W. Davis Parker

Mitochondrial abnormalities in non-alcoholic steatohepatitis

BACKGROUND/AIMS We assessed mitochondrial morphology by electron microscopy and the prevalence of a mitochondrial gene deletion in patients with non-alcoholic steatohepatitis (NASH), alcohol-related liver disease and non-fatty liver diseases. Respiratory chain function using a cytoplasmic hybrid (cybrid) assay was further studied in NASH patients and healthy controls. METHODS Electron microscopy was performed in 26 specimens. Fifteen patients were studied by polymerase chain reaction to detect a 520-bp deletion product of the mitochondrial genome (dmtDNA). Cybrids were created by fusion of platelets with anaerobic neuroblastoma cells in six NASH patients and 12 controls. RESULTS Eight of ten NASH, one of seven alcoholics and two of nine other patients had linear crystalline inclusions in megamitochondria (p<0.05). Three of five patients with alcohol-related liver disease had dmtDNA compared to one of five NASH patients and one of five non-steatohepatitis controls. Cybrid respiratory chain function in platelets was not different from that of controls. CONCLUSIONS Respiratory chain dysfunction, if present in NASH, is not expressed in platelet-derived mitochondria. In contrast to alcohol-related liver disease with active drinking, NASH patients do not commonly express the 5-kb mitochondrial DNA gene deletion in liver tissue. As previously described in early alcohol-related liver disease, crystalline inclusions of unknown composition are seen in hepatic mitochondria in NASH. Their presence suggests either an adaptive process or mitochondrial injury.

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinsons disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.

Complex I deficiency in Parkinson's disease frontal cortex.

A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinsons disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a number of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue preparations. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodological issues are critical in demonstrating this loss of activity.

Neurotoxic Aβ peptides increase oxidative stress in vivo through NMDA-receptor and nitric-oxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro

Beta amyloid (Aβ) peptides accumulate in Alzheimers disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Aβ to increase oxygen radical production in vivo. The 1–40 Aβ fragment increased 2,3‐dehydroxybenzoic acid efflux more than the 1–28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Aβ peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Aβ(25–35) and Aβ(35–25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N‐ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Aβ aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Aβ(25–35) but not by Aβ(35–25). Neurotoxic Aβ peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Aβ levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimers disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed‐forward hypothesis of Aβ neurotoxicity.

Mitochondria in Sporadic Amyotrophic Lateral Sclerosis

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.

Interaction Among Mitochondria, Mitogen‐Activated Protein Kinases, and Nuclear Factor‐κB in Cellular Models of Parkinson's Disease

Abstract: Oxidative stress induced by acute complex I inhibition with 1‐methyl‐4‐phenylpyridinium ion activated biphasically the stress‐activated c‐Jun N‐terminal kinase (JNK) and the early transcription factor nuclear factor‐κB (NF‐κB) in SH‐SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late‐phase JNK activation and the cleavage of signaling proteins Raf‐1 and mitogen‐activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)‐1 appeared to be ANT‐independent. Early NF‐κB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinsons disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF‐κB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF‐κB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf‐1 and cleavage/activation of MEKK‐1, processes reported in other models to be caspase‐mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF‐κB, may offer neuroprotection in this debilitating disease.

High frequency of mitochondrial complex I mutations in Parkinson's disease and aging

Idiopathic Parkinsons disease (PD) involves a systemic loss of activity of complex I of the mitochondrial electron transport chain. This biochemical lesion plays a key pathogenic role. Transfer of PD mitochondrial DNA recapitulates this loss of activity and several other pathogenic features of PD suggesting that this lesion may arise, at least in part, from mitochondrial DNA. We investigated this possibility by an extensive clonal sequencing of the seven mitochondrial genes encoding complex I subunits in PD and age-matched control frontal cortex. Each gene was completely sequenced an average of 94.4 times for each subject. Aminoacid-changing mutations were found at the frequency of 59.3 per million bases in both PD and controls, corresponding to approximately 32% of the mitochondrial genomes in the average sample having at least one mutation in a complex I gene. Individual low frequency mutations had an abundance of 1-10%. Significant interindividual variation in mutation frequency was observed. Several aminoacid-changing mutations were identified and multiple PD brains but not in controls. Genetic algorithm analysis detected areas in ND genes with a higher mutation frequency in PD that allowed differentiation of PD from controls. Total mutational burden due to low-abundance heteroplasmy is high and may play a role in human disease.

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies

Many models of Parkinsons disease (PD) have succeeded in replicating dopaminergic neuron loss or α‐synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long‐term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to α‐synuclein, ubiquitin, parkin, synphilin‐1, neurofilament, β‐tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Creation and Characterization of Mitochondrial DNA-Depleted Cell Lines with “Neuronal-Like” Properties

Abstract: Mitochondrial dysfunction and attendant bioenergetic defects are increasingly recognized as playing an important role in neurodegenerative disorders. The increased attention on mitochondrial involvement points to the need for developing cell lines that have neuron‐like characteristics for the genetic analysis and modeling of these diseases. We describe the creation of respiratory‐deficient SH‐SY5Y neuroblastoma cell lines (ρ064/5) by selectively depleting mitochondrial DNA through prolonged exposure to ethidium bromide. Oxygen consumption in these cells and activities of the electron transport chain enzyme complexes I and IV that contain subunits encoded by the mitochondrial genome are eliminated. In contrast, the function of complex II, a nuclear‐encoded electron transport chain component, is largely intact in these cells. The ρ064/5 cells retain the ability to differentiate into cells with neuron‐like phenotypes following treatment with phorbol ester or retinoic acid. Normal respiratory function is recovered by repopulation of ρ064/5 cells with exogenous human platelet mitochondria. The ρ064/5 cell line serves as a valuable model for the study of neurologic diseases suspected of involving mitochondrial dysfunction.

Mitochondrial Dysfunction in Idiopathic Parkinson Disease

Disordered mitochondrial metabolism may play an important role in a number of idiopathic neurodegenerative disorders. The question of mitochondrial dysfunction is particularly attractive in the case of idiopathic Parkinson disease (PD), since Vyas et al. recognized in the 1980s that the parkinsonism-inducing compound N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is a mitochondrial toxin. The unique genetic properties of mitochondria also make them worthy of consideration for a pathogenic role in PD, as well as in other late-onset, sporadic neurodegenerative disorders. Although affected persons occasionally do provide family histories that suggest Mendelian inheritance, the vast majority of the time these diseases appear sporadically. Because of unique features such as heteroplasmy, replicative segregation, and threshold effects, mitochondrial inheritance can allow for the apparent sporadic nature of these diseases.