A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders.

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms.

Parkinson's disease neurons exhibit alterations in mitochondrial quality control proteins.

Mitochondrial dysfunction has been suggested to contribute to Parkinson's disease pathogenesis, though an understanding of the extent or exact mechanism of this contribution remains elusive. This has been complicated by challenging nature of pathway-based analysis and an inability simultaneously study multiple related proteins within human brain tissue. We used imaging mass cytometry (IMC) to overcome these challenges, measuring multiple protein targets, whilst retaining the spatial relationship between targets in post-mortem midbrain sections.

Disease Diagnostics and Potential Coinfections by During an Ongoing Coral Disease Outbreak in Florida.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

Objectives: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing.

A subset of five human mitochondrial formyl peptides mimics bacterial peptides and functionally deactivates human neutrophils.

Background: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs).

A rapid, noninvasive immunoassay for frataxin: utility in assessment of Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by reduced amounts of the mitochondrial protein frataxin. Frataxin levels in research studies are typically measured via Western blot analysis from patient fibroblasts, lymphocytes, or muscle biopsies; none of these is ideal for rapid detection in large scale clinical studies. Recently, a rapid, noninvasive lateral flow immunoassay was developed to accurately measure picogram levels of frataxin protein and shown to distinguish lymphoblastoid cells from FRDA carriers, patients and controls.

Novel antibody-based strategies for the rapid diagnosis of mitochondrial disease and dysfunction.

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).

Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis.

Drug-induced mitochondrial toxicity can occur as a result of inhibition of mitochondrial DNA (mtDNA) replication as with certain nucleoside reverse transcriptase inhibitors or inhibition of mtDNA-encoded protein synthesis as with certain antibacterials. Both types of dysfunction have the overall effect of reducing the level of proteins encoded by mtDNA. A lateral-flow immunoassay which measures the levels of both a mtDNA-encoded protein and a nuclear DNA-encoded protein allows simple and rapid determination of the ratio of these 2 proteins and, hence, identifies changes in mtDNA-encoded protein levels.

Mitochondrial oxidative phosphorylation protein levels in peripheral blood mononuclear cells correlate with levels in subcutaneous adipose tissue within samples differing by HIV and lipoatrophy status.

Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls.

Monitoring oxidative and nitrative modification of cellular proteins; a paradigm for identifying key disease related markers of oxidative stress.

High levels of free radicals produced by the mitochondrial respiratory chain, with subsequent damage to mitochondria have been implicated in a large and growing number of diseases. The underlying pathology of these diseases is oxidative damage to mitochondrial DNA, lipids and proteins which accumulate over time to produce a metabolic deficiency. We are developing an antibody based immunocapture array for many important mitochondrial proteins involved in free radical production, detoxification and mitochondrial energy production.

Lateral-flow immunoassay for the frataxin protein in Friedreich's ataxia patients and carriers.

Friedreich's Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials.

The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11.

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously.

Angiostatin-like activity of a monoclonal antibody to the catalytic subunit of F1F0 ATP synthase.

The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase.

Small-scale immunopurification of cytochrome c oxidase for a high-throughput multiplexing analysis of enzyme activity and amount.

COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membrane-embedded 13-subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments.

Immunological approaches to the characterization and diagnosis of mitochondrial disease.

Monoclonal antibodies (mAbs) are important tools in the diagnosis and characterization of mitochondrial diseases. They can be used in immunohistochemical and/or Western blotting approaches to identify misassembled OXPHOS complexes or pyruvate dehydrogenase deficiencies where the intact complex is not formed which is the great majority of cases. The advantage of antibody based approaches is that they can be quantitative, require very small amounts of tissue sample and are fast, simple and relatively cheap to perform.

Focused proteomics: towards a high throughput monoclonal antibody-based resolution of proteins for diagnosis of mitochondrial diseases.

The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J.

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes.

Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex.

Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates.

The subunit composition of the human NADH dehydrogenase obtained by rapid one-step immunopurification.

Defects of the NADH dehydrogenase complex are predominantly manifested in mitochondrial diseases and are significantly associated with the development of many late onset neurological disorders such as Parkinson's disease. Here we describe an immunocapture procedure for isolating this multisubunit membrane-bound complex from human tissue. Using small amounts of immunoisolated protein, one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass finger printing (PMF), and nanoflow liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), we can resolve and identify the human homologues of 42 polypeptides detected so far in the more extensively studied beef heart complex I.

An immunocytochemical approach to detection of mitochondrial disorders.

Mitochondrial disorders can lead to a confusing array of symptoms, which frequently makes a diagnosis difficult. Traditional approaches to such diagnoses are based on enzyme activity assays, with further characterization provided by genetic analysis. However, these methods require relatively large sample sizes, are time-consuming, labor-intensive, and show variability between laboratories.

A replicating module as the unit of mitochondrial structure and functioning.

The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.

A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies.

Human mitochondrial F(1)F(0) ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F(1) in a 96-well microplate activity assay system, to establish a method for fast high throughput screening of inhibitors, toxins, and drugs with very small amounts of enzyme. For preparative purification, mitochondria from human heart tissue as well as cultured fibroblasts were solubilized with dodecyl-beta-d-maltoside, and the F(1)F(0) was isolated with anti-F(1) monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F(1)F(0) contained a full complement of subunits that were identified with specific antibodies against five of the subunits (alpha, beta, OSCP, d, and IF(1)) and by MALDI-TOF and/or LC/MS/MS for all subunits except subunit c, which could not be resolved by these methods because of the limits of detection.

Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts.

Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency.