Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer's disease

Although oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative diseases such as Alzheimer's disease (AD), it remains unclear how mitochondrial oxidative stress may induce neuronal death. In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage are associated with, and may indeed promote cell death and degeneration. In this review, we examine current evidence supporting the involvement of mitochondria and mitochondrially generated stress signaling in AD and discuss potential implications for the mechanism of pathogenesis of this disease. Mitochondria are pivotal in controlling cell life and death not only by producing ATP, and sequestering calcium, but by also generating free radicals and serving as repositories for proteins which regulate the intrinsic apoptotic pathway. Perturbations in the physiological function of mitochondria inevitably disturb cell function, sensitize cells to neurotoxic insults and may initiate cell death, all significant phenomena in the pathogenesis of a number of neurodegenerative disorders including AD.     

Mitochondria, mitochondrial DNA and Alzheimer's disease. What comes first?

To date, the beta amyloid (Abeta) cascade hypothesis remains the main pathogenetic model of Alzheimer's disease (AD), but its role in the majority of sporadic AD cases is unclear. The mitochondria play central role in the bioenergetics of the cell and apoptotic cell death. In the past 20 years research has been directed at clarifying the involvement of mitochondria and defects in mitochondrial oxidative phosphorylation in late-onset neurodegenerative disorders, including AD. Morphological, biochemical and genetic abnormalities of the mitochondria in several AD tissues have been reported. Impaired mitochondrial respiration, particularly COX deficiency, has been observed in brain, platelets and fibroblasts of AD patients. The "mitochondrial cascade hypothesis" could explain many of the biochemical, genetic and pathological features of sporadic AD. Somatic mutations in mitochondrial DNA (mtDNA) could cause energy failure, increased oxidative stress and accumulation of Abeta, which in a vicious cycle reinforces the mtDNA damage and the oxidative stress. Despite the evidence of mitochondrial dysfunction in AD, no causative mutations in the mtDNA have been detected so far. Indeed, results of studies on the role of mtDNA haplogroups in AD are controversial. In this review we discuss the role of the mitochondria in the cascade of events leading to AD, and we will try to provide an answer to the question "what comes first".     

Human serum transthyretin levels correlate inversely with Alzheimer's disease

Alzheimer's disease (AD) is histopathologically characterized by the presence of senile plaques, neurofibrillary tangles, and synapse loss. The main component of senile plaques is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress in in vitro and in vivo studies. AD is associated with elevated levels of oxidative damage in brain and peripheral lymphocytes. Further Aβ has been found to be accumulated in mitochondria, which might contribute to the reported alterations in the mitochondrial morphology, and impaired mitochondrial energy metabolism in AD brain. Biomarkers are desperately needed for earlier diagnosis of AD and to monitor efficacy of new therapies. Hence, in the present study we show that markers of oxidative damage are elevated in mitochondria isolated from AD lymphocytes suggesting that these oxidative stress indices potentially could serve as a viable biomarker for AD.     

Mitochondrion-specific antioxidants as drug treatments for Alzheimer disease

Age-related dementias such as Alzheimer disease (AD) have been linked to vascular disorders like hypertension, diabetes and atherosclerosis. These risk factors cause ischemia, inflammation, oxidative damage and consequently reperfusion, which is largely due to reactive oxygen species (ROS) that are believed to induce mitochondrial damage. At higher concentrations, ROS can cause cell injury and death which occurs during the aging process, where oxidative stress is incremented due to an accelerated generation of ROS and a gradual decline in cellular antioxidant defense mechanisms. Neuronal mitochondria are especially vulnerable to oxidative stress due to their role in energy supply and use, causing a cascade of debilitating factors such as the production of giant and/or vulnerable young mitochondrion who's DNA has been compromised. Therefore, mitochondria specific antioxidants such as acetyl-L-carnitine and R-alphalipoic acid seem to be potential treatments for AD. They target the factors that damage mitochondria and reverse its effect, thus eliminating the imbalance seen in energy production and amyloid beta oxidation and making these antioxidants very powerful alternate strategies for the treatment of AD.     

