Mitochondrial dysfunction in sporadic and genetic Alzheimer's disease

Experimental Gerontology 41 (2006) 668–673 www.elsevier.com/locate/expgero

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Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease Susanne Hauptmann a,*, Uta Keil a, Isabel Scherping a, Astrid Bonert a, Anne Eckert a,b, Walter E. Mu¨ller a a

Department of Pharmacology Biocentre, University of Frankfurt, 60439 Frankfurt, Germany Neurobiology Research Laboratory, Psychiatric University Clinic, 4025 Basel, Switzerland

b

Received 8 November 2005; received in revised form 15 March 2006; accepted 17 March 2006 Available online 4 May 2006

Abstract Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of many common age-related neurodegenerative diseases, including Alzheimer’s disease (AD). AD is the most common neurodegenerative disorder characterized by dementia, memory loss, neuronal apoptosis and eventually death of the affected individuals. AD is characterized by two pathologic hallmark lesions that consist of extracellular plaques of amyloid-b peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated microtubular protein tau. Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer’s disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein and tau alterations in this type of dementia remains controversial. q 2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Mitochondrial dysfunction; b-Amyloid; Neurofibrillary tangles

1. Alzheimer’s disease Mitochondria, increasingly implicated as sensors and executioners in the cell’s decision to live or die, are involved in the pathogenesis of most neurodegenerative diseases including Alzheimer’s disease (AD), considered as the most common age-related neurodegenerative disorder. The clinical symptoms of AD include a progressive loss of memory and impairment of cognitive ability. The AD brain is marked by severe neurodegenerative alterations, such as the loss of synapses and neurons, atrophy and the selective depletion of neurotransmitter systems (e.g. acetylcholine) in the hippocampus and cerebral cortex. Such defects are mainly observed in the late stage of the disease, and have also been partially demonstrated using transgenic animal models of AD. AD can be classified into sporadic AD, which is by far the most common form and where aging itself is the only important risk factor known, and the familial form (FAD), that represents only a small fraction of all AD cases and typically patients present with ages onset of younger than 65 years, showing autosomal dominant transmission within affected families. To date, * Corresponding author. Tel.: C49 69 79829380; fax: C49 69 79829374. E-mail address: [email protected] (S. Hauptmann).

0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.03.012

mutations in the following genes have been described to be causative for FAD–presenilin 1 gene on chromosome 14, presenilin 2 gene on chromosome 1 and amyloid precursor protein gene on chromosome 21 (Goate et al., 1991; Sherrington et al., 1995; Rogaev et al., 1995). Patients with either sporadic AD or FAD share common clinical and neuropathological features, including the two major histopathological hallmarks, extracellular plaques and intracellular neurofibrillary tangles (NFT). The extracellular plaques are composed of amyloid b peptide (Ab), which is derived from the amyloid precursor protein (APP) through an initial b-secretase cleavage followed by an intramembraneous cut of the presenilin 1 (PS1)dependent g-secretase complex. As mentioned above, autosomal dominant forms associated with FAD are caused by mutations in genes encoding for APP, presenilin 1 and presenilin 2. Among the mutations in APP associated with early-onset familial AD are mutations in amino acid 717 of APP (London mutation) close to the g-secretase cleavage site that lead to increased production of Ab1–42 (Scheuner et al., 1996). Moreover, a double mutation in codons 670 and 671 of the APP gene on chromosome 21 has been found in a Swedish family with early-onset familial AD. This mutation results in increased production of Ab1–40 and Ab1–42 because b-secretase cleavage is enhanced (Citron et al., 1992). Remarkably, all of the more than 60 FAD mutations, including all PS1 and PS2

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mutations, apparently result in the overproduction of Ab, further providing evidence that Ab plays a crucial role in the pathogenesis of AD. However, there is some evidence, that some PS1 mutations might directly and independently of Ab disturb neuronal and mitochondrial function. Interestingly, we observed increased mitochondrial ROS formation and a hypersensitivity to cell death in transgenic mice bearing the M146L mutation in the PS1 gene (Schuessel et al., 2006) (Fig. 1). Neurofibrillary tangles are intracellular filamentous inclusions that are composed of hyperphosphorylated forms of the microtubule-associated tau protein. These filamentous inclusions accumulate in selective neurons in brain of individuals with AD, but they also occur in other neurodegenerative disorders, including the frontal temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and Pick’s disease. During the last decade, several hypotheses have been set forth to explain the pathophysiology of AD. Currently, the main hypothesis concerning the origin of the disease is based on neurotoxic effects of amyloid beta leading to disruption of calcium homeostasis, energy failure, induction of oxidative stress, mitochondrial and consequently synaptic dysfunction and most recently to hyperphosphorylation of tau protein. Despite the great diversity of numerous conflicting results in the literature that originate from the comparison of studies performed in different experimental conditions and the difficulties to work with Ab solutions, the mechanism underlying AD and the events responsible for its progression

