Janice K. Parks

Mitochondrial abnormalities in non-alcoholic steatohepatitis

BACKGROUND/AIMSnWe 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.nnnMETHODSnElectron 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.nnnRESULTSnEight 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.nnnCONCLUSIONSnRespiratory 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.

Calcium Homeostasis and Reactive Oxygen Species Production in Cells Transformed by Mitochondria from Individuals with Sporadic Alzheimer’s Disease

Alzheimer’s disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to ρ0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4,5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. β-amyloid (25–35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.

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 1–40u2003Aβ 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. Cyclosporinu2003A 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. Cytochromeu2003c 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 complexu2003IV 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.

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.

Chronic reduction in complex I function alters calcium signaling in SH-SY5Y neuroblastoma cells.

Sporadic, non-familial Parkinsons disease is characterized by a 15-30% reduction in complex I activity of the electron transport chain. A pharmacological model of reduced complex I activity was created by prolonged treatment of SH-SY5Y cells with low doses (5-20 nM) of rotenone, a selective inhibitor of complex I. Short-term (less than 2 week) exposure to rotenone did not influence calcium signaling, production of reactive oxygen species, or mitochondrial morphology. However, following 2 weeks of rotenone exposure, SH-SY5Y cells showed unusual calcium dynamics, specifically multiple calcium responses to carbachol, a muscarinic agonist. These secondary calcium responses were not seen in control SH-SY5Y cells and were dependent upon calcium influx. Mitochondrial membrane potential was also reduced in low dose rotenone-treated cells. These results demonstrate that a chronic, partial reduction in complex I activity, such as that seen in Parkinsons disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Mitochondrial DNA-depleted neuroblastoma (Rho‡) cells exhibit altered calcium signaling

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Mitochondrial Dysfunction in Parkinson’s Disease

Parkinson’s disease (PD, also referred to as sporadic or idiopathic PD and primary parkinsonism) is a progressive motor disorder that occurs in old age and is characterized by resting tremor, rigidity and slowness of movement. It is named after James Parkinson, the physician whose description of PD is one of the early authoritative accounts of the disease(Parkinson 1817). He called it the shaking palsy or paralysis agitans. Earlier references to nthe symptoms of PD may be found in Indian and Egyptian texts (Garcia Ruiz 2004).Parkinson, referring to previous accounts of tremor and palsy by Galen, Sylvius de la Boe, nBoissier de Sauvages and Gaubius, stressed on the distinctive tremor that occurs in the absence of voluntary activity in PD patients (resting tremor) and the tendency of patients to run when attempting to walk (festinating gait). He also described the slowness in initiating nmovement (bradykinesia), freezing (akinesia), postural instability, difficulty in performing complex activities like writing, sleep disturbance and drooling seen in PD. Jean-Martin Charcot (1877) gave PD its present name and added to the description given by Parkinson the symptoms of masked facies, rigidity and the non-motor symptoms (reviewed by Elmer 2005). The secondary non-motor symptoms of PD include autonomic dysfunction, depression and dementia.