Mouse models of age-related mitochondrial neurosensory hearing loss

Hearing loss is the most common sensory disorder in the elderly population. Overall, 10% of the population has a hearing loss in the US, and this age-related hearing disorder is projected to afflict more than 28 million Americans by 2030. Age-related hearing loss is associated with loss of sensory hair cells (sensory hearing loss) and/or spiral ganglion neurons (neuronal hearing loss) in the cochlea of the inner ear. Many lines of evidence indicate that oxidative stress and associated mitochondrial dysfunction play a central role in age-related neurodegenerative diseases and are a cause of age-related neurosensory hearing loss. Yet, the molecular mechanisms of how oxidative stress and/or mitochondrial dysfunction lead to hearing loss during aging remain unclear, and currently there is no treatment for this age-dependent disorder. Several mouse models of aging and age-related diseases have been linked to age-related mitochondrial neurosensory hearing loss. Evaluation of these animal models has offered basic knowledge of the mechanism underlying hearing loss associated with oxidative stress, mitochondrial dysfunction, and aging. Here we review the evidence that specific mutations in the mitochondrial DNA or nuclear DNA that affect mitochondrial function result in increased oxidative damage and associated loss of sensory hair cells and/or spiral ganglion neurons in the cochlea during aging, thereby causing hearing loss in these mouse models. Future studies comparing these models will provide further insight into fundamental knowledge about the disordered process of hearing and treatments to improve the lives of individuals with communication disorders. This article is part of a Special Issue entitled ‘Mitochondrial function and dysfunction in neurodegeneration’.     

Mitochondria as a therapeutic target for aging and neurodegenerative diseases

Mitochondria are cytoplasmic organelles responsible for life and death. Extensive evidence from animal models, postmortem brain studies of and clinical studies of aging and neurodegenerative diseases suggests that mitochondrial function is defective in aging and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Several lines of research suggest that mitochondrial abnormalities, including defects in oxidative phosphorylation, increased accumulation of mitochondrial DNA defects, impaired calcium influx, accumulation of mutant proteins in mitochondria, and mitochondrial membrane potential dissipation are important cellular changes in both early and late-onset neurodegenerative diseases. Further, emerging evidence suggests that structural changes in mitochondria, including increased mitochondrial fragmentation and decreased mitochondrial fusion, are critical factors associated with mitochondrial dysfunction and cell death in aging and neurodegenerative diseases. This paper discusses research that elucidates features of mitochondria that are associated with cellular dysfunction in aging and neurodegenerative diseases and discusses mitochondrial structural and functional changes, and abnormal mitochondrial dynamics in neurodegenerative diseases. It also outlines mitochondria-targeted therapeutics in neurodegenerative diseases.     

Mitochondrial failures in Alzheimer's disease

Mitochondrial dysfunction and free radical-induced oxidative damage have been implicated in the pathogenesis of several different neurodegenerative diseases such as Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and Alzheimer's disease (AD). The defective adenosine triphosphate (ATP) production and increased oxygen radicals may induce mitochondria-dependent cell death because damaged mitochondria are unable to maintain the energy demands of the cell. The role of vascular hypoperfusion-induced mitochondria failure in the pathogenesis of AD now has been widely accepted. However, the exact cellular mechanisms behind vascular lesions and their relation to oxidative stress markers identified by RNA oxidation, lipid peroxidation, or mitochondrial DNA (mtDNA) deletion remain unknown. Future studies comparing the spectrum of mitochondrial damage and the relationship to oxidative stress-induced damage during the aging process or, more importantly, during the maturation of AD pathology are warranted.     

Oxidative stress, mitochondrial damage and neurodegenerative diseases****

Oxidative stress and mitochondrial damage have been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Oxidative stress is characterized by the overproduction of reactive oxygen species, which can induce mitochondrial DNA mutations, damage the mitochondrial respiratory chain, alter membrane permeability, and influence Ca2＋ homeostasis and mitochondrial defense systems. All these changes are implicated in the development of these neurodegenerative diseases, mediating or amplifying neuronal dysfunction and triggering neurodegeneration. This paper summarizes the contribution of oxidative stress and mitochondrial damage to the onset of neurodegenerative diseases and discusses strategies to modify mitochondrial dysfunction that may be attractive therapeutic interventions for the treatment of various neurodegenerative diseases.     

