Novel antibody-based strategies for the rapid diagnosis of mitochondrial disease and dysfunction.
ABSTRACT We are developing rapid immunoassays to measure the protein levels, enzymatic activities and post-translational modifications of mitochondrial proteins. These assays can be arrayed in multi-analyte panels for biomarker discovery and they can also be used individually at point of care where the level or activity of a small number proteins or even a single protein is highly informative. For example, we have characterized OXPHOS deficits associated with lipoatrophy, an adverse metabolic side-effect of anti-retroviral therapy, and have shown that OXPHOS deficits observed in vitro are also exhibited not only in clinically affected tissue (peripheral fat) but also in more easily accessible tissue (peripheral blood mononucleated cells). Similarly, we have shown that a small set of assays can be used to identify almost all patients with genetic deficits in OXPHOS complexes I or IV, the most common cause of inherited mitochondrial disease. Finally, we recently reported that Friedreich's Ataxia (FA) patients and carriers can be identified on the basis of a simple dipstick test to measure levels of a single protein, frataxin, an iron regulatory protein whose disrupted expression is the proximal cause of neurodegeneration in FA. Because each of these tests can be performed in an extremely simple, rapid dipstick format using non-invasive samples such as cheek swabs and fingerprick blood, they have potential for use as point of care diagnostics for mitochondrial disease and as front-line screening tools to help guide drug therapies and minimize adverse off-target drug effects.
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ABSTRACT: Here, we demonstrate a role for the mitochondrial NAD-dependent deacetylase Sirt3 in the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport. We note that Sirt3(-/-) mouse embryonic fibroblasts have a reduction in basal ATP levels. Reconstitution with wild-type but not a deacetylase-deficient form of Sirt3 restored ATP levels in these cells. Furthermore in wild-type mice, the resting level of ATP correlates with organ-specific Sirt3 protein expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in the heart, kidney, and liver were reduced >50%. We further demonstrate that mitochondrial protein acetylation is markedly elevated in Sirt3(-/-) tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3(-/-) animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions in vivo to regulate and maintain basal ATP levels.Proceedings of the National Academy of Sciences 10/2008; 105(38):14447-52. · 9.74 Impact Factor
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ABSTRACT: Mitochondrial respiratory chain abnormalities are an important cause of neuromuscular disease and may be due to defects of either the mitochondrial or nuclear genome. On account of the clinical and genetic heterogeneity exhibited by the mitochondrial myopathies, their investigation and diagnosis remains a challenge, requiring a combination of techniques including muscle histochemistry, biochemical assessment of respiratory chain function and molecular genetic studies. Here, we describe a step-by-step approach to the clinical and laboratory diagnosis of mitochondrial muscle disease, highlighting the many potential problems that can hinder reaching the correct diagnosis.Neuromuscular Disorders 05/2004; 14(4):237-45. · 3.46 Impact Factor
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ABSTRACT: Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.Electrophoresis 11/1997; 18(11):2059-64. · 3.26 Impact Factor