Aberrant mitochondrial RNA in the role of aging and aging associated diseases.

Accumulations of mitochondrial DNA (mtDNA) mutations associated with aging are evident in multiple human tissues. The role of mtDNA mutations can be observed in an aging animal model such as homozygous knock-in PolgA mice, which have a large colonial expansion of mtDNA mutations. They develop reduced lifespan and premature onset of age-related phenotypes, that are also observed in clinical practice like mitochondrial aging acceleration with anti-retroviral therapy through clonal expansion of mtDNA mutations. These clonally expanded mtDNA mutations maintain transcription ability which could result in an accumulation of abnormal mitochondrial RNA (mtRNA) in the affected cells. Compensation-effect doctrine states that accumulated mtDNA mutations in the cell must reach a set threshold before they have a negative effect on cell function due to compensation effects from normal cellular mtDNA. In contrast to this theory, we suggest that an accumulation of aberrant mtRNA transcribed from mtDNA mutations negatively influences cellular function through complex internal and external mitochondrial pathways, and might be an important cause of aging and aging-associated diseases.

Polymorphic variation in cytochrome oxidase subunit genes.

Cytochrome oxidase (COX) activity varies between individuals and low activities associate with Alzheimer's disease. Whether genetic heterogeneity influences function of this multimeric enzyme is unknown. To explore this we sequenced three mitochondrial DNA (mtDNA) and ten nuclear COX subunit genes from at least 50 individuals. 20% had non-synonymous mtDNA COX gene polymorphisms, 12% had a COX4I1 non-synonymous G to A transition, and other genes rarely contained non-synonymous polymorphisms. Frequent untranslated region (UTR) polymorphisms were seen in COX6A1, COX6B1, COX6C, and COX7A1; heterogeneity in a COX7A1 5' UTR Sp1 site was extensive. Synonymous polymorphisms were common and less frequent in the more conserved COX1 than the less conserved COX3, suggesting at least in mtDNA synonymous polymorphisms experience selection pressure and are not functionally silent. Compound gene variations occurred within individuals. To test whether variations could have functional consequences, we studied the COX4I1 G to A transition and an AGCCCC deletion in the COX7A1 5' UTR Sp1 site. Cells expressing the COX4I1 polymorphism had reduced COX Vmax activity. In reporter construct-transduced cells where green fluorescent protein expression depended on the COX7A1 Sp1 site, AGCCCC deletion reduced fluorescence. Our findings indicate COX subunit gene heterogeneity is pervasive and may mediate COX functional variation.

Effects of memantine on mitochondrial function.

Because NMDA complex and mitochondrial function are related, we hypothesized memantine would influence mitochondrial function. We addressed this in vitro by studying the effects of chronic and acute memantine exposures on mitochondrial function. For acute exposure experiments, mitochondria were isolated from NT2 cells and assayed for electron transport chain (ETC) enzyme function and peroxide production in buffers containing up to 60uM memantine. For chronic exposure experiments, NT2 cells were maintained for at least two weeks in medium containing up to 60uM memantine, following which we assayed cells or their mitochondria for ETC enzyme activities, cytochrome oxidase protein levels, oxidative stress, calcium levels, and mitochondrial DNA levels. The ability of the NMDA receptor antagonist aminophosphonovaleric acid (APV) to modify memantine's mitochondrial effects was evaluated. Acute and chronic memantine similarly affected complex I (increased at high concentrations) and IV (decreased at high concentrations) V(max) activities. APV did not alter the effects of chronic memantine exposure on citrate synthase and complex IV. We detected a lower mitochondrial peroxide production rate with acute exposure, and an increased mitochondrial peroxide production rate with chronic exposure. Micromolar memantine concentrations affect mitochondria, some of these effects are directly mediated, and acute and chronic effects may differ.

Complex I deficiency in Parkinson's disease frontal cortex.

A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's 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.

Genetic algorithm for analysis of mutations in Parkinson's disease.

OBJECTIVE: Mitochondrial genetics has unique features that impede analysis of the biological significance of mitochondrial mutations. Simple searches for differences in total mutational load between normal and pathological samples have been frequently unrewarding, raising the possibility that more complex patterns of mutations may be responsible for some conditions. We explore this possibility in the context of Parkinson's disease (PD). METHODS AND MATERIALS: We report the development of a modified genetic algorithm suited for detection of biologically meaningful patterns of mitochondrial mutations. The algorithm is applied to a database of mutations derived from biological samples, and verified by the use of shuffled data, and repeated leave-one-out testing. RESULTS: It is possible to derive, from a very small sample, multiple accurate classifier functions that correlate with biological features. The methodology is validated statistically through experiments with fabricated data. CONCLUSION: This algorithm might be generally applicable to conditions where interactions among multiple mitochondrial DNA mutations are important. The patterns embodied in the classifier functions obtained should be the subject of further experimental study.

Mitochondrial ND5 mutations in idiopathic Parkinson's disease.

Idiopathic Parkinson's disease (PD) is characterized by a systemic loss of activity of complex I (NADH:ubiquinone oxidoreductase), the target enzyme of the parkinsonism producing neurotoxin, MPTP. Cybrid experiments strongly suggest that the loss of complex I activity arises from mitochondrial DNA. We prospectively evaluated low frequency, amino acid changing, heteroplasmic mutations in a narrow region of ND5, a mitochondrial gene encoding a complex I subunit, in brain tissue from PD and controls. The presence or absence of amino acid changing mutations correctly classified 15 of 16 samples. Heteroplasmic mutations in a specific region of ND5 largely segregate PD from controls and may be of major pathogenic importance in idiopathic PD.

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

Idiopathic Parkinson's 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 Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-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 alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-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.

Mitochondrial abnormalities in cybrid cell models of sporadic Alzheimer's disease worsen with passage in culture.

We created and studied new cybrid cell lines from sporadic Alzheimer's disease (SAD) or control (CTL) subjects to assess mitochondrial abnormalities just after metabolic selection ("early passage") and again six passages later ("late passage"). Cytochrome oxidase (CO) activities in early passage SAD cybrids created independently from the same platelet samples were highly correlated. Early passage SAD and CTL cybrids showed equivalent mitochondrial morphologies. Late passage SAD cybrids showed increased mitochondrial number, reduced mitochondrial size, and an approximately eightfold increase in morphologically abnormal mitochondria. Deficiency of SAD cybrid mitochondrial membrane potentials (DeltaPsi(M)) increased with passage. Mitochondrial bromodeoxyuridine (BrdU) uptake to estimate mitochondrial DNA (mtDNA) synthesis did not change with passage in CTL but increased in SAD cybrids. With time in culture, SAD mtDNA appears to replicate faster in cybrids, yielding cells with relative worsening of bioenergetic function. Metabolically deleterious SAD mitochondrial genes, like those in yeast, may have a replicative advantage over nondeleterious mitochondrial genes that assume dominance in CTL cybrids.