Mitochondrial Medicine for Aging and Neurodegenerative Diseases

Mitochondria are key cytoplasmic organelles, responsible for generating cellular energy, regulating intracellular calcium levels, altering the reduction-oxidation potential of cells, and regulating cell death. Increasing evidence suggests that mitochondria play a central role in aging and in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Freidriech ataxia. Further, several lines of evidence suggest that mitochondrial dysfunction is an early event in most late-onset neurodegenerative diseases. Biochemical and animal model studies of inherited neurodegenerative diseases have revealed that mutant proteins of these diseases are associated with mitochondria. Mutant proteins are reported to block the transport of nuclear-encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins and disrupt the electron transport chain, induce free radicals, cause mitochondrial dysfunction, and, ultimately, damage neurons. This article discusses critical issues of mitochondria causing dysfunction in aging and neurodegenerative diseases, and discusses the potential of developing mitochondrial medicine, particularly mitochondrially targeted antioxidants, to treat aging and neurodegenerative diseases.     

Mitochondrial DNA deletions in Alzheimer's brains: A review

Mitochondrial dysfunction and increased oxidative stress have been associated with normal aging and are possibly implicated in the etiology of late-onset Alzheimer's disease (AD). DNA deletions, as well as other alterations, can result from oxidative damage to nucleic acids. Many studies during the past two decades have investigated the incidence of mitochondrial DNA deletions in postmortem brain tissues of late-onset AD patients compared with age-matched normal control subjects. Published studies are not entirely concordant, but their differences might shed light on the heterogeneity of AD itself. Our understanding of the role that mitochondrial DNA deletions play in disease progression may provide valuable information that could someday lead to a treatment.     

New insights into progressive supranuclear palsy

Increased oxidative damage and mitochondrial dysfunction have been suggested to play crucial roles in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. In this review, we will focus on progressive supranuclear palsy (PSP), a rare parkinsonian disorder with tau pathology. Particular emphasis is placed on the genetic and biochemical data that has emerged, offering new perspectives into the pathogenesis of this devastating disease, especially the contributory roles of oxidative damage and mitochondrial dysfunction.     

Oxidative stress treatment for clinical trials in neurodegenerative diseases

Oxidative stress is a metabolic condition arising from imbalance between the production of potentially reactive oxygen species and the scavenging activities. Mitochondria are the main providers but also the main scavengers of cell oxidative stress. The role of mitochondrial dysfunction and oxidative stress in the pathogenesis of neurodegenerative diseases is well documented. Therefore, therapeutic approaches targeting mitochondrial dysfunction and oxidative damage hold great promise in neurodegenerative diseases. Despite this evidence, human experience with antioxidant neuroprotectants has generally been negative with regards to the clinical progress of disease, with unclear results in biochemical assays. Here we review the antioxidant approaches performed so far in neurodegenerative diseases and the future challenges in modern medicine.     

The mitochondrial impairment, oxidative stress and neurodegeneration connection: reality or just an attractive hypothesis?

Aging is the most important risk factor for common neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Aging in the central nervous system has been associated with elevated mutation load in mitochondrial DNA, defects in mitochondrial respiration and increased oxidative damage. These observations support a 'vicious cycle' theory which states that there is a feedback mechanism connecting these events in aging and age-associated neurodegeneration. Despite being an extremely attractive hypothesis, the bulk of the evidence supporting the mitochondrial vicious cycle model comes from pharmacological experiments in which the modes of mitochondrial enzyme inhibition are far from those observed in real life. Furthermore, recent in vivo evidence does not support this model. In this review, we focus on the relationship among the components of the putative vicious cycle, with particular emphasis on the role of mitochondrial defects on oxidative stress.     

Detection of DNA base-excision repair activity for oxidative lesions in adult rat brain mitochondria