Expression of 8-oxoguanine DNA glycosylase is reduced and associated with neurofibrillary tangles in Alzheimer's disease brain

Recent studies have confirmed the role of reactive oxygen species in the pathogenesis of Alzheimer's disease (AD). 8-Oxo-2'-deoxyguanosine accumulation in AD brain has been discussed, but few studies of DNA repair enzymes in AD brain have been done. Further, a relationship between mitochondrial function and oxidative stress has been noticed. In this study, to evaluate the repair mechanism for oxidative DNA damage in AD brain, we investigated brain tissues from autopsy cases of AD and control cases using an antibody against the mitochondrial form of 8-oxoguanine DNA glycosylase (hOGG1-2a), an enzyme that repairs 8-oxo-2'-deoxyguanosine. hOGGI-2a is expressed mainly in the neuronal cytoplasm in both AD and control cases in regionally different manners. Expression of hOGG1-2a is decreased in the orbitofrontal gyrus and entorhinal cortex in AD compared to that in control cases. Immunoreactivity to hOGG1-2a is associated with neurofibrillary tangles, dystrophic neurites and reactive astrocytes in AD. Our results indicate that the repair enzyme for oxidative damage in mitochondrial DNA may not function appropriately in AD, and thus oxidative DNA damage in mitochondria may be involved in the pathomechanism of AD.     

Mitochondria in the aetiology and pathogenesis of Parkinson's disease

Several biochemical abnormalities have been described in the brains of patients with Parkinson's disease (PD), including oxidative stress and mitochondrial dysfunction. The identification of specific gene mutations that cause PD has reinforced the relevance of oxidative stress and mitochondrial dysfunction in the familial and the sporadic forms of the disease. The proteins that are associated with familial PD--PTEN-induced putative kinase 1 (PINK1), DJ-1, alpha-synuclein, leucine-rich repeat kinase 2, and, possibly, parkin--are either mitochondrial proteins or are associated with mitochondria, and all interface with the pathways of oxidative stress and free radical damage. Insights into the aetiology and pathogenesis of PD provide hope that drugs or cocktails of drugs that might successfully intervene in the pathogenesis and slow the progression of the disease can be derived from the study of the converging rather than diverging pathways to cell dysfunction and death.     

Role of vascular hypoperfusion-induced oxidative stress and mitochondria failure in the pathogenesis of Alzheimer disease

Chronic vascular hypoperfusion induces oxidative stress and brain energy failure, and leads to neuronal death, which manifests as cognitive impairment and the development of brain pathology as in Alzheimer disease (AD). It is becoming more widely accepted that AD is characterized by impairments in energy metabolism. We hypothesize that hypoperfusion-induced mitochondrial failure plays a central role in the generation of reactive oxygen species, resulting in oxidative damage to brain cellular compartments, especially in the vascular endothelium and neuronal cell bodies in AD. All of these changes have been found to occur before pathology and coexist during the progression of AD. In this review we have summarized recent evidence and our own knowledge regarding the relationship between the hypoperfusion-induced vascular damage that initiates oxidative stress and mitochondrial abnormalities that appear to be a key target for the development of AD pathology. Future investigations into both the mechanisms behind amyloid beta (Abeta) deposition and the possible accelerating effects of environmental factors, such as chronic hypoxia/reperfusion may open the door for effective pharmacological treatments of AD. We hypothesize that an imbalance between endothelium derived vasoconstrictors and vasodilators, along with an antioxidant system deficiency and mitochondria lesions are prominent in AD. Future studies examining the importance of mitochondrial pathophysiology in different brain cellular compartments may provide insight not only into neurodegenerative and/or cerebrovascular disease pathobiology but may also provide targets for treating these conditions.     

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.     

Modulation of mitochondrial bioenergetics as a therapeutic strategy in Alzheimer's disease

Alzheimer's disease(AD) is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.     

