced to 60% of controls. Despite the biochemical phenotype, no oxidative damage, neuronal death or gliosis were detected in the Ndufa5 CNS-KO brain at this age. These results showed that a partial defect in CI in neurons can lead to late-onset motor phenotypes without neuronal loss or oxidative damage.

Long-Term Bezafibrate Treatment Improves Skin and Spleen Phenotypes of the mtDNA Mutator Mouse

Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent’s liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.

Sustained AMPK activation improves muscle function in a mitochondrial myopathy mouse model by promoting muscle fiber regeneration

Acute pharmacological activation of adenosine monophosphate (AMP)-kinase using 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) has been shown to improve muscle mitochondrial function by increasing mitochondrial biogenesis. We asked whether prolonged AICAR treatment is beneficial in a mouse model of slowly progressing mitochondrial myopathy (Cox10-Mef2c-Cre), and whether the compensatory mechanism is indeed an increase in mitochondrial biogenesis. We treated the animals for 3 months and found that sustained AMP-dependent kinase activation improved cytochrome c oxidase activity, rescued the motor phenotype and delayed the onset of the myopathy. This improvement was observed whether treatment started before or after the onset of the disease. We found that AICAR increased skeletal muscle regeneration thereby decreasing the levels of deleted Cox10-floxed alleles. We conclude that although increase in mitochondrial biogenesis and other pathways may contribute, the main mechanism by which AICAR improves the myopathy phenotype is by promoting muscle regeneration.

A 3′ UTR Modification of the Mitochondrial Rieske Iron Sulfur Protein in Mice Produces a Specific Skin Pigmentation Phenotype