Endogenous oxidative damage to brain mitochondrial DNA and consequential disturbances of gene expression and mitochondrial dysfunction have long been implicated in aging and the pathogenesis of neurodegenerative diseases. It has yet to be determined, however, whether mitochondria in brain cells contain an active DNA repair system and, if so, how this system functions. Therefore, the capacity for the repair of defined types of oxidative DNA lesions has been investigated in adult rat brain mitochondria. Using in vitro DNA incorporation repair assay, we have detected base excision repair (BER) activity for the common oxidative DNA adduct 8-hydroxyl-2'-deoxyguanine (8-oxodG) in mitochondria protein extracts from cortical tissues and cultured primary cortical neurons and astrocytes. The levels of BER activity were both protein concentration-dependent and repair-incubation time-dependent. To resolve the BER pathway, the activity of essential BER enzymes was examined in mitochondria using oligonucleotide incision assay, DNA polymerase assay, and DNA ligase assay employing specific DNA substrates. Mitochondrial extracts were able to remove specifically 8-oxodG, uracil, and the apurinic/apyrimidinic abasic site from substrates. Moreover, a gamma-like DNA polymerase activity and a DNA ligase activity were detected in mitochondiral extracts, based on the formation of specific repair products. These results demonstrate that adult brain mitochondria possess an active BER system for repairing oxidative DNA lesions. This repair system appears to function by sequential actions of DNA repair enzymes that are homologous to, but not identical to, that in the nucleus. Thus, BER may represent an endogenous protective mechanism against oxidative damage to mitochondrial, as well as nuclear, genomes in brain cells.     

Aging,energy, and oxidative stress in neurodegenerative diseases

The etiology of neurodegenerative diseases remains enigmatic; however, evidence for defects in energy metabolism, excitotoxicity, and for oxidative damage is increasingly compelling. It is likely that there is a complex interplay between these mechanisms. A defect in energy metabolism may lead to neuronal depolarization, activation of N-methyl-D-aspartate excitatory amino acid-receptors, and increases in intracellular calcium, which are buffered by mitochondria. Mitochondria are the major intracellular source of free radicals, and increased mitochondrial calcium concentrations enhance free radical generation. Mitochondrial DNA is particularly susceptible to oxidative stress, and there is evidence of age-dependent damage and deterioration of respiratory enzyme activities with normal aging. This may contribute to the delayed onset and age dependence of neurodegenerative diseases. There is evidence for increased oxidative damage to macromolecules in amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Potential therapeutic approaches include glutamate release inhibitors, excitatory amino acid antagonists, strategies to improve mitochondrial function, free radical scavengers, and trophic factors. All of these approaches appear promising in experimental studies and are now being applied to human studies.     

Bcl-2 facilitates recovery from DNA damage after oxidative stress.

Oxidative stress is a major factor affecting the brain during aging and neurodegenerative diseases such as Alzheimer's disease (AD). Understanding the mechanisms by which neurons can be protected from oxidative stress, therefore, is critical for the prevention and treatment of such degeneration. Previous studies have shown that bcl-2 expression is increased in neurons with DNA damage in AD and bcl-2 has an antioxidant effect. The goal of this study is to document the effects of oxidative insults on mitochondrial and nuclear DNA in PC12 cells and determine the extent to which bcl-2 prevents damage or facilitates repair. Using extralong PCR to amplify nuclear and mitochondrial DNA, the time course of DNA damage and repair was determined. Within minutes after exposure of cells to low concentrations of hydrogen peroxide and peroxynitrite, significant mitochondrial and nuclear DNA damage was evident. Mitochondrial DNA was damaged to a greater degree than nuclear DNA. Expression of bcl-2 in PC12 cells inhibited nitric oxide donor (sodium nitroprusside)- and peroxynitrite-induced cell death. Although oxidative insults caused both genomic and mitochondrial DNA damage in cells expressing bcl-2, recovery from DNA damage was accelerated in these cells. These results suggest that neuronal up-regulation of bcl-2 may facilitate DNA repair after oxidative stress.     