Role of oxidative stress on beta-amyloid neurotoxicity elicited during impairment of energy metabolism in the hippocampus: protection by antioxidants

Age-associated oxidative stress has been implicated in neuronal damage linked with Alzheimer's disease (AD). In addition to the role of beta-amyloid peptide (Abeta) in the pathogenesis of AD, reduced glucose oxidative metabolism and decreased mitochondrial activity have been suggested as associated factors. However, the relationship between Abeta toxicity, metabolic impairment, and oxidative stress is far from being understood. In vivo neurotoxicity of Abeta25-35 peptide has been conflicting. However, in previous studies, we have shown that Abeta25-35 consistently induces synaptic toxicity and neuronal death in the hippocampus in vivo, when administered during moderate glycolytic or mitochondrial inhibition. In the present study, we have investigated whether enhancement of Abeta neurotoxicity during these conditions involves oxidative stress. Results show increased lipoperoxidation (LPO) when Abeta is administered in the hippocampus of rats previously treated with the glycolysis inhibitor, iodoacetate. Neuronal damage and LPO are efficiently prevented by vitamin E, while the spin trapper, alpha-phenyl-N-tert-butyl nitrone, shows partial protection. Abeta stimulates LPO in synaptosomes, but toxicity is only observed in the presence of metabolic inhibitors. Damage and LPO are efficiently prevented by vitamin E. The present results suggest an interaction between oxidative stress and metabolic impairment in the Abeta neurotoxic cascade.     

Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria

Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.     

Mitochondrial Dysfunction and Biogenesis in Neurodegenerative diseases: Pathogenesis and Treatment

Neurodegenerative diseases are a heterogeneous group of disorders that are incurable and characterized by the progressive degeneration of the function and structure of the central nervous system (CNS) for reasons that are not yet understood. Neurodegeneration is the umbrella term for the progressive death of nerve cells and loss of brain tissue. Because of their high energy requirements, neurons are especially vulnerable to injury and death from dysfunctional mitochondria. Widespread damage to mitochondria causes cells to die because they can no longer produce enough energy. Several lines of pathological and physiological evidence reveal that impaired mitochondrial function and dynamics play crucial roles in aging and pathogenesis of neurodegenerative diseases. As mitochondria are the major intracellular organelles that regulate both cell survival and death, they are highly considered as a potential target for pharmacological‐based therapies. The purpose of this review was to present the current status of our knowledge and understanding of the involvement of mitochondrial dysfunction in pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) and the importance of mitochondrial biogenesis as a potential novel therapeutic target for their treatment. Likewise, we highlight a concise overview of the key roles of mitochondrial electron transport chain (ETC.) complexes as well as mitochondrial biogenesis regulators regarding those diseases.     

Mitochondrial aging and dysfunction in Alzheimer's disease

Disruptions in energy metabolism have been suggested to be a prominent feature, perhaps even a fundamental component, of Alzheimer's disease (AD). These abnormalities in cerebral metabolism precede the onset of neurological dysfunction as well as gross neuropathology of AD. These changes may stem from inhibition of mitochondrial enzymes including pyruvate dehydrogenase, cytochrome c oxidase, and alpha-ketoglutarate dehydrogenase. Several lines of evidence also suggest a role for oxidative stress in the neuropathology associated with the disease state. Because mitochondria are the major site of free radical production in cells, they are also a primary target for oxidative damage and subsequent dysfunction. This link between mitochondrial dysfunction and the pathophysiology of AD is supported by several lines of evidence.     

Mitochondrial dysfunction and accumulation of the β-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1^+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1^+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1^+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1^+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.     