TO THE EDITOR The role of mitochondrial proteins in melanocyte function and pigmentation has been brought into the spotlight by a recent article in the Journal of Investigative Dermatology (Ni-Komatsu and Orlow, 2007). Using a zebrafish screening, two oxidative phosphorylation-related factors, prohibitin and the complex V (F1/F0 ATPase), were identified as modulators of pigmentation. In addition, alterations in skin pigmentation have previously been reported in some patients with mitochondrial disorders (Bodemer et al., 1999; Kubota et al., 1999). During our studies on mitochondrial function, we developed a mouse with a knock-in Rieske iron sulfur protein (RISP). The production of mice with the RISP genetic modification was approved by the University of Miami Institutional Animal Care and Use Committee. The RISP is a subunit of the mitochondrial oxidative phosphorylation complex III. The knocked-in gene was flanked by loxP sites for tissue-specific ablation. The construct contained a full-length, intact gene, but included a neomycin/thymidine kinase selection cassette in the 3′-untranslated region. Therefore, the knock-in gene should be functional, and defective only when exposed to the Cre recombinase, which would delete exon 2 of the RISP. This can be accomplished by crossing the knock-in mice with Cre-expressing transgenic mice (Diaz et al., 2005). Not surprisingly, mice homozygous for the knock-in allele were healthy and lived a normal life, as modification of the knock-in gene did not affect the coding sequence or splicing of the RISP gene. However, mice heterozygous or homozygous for the knock-in allele showed change in coat color starting at about 4–7 months of age. In mice, the coat color depends on the ratio and distribution of two melanin pigments, the eumelanins (black to brown pigments) and the pheomelanins (yellow to red pigments), derived from a common precursor (dopaquinone), which are synthesized by follicular melanocytes. The heterozygous mice acquired dark patches in the dorsal brown coat. With age, the dark patches eventually filled almost all the dorsal coat, although it remained unchanged in the ventral region (Figure 1a). In homozygous mice, the dark patches eventually turned gray (Figure 1c and d). This phenotype segregated with the presence and dosage of the knock-in allele. Figure 1 Coat color changes of mice with an RISP-knock-in gene Analyses of RNAs by northern blots showed that, as expected, the knock-in allele was larger than the endogeneous RISP transcript (Figure 2a and b). Western blot analyses showed that in the skin, the levels of RISP were markedly decreased in homozygous knock-in mice (Figure 2c). The secondary antibody against mouse immunoglobulins detected non-specific bands in the region of RISP in skin homogenates (endogenous immunoglobulins), but we were able to distinguish those from the RISP by analyzing a heart mitochondrial sample in parallel (Figure 2c). The RISP levels were not significantly altered in muscle, brain, heart, or liver (Figure 2d), demonstrating that the knock-in transcripts were correctly translated into a functional protein in most tissues. Figure 2 Characterization of the RISP-knock-in gene expression We isolated fibroblasts from homozygous floxed mice, but detected neither a complex III defect (not shown) nor a significant decrease in RISP (Figure 2e). As an additional control, we deleted the RISP gene in fibroblast cultures using a plasmid encoding the Cre-recombinase. The knockout fibroblasts showed no RISP and a reduction in subunit core 2 of complex III (lane 2 in Figure 2e). Compared with floxed fibroblasts, isolated melanocytes showed proportionally higher decrease in RISP when normalized to a mitochondrial marker (VDAC1; Figure 2e), suggesting that melanocytes are more sensitive to the knock-in allele. Unfortunately, the yield and life span of the isolated primary melanocytes were very limited, precluding further experiments with them. From our observations, we speculate that melanocytes have a specific regulation (at the transcriptional or translational level) of RISP expression. Although it is unclear how a pigmentation phenotype developed in these mice, defects in complex III are commonly associated with an increase in reactive oxygen species (ROS) production in the mitochondria. ROS are not only known mediators of UV-induced hyperpigmentation and can eventually lead to melanocyte apoptosis and hypopigmentation (Costin and Hearing, 2007), but also participate in the metabolism of dopaquinone (Mastore et al., 2005). However, we did not find increased ROS production in RISP-deficient fibroblasts (not shown) and the knockdown reductions in the RISP protein have been associated with a decrease in ROS (Bell et al., 2007). Accordingly, treatment of homozygous or heterozygous RISP knock-in mice with N-acetyl cysteine for 60 days (starting at age 2 months) failed to prevent changes in coat color (not shown). Staining of skin sections for melanin (using the Fontana–Masson silver method) did not show major differences between wild-type and homozygous knock-in mice. The fact that darkening of the coat is not observed in the ventral part suggests that the agouti could be involved in this phenomenon. Agouti protein antagonizes the effects of α-melanocyte-stimulating hormone, switching the melanogenesis from eumelanin to phaeomelanin. Differences in agouti expression along the dorsal–ventral axis, have been found (Vrieling et al., 1994). Although we cannot rule out the participation of ROS in the coat color phenotype, mitochondrial function may have a broader role in the modulation of skin pigmentation, as the findings of Ni-Komatsu and Orlow suggest. In any case, the mice described here provide a unique model for the study of pigmentation abnormalities in mitochondrial disorders.

Overexpression of PGC-1α in aging muscle enhances a subset of young-like molecular patterns

PGC‐1α is a transcriptional co‐activator known as the master regulator of mitochondrial biogenesis. Its control of metabolism has been suggested to exert critical influence in the aging process. We have aged mice overexpressing PGC‐1α in skeletal muscle to determine whether the transcriptional changes reflected a pattern of expression observed in younger muscle. Analyses of muscle proteins showed that Pax7 and several autophagy markers were increased. In general, the steady‐state levels of several muscle proteins resembled that of muscle from young mice. Age‐related mtDNA deletion levels were not increased by the PGC‐1α‐associated increase in mitochondrial biogenesis. Accordingly, age‐related changes in the neuromuscular junction were minimized by PGC‐1α overexpression. RNA‐Seq showed that several genes overexpressed in the aged PGC‐1α transgenic are expressed at higher levels in young when compared to aged skeletal muscle. As expected, there was increased expression of genes associated with energy metabolism but also of pathways associated with muscle integrity and regeneration. We also found that PGC‐1α overexpression had a mild but significant effect on longevity. Taken together, overexpression of PGC‐1α in aged muscle led to molecular changes that resemble the patterns observed in skeletal muscle from younger mice.