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remain unclear. Therefore the main challenge in AD research is to understand the relationship between these different observations. 2. Effects of Ab peptide on mitochondria There are many links between mitochondrial dysfunction and Alzheimer’s disease. Early defects in glucose utilization in the brains of AD Patients suggest possible abnormalities in mitochondrial function in AD (Hoyer, 2000; Blass et al., 2002). Several studies have indicated that Ab may be directly toxic to isolated mitochondria. Interestingly, the activities of those enzymes, which are reduced in the brains of AD patients, such as COX, a-ketoglutarate dehydrogenase and pyruvate dehydrogenase, were inhibited by Ab (Casley et al., 2002). In support of this possibility, a range of reports has shown altered mitochondrial properties in the brain and peripheral tissues of AD patients. Recently, it has been demonstrated that Ab can be formed intracellularly in neurons (Petanceska et al., 2000). A central controversy surrounds the identity of the toxic form of Ab in vivo and whether extracellular Ab deposition or intracellular Ab overproduction precedes the disease process. According to the ‘amyloid hypothesis’, extracellular accumulations of Ab in the brain are the primary influence driving AD pathogenesis. Furthermore, the formation of neurofibrillary tangles containing tau protein, is proposed to result from an imbalance between Ab production and Ab clearance. Although the initial ‘amyloid cascade hypothesis’ (Selkoe, 1997) offers a broad framework to explain AD pathogenesis, it is currently

Fig. 1. Mitochondrial dysfunction as an early common pathological pathway of aging, tau pathology and other unknown riskfactors of sporadic AD as well as PS1 and APP mutations in genetic AD. Aging and PS1-mutations may in addition directly impair mitochondrial function independently of the Ab cascade. Amyloid precursor protein (APP), Presenilin 1 (PS1), reactive oxygen species (ROS).

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lacking in detail, and certain observations do not fit easily with the simplest version of the hypothesis, such as observations of synaptic and neuronal dysfunction appearing before senile plaque and tangle pathology (de la Torre, 2004). In contrast the so-called ‘intracellular hypothesis’ suggests that neuronal dysfunction and degeneration could be caused by an intraneuronal gradual accumulation of Ab rather than by an extracellular process (Fernandez-Vizarra et al., 2004). Some recent studies support this hypothesis, such as generation of Ab within neurons, accumulation of Ab inside these neurons (Fernandez-Vizarra et al., 2004) and the release of accumulated Ab by neuronal lysis to form the senile plaque (D’Andrea et al., 2001). The relevance of very early synaptic and mitochondrial alterations, long before plaque formation, has been acknowledged by the modified ‘ amyloid cascade hypothesis’ (Selkoe, 2002). A large amount of evidence suggests that mitochondria could intervene in the mechanism by which intraneuronal Ab triggers neuronal dysfunction and degeneration. This is supported by the in vivo demonstration of Ab accumulation within mitochondria in brains from AD patients (FernandezVizarra et al., 2004; Lustbader et al., 2004) and the observation of mitochondrial structural abnormalities (Hirai et al., 2001). In addition, impairment of mitochondrial oxidative phosphorylation was also extensively reported in the brain of AD patients (Chagnon et al., 1995) as well as that the degree of impairment being proportional to the clinical disability (Blass, 2003). Further observations revealed a deficient mitochondrial respiration with a complex IV (cytochrome c oxidase) enzymatic defect. Studies with cybrid cells containing mitochondrial DNA from sporadic AD patients show characteristics compatible with the disease, including increased secretion and intracellular accumulation of Ab, decreased mitochondrial membrane potential, oxidative stress, decreased cytochrome c oxidase activity, and altered mitochondrial morphology (Trimmer et al., 2000; Cardoso et al., 2004). Although, these observations suggest the possibility of a direct link between mitochondrial function and AD, the exact mechanism of Ab-mediated mitochondrial dysfunction, potentially contributing to neuronal perturbation, have yet to be clarified. In addition studies with cybrids were criticized because the results indicating a possible link between a particular haplotype and AD are limited by the number of subjects analyzed (Schon et al., 1998). Aleardi et al. (2005) proposed several in vitro mechanisms of Ab induced mitochondrial toxicity. They suppose that the primary mechanism for mitochondrial toxicity at low concentrations might be the membrane destabilizing property of Ab, which has been demonstrated for many tissues (Mueller et al., 2001). Furthermore, they observed a further effect of Ab on mitochondrial function which was a direct inhibