James A. Dykens

Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization

OBJECTIVEnIncreased chondrocyte nitric oxide (NO) and peroxynitrite production appears to modulate decreased matrix synthesis and increased mineralization in osteoarthritis (OA). Because NO inhibits mitochondrial respiration, this study was undertaken to directly assess the potential role of chondrocyte mitochondrial oxidative phosphorylation (OXPHOS) in matrix synthesis and mineralization.nnnMETHODSnWe studied cultured human articular chondrocytes and immortalized costal chondrocytes (TC28 cells). We also assessed the effects of antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) on chondrocyte mitochondrial respiration, ATP synthesis, and inorganic pyrophosphate (PPi) generation, and the mineralizing potential of released matrix vesicles (MV).nnnRESULTSnArticular chondrocytes and TC28 cells respired at comparable rates. Peroxynitrite and NO donors markedly suppressed respiration and ATP generation in chondrocytes. Because NO exerts multiple effects on chondrocytes, we investigated the primary functions of mitochondrial respiration and OXPHOS. To do so, we identified minimally cytotoxic doses of antimycin and oligomycin, which both induced intracellular ATP depletion (by 50-80%), attenuated collagen and proteoglycan synthesis, and blocked transforming growth factor beta from increasing intracellular ATP and elaboration of PPi, a critical inhibitor of hydroxyapatite deposition. Antimycin and oligomycin also abrogated the ability of the ATP-hydrolyzing enzyme plasma cell membrane glycoprotein 1 (PC-1) to increase chondrocyte PPi generation. Finally, MV from cells treated with antimycin or oligomycin contained less PPi and precipitated >50% more 45Ca.nnnCONCLUSIONnChondrocyte mitochondrial reserve, as NO-sensitive mitochondrial respiration-mediated ATP production, appears to support matrix synthesis and PPi elaboration and to regulate MV composition and mineralizing activity. NO-induced depression of chondrocyte respiration could modulate matrix loss and secondary cartilage mineralization in OA.

Development of 17α-estradiol as a neuroprotective therapeutic agent : Rationale and results from a phase I clinical study

Abstract: 17α‐estradiol (17α‐E2) differs from its isomer, the potent feminizing hormone 17β‐estradiol (17β‐E2), only in the stereochemistry at one carbon, but this is sufficient to render it at least 200‐fold less active as a transactivating hormone. Despite its meager hormonal activity, 17α‐E2 is as potent as 17β‐E2 in protecting a wide variety of cell types, including primary neurons, from a diverse array of lethal and etiologically relevant stressors, including amyloid toxicity, serum withdrawal, oxidative stress, excitotoxicity, and mitochondrial inhibition, among others. Moreover, both estradiol isomers have shown efficacy in animal models of stroke, Alzheimers disease (AD), and Parkinsons disease (PD). Data from many labs have yielded a mechanistic model in which 17α‐E2 intercalates into cell membranes, where it terminates lipid peroxidation chain reactions, thereby preserving membrane integrity, and where it in turn is redox cycled by glutathione or by NADPH through enzymatic coupling. Maintaining membrane integrity is critical to mitochondrial function, where loss of impermeability of the inner membrane initiates both necrotic and apoptotic pathways. Thus, by serving as a mitoprotectant, 17α‐E2 forestalls cell death and could correspondingly provide therapeutic benefit in a host of degenerative diseases, including AD, PD, Friedreichs ataxia, and amyotrophic lateral sclerosis, while at the same time circumventing the common adverse effects elicited by more hormonally active analogues. Positive safety and pharmacokinetic data from a successful phase I clinical study with oral 17α‐E2 (sodium sulfate conjugate) are presented here, and several options for its future clinical assessment are discussed.

High-throughput assessment of mitochondrial membrane potential in situ using fluorescence resonance energy transfer.

Mitochondrial dysfunction causes dozens of debilitating diseases, and is implicated in the etiology of type 2 diabetes, Parkinsons, and Alzheimers diseases, among others. However, development of mitochondrially targeted therapeutic agents has been impeded by the lack of high-throughput screening techniques that are capable of distinguishing in intact cells the mitochondrial membrane potential (deltapsi(m)) from the plasma membrane potential, (deltapsi(p)). We report here a fluorescence resonance energy transfer (FRET) assay that specifically monitors deltapsi(m) that is not confounded by background signal arising from potentiometric dye responding to deltapsi(p). The technique relies on energy transfer between nonyl acridine orange (NAO), which stains diphosphatidyl glycerol (cardiolipin) that is indigenous to the inner mitochondrial membrane, and tetramethylrhodamine methyl ester (TMR), a potentiometric dye that is sequestered by mitochondria as a Nernstian function of deltapsi(m) and concentration. FRET occurs only when both dyes co-localize to the mitochondria, and results in quenching of NAO emission by TMR in proportion to deltapsi(m). Validation studies using compounds with well-characterized mitochondrial effects, including oligomycin, CCCP+, bongkrekic acid, cyclosporin A, nigericin, ADP, and ruthenium red, demonstrate that the FRET-based deltapsi(m) assay responds in accord with the known pharmacology. Validation studies assessing the suitability of the technique for high-throughput compound screening indicate that the assay provides a sensitive and robust assessment not only of mitochondrial integrity in situ, but also, when used in conjunction with agents such as cyclosporin A, an indicator of permeability transition.

Introduction to mitochondrial function and genomics

In virtually all plant and animal cells, mitochondria are the primary providers of energy but also are the major producers of free radicals and important inducers of programmed cell death pathways. As such, mitochondria are crucial to the proper growth and functioning of the cell, but they also play fundamental roles in numerous pathologic conditions when they become dysfunctional. Mitochondria contain their own DNA, but because they are solely inherited maternally, contain multiple copies of the genome, and replicate independently of cell division, mitochondrial genetics is more akin to population genetics than the Mendelian genetics characteristic of the nuclear genome. Oxidation reactions in the core of the mitochondria yield electrons that are passed sequentially through three respiratory complexes in the inner mitochondrial membrane to generate potential energy for ATP formation. Although numerous diseases are associated with well‐defined mutations in the mitochondrial genome, the etiology of the dysfunction in the respiratory complexes characteristic of Alzheimers and Parkinsons diseases (defects in Complexes IV and I, respectively) is still under investigation. Regardless of underlying cause, improving mitochondrial function represents a novel therapeutic strategy in late‐onset, sporadically occurring degenerative diseases such as Alzheimers. Moreover, elucidating mechanisms that contribute to mitochondrial dysfunction will provide avenues for development of better therapeutic and diagnostic tools for these diseases. Drug Dev. Res. 46:2–13, 1999.

Mitochondrial Drugs Come of Age

Keywords: n nbioenergetics; ndiagnostics; ndisease; ndrugs; nefficacy; ngenetics; nmetabolism; nmitochondria; ntoxicity