Mitochondria and vascular lesions as a central target for the development of Alzheimer's disease and Alzheimer disease-like pathology in transgenic mice

Accumulating evidence strongly suggests that the AD brain is characterized by impairments in energy metabolism, and vascular hypoperfusion, whereby oxidative stress appears to be an especially important contributor to neuronal death and development of AD pathology. We hypothesized that mitochondria play a key role in the generation of reactive oxygen species, resulting in oxidative damage to neuronal cell bodies, as well as other cellular compartments in the AD brain. All of these changes have been found to accompany AD pathology. In this review we have outlined recent evidence from the literature and our own original studies concerning the role of mitochondrial abnormalities and vascular damage in the pathogenesis of AD and AD-like pathology in transgenic mice (as a model for human AD). We examined ultrastructural features of vascular lesions and mitochondria from vascular wall cells in human AD brain biopsies, in human short post-mortem brain tissues and in yeast artificial chromosome (YAC) and C57B6/SJL transgenic positive (Tg+) mice overexpressing amyloid beta precursor protein (A beta PP). In situ hybridization using mitochondrial DNA (mtDNA) probes for human wild type, 5kb deleted and mouse mtDNA was performed along with immunocytochemistry using antibodies against amyloid beta precursor protein (A beta PP), 8-hydroxy-2'-guanosine (8OHG) and cytochrome C oxidase (COX) were studied at the electron microscopic levels. There was a higher degree of amyloid deposition in the vascular walls of the human AD, YAC and C57B6/SJL Tg(+) mice compared to aged-matched controls. In addition, vessels with more severe lesions showed immunopositive staining for APP and possessed large, lipid-laden vacuoles in the cytoplasm of endothelial cells (EC). Significantly more mitochondrial abnormalities were seen in human AD, YAC and C57B6/SJL Tg(+) mouse microvessels where lesions occurred. In situ hybridization using wild and chimera (5 kB) mtDNA probes revealed positive signals in damaged mitochondria from the vascular endothelium and in perivascular cells of lesioned microvessels close to regions of large amyloid deposition. These features were absent in undamaged regions of human AD tissues, YAC and C57B6/SJL Tg(+) mouse tissues and in aged-matched control subjects. In addition, vessels with atherosclerotic lesions revealed endothelium and perivascular cells possessing clusters of wild and deleted mtDNA positive probes. These mtDNA deletions were accompanied by increased amounts of immunoreactive APP, 8OHG and COX in the same cellular compartment. Our observations first time demonstrate that vascular wall cells, especially their mitochondria, appear to be a central target for oxidative stress induced damage.     

Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinson's, Alzheimer's, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FRDA pathogenesis. Brains of FRDA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. This may open up new perspectives in medicine, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms, such as the heat shock response, through nutritional antioxidants, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.     

Targeting the Prodromal Stage of Alzheimer’s Disease: Bioenergetic and Mitochondrial Opportunities

Alzheimer’s disease (AD) has a complex and progressive neurodegenerative phenotype, with hypometabolism and impaired mitochondrial bioenergetics among the earliest pathogenic events. Bioenergetic deficits are well documented in preclinical models of mammalian aging and AD, emerge early in the prodromal phase of AD, and in those at risk for AD. This review discusses the importance of early therapeutic intervention during the prodromal stage that precedes irreversible degeneration in AD. Mechanisms of action for current mitochondrial and bioenergetic therapeutics for AD broadly fall into the following categories: 1) glucose metabolism and substrate supply; 2) mitochondrial enhancers to potentiate energy production; 3) antioxidants to scavenge reactive oxygen species and reduce oxidative damage; 4) candidates that target apoptotic and mitophagy pathways to either remove damaged mitochondria or prevent neuronal death. Thus far, mitochondrial therapeutic strategies have shown promise at the preclinical stage but have had little-to-no success in clinical trials. Lessons learned from preclinical and clinical therapeutic studies are discussed. Understanding the bioenergetic adaptations that occur during aging and AD led us to focus on a systems biology approach that targets the bioenergetic system rather than a single component of this system. Bioenergetic system-level therapeutics personalized to bioenergetic phenotype would target bioenergetic deficits across the prodromal and clinical stages to prevent and delay progression of AD.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0324-8) contains supplementary material, which is available to authorized users.Key Words: Alzheimer’s disease (AD) mitochondria, prodromal, therapeutics, neurodegeneration, bioenergetics