The oxidative damage theory of aging

The oxidative stress theory of aging postulates that age-associated reductions in physiologic functions are caused by a slow steady accumulation of oxidative damage to macromolecules, which increases with age and which is associated with life expectancy of organisms. A corollary is that the rate of aging should be retarded by attenuation of oxidative damage. A large body of evidence has accumulated in support of this hypothesis. Increases in oxidative damage to DNA, proteins, and lipids have all been found with normal aging. Genetic manipulation of oxidative damage can increase life expectancy in animals. Overexpression of Cu/Zn superoxide dismutase or manganese superoxide dismutase appears to extend life span. Overexpression of methionine sulfoxide reductase in Drosophila resulted in a 70% increase in survival, and a 50% reduction in methionine sulfoxide reductase in mice resulted in a 30% reduction in life span. Caloric restriction, which extends life span, also reduces oxidative stress. Manipulation of gene expression in Drosophila with phenylbutyrate significantly increases lifespan, and is associated with a 50-fold increase in expression of manganese superoxide dismutase. We recently further examined the mitochondrial DNA theory of aging, which proposes that mitochondrial DNA accumulates mutations with age and that these contribute to the physiological decline in aging. Using a PCR-cloning-sequencing strategy, we found a significant increase in aggregate burden of mitochondrial DNA point mutations in brain in elderly subjects compared to younger subjects. The average aggregate mutational burden in elderly subjects was 2×10⁻⁴ mutations per base. The bulk of these mutations were individually rare point mutations, and 60% changed an amino acid. Cytochrome oxidase activity correlated negatively with increased mutational burden. These findings bolster the possibility that oxidative damage to mitochondrial DNA may play a significant role in normal aging.     

Mitochondrial abnormalities in muscle and other aging cells: classification,causes, and effects

The involvement of mitochondria and of mitochondrial DNA (mtDNA) in the aging process has generated much interest and even more controversy. The mitochondrial theory of aging considers a vicious circle consisting of: (1) accumulation of somatic mtDNA mutations; (2) impairment of respiratory chain function; (3) increased production of reactive oxygen species (ROS) in mitochondria; and (4) further damage to mtDNA. We review the evidence for and against the belief that these steps occur in aging muscle and brain, considering separately morphological, biochemical, and molecular data. The relationship between mitochondrial aging and late-onset neurodegenerative diseases is briefly reviewed. We conclude that mitochondrial dysfunction does play a crucial role in the aging process of both muscle and brain, but it remains unclear whether mitochondria are the culprits or mere accomplices.     

Mitochondrial membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain

Oxidative damage on biological molecules has been proposed as a major cause of alterations observed in aging brain as well as in neurodegenerative diseases. In this study, we measured membrane fluidity in mitochondria extracted from three cerebral regions and cerebellum of Alzheimer disease (AD) patients and age-matched controls by means of fluorescence polarization technique. A significant reduction of mitochondrial membrane fluidity was found in AD, except in cerebellum. In controls, a decrease of membrane fluidity was observed along with age, and it was also related to the content of the oxidized nucleoside 8-hydroxy-2′-deoxyguanosine (OH⁸dG) in mitochondrial DNA (mtDNA). Alteration in membrane fluidity seems to be a result of lipid peroxidation, since it dramatically decreased when mitochondria were exposed to FeCl₂ and H₂O₂. The parallel increase of viscosity in mitochondrial membrane and the amount of OH⁸dG in mtDNA is suggestive of a relationship between these biological markers of oxidative stress. These results provide further evidence that oxidative stress may play a role in the pathogenesis of AD.     

Oxidative damage to mitochondrial DNA is increased in Alzheimer's disease

Oxidative damage to DNA may play a role in both normal aging and in neurodegenerative diseases. We examined whether Alzheimer's disease (AD) is associated with increased oxidative damage to both nDNA and mtDNA in postmortem brain tissue. We measured the oxidized nucleoside, 8-hydroxy-2′-deoxyguanosine (OH⁸dG), in DNA isolated from three regions of cerebral cortex and cerebellum in 13 AD and 13 age-matched controls. There was a significant threefold increase in the amount of OH⁸dG in mtDNA in parietal cortex of AD patients compared with controls. In the entire group of samples there was a small significant increase in oxidative damage to nDNA and a highly significant threefold increase in oxidative damage to mtDNA in AD compared with age-matched controls. These results confirm that mitochondrial DNA is particularly sensitive to oxidative damage, and they show that there is increased oxidative damage to DNA in AD, which may contribute to the neurodegenerative process.     

Mitochondrial DNA mutations increase in early stage Alzheimer disease and are inconsistent with oxidative damage

Mitochondrial dysfunction and oxidative damage are commonly associated with early stage Alzheimer disease (AD). The accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been hypothesized to be a driver of these phenotypes, but the detection of increased mutation loads has been difficult due to a lack of sensitive methods. We used an ultrasensitive next generation sequencing technique to measure the mutation load of the entire mitochondrial genome. Here, we report a significant increase in the mtDNA mutation frequency in the hippocampus of early stage AD, with the cause of these mutations being consistent with replication errors and not oxidative damage. Ann Neurol 2016;80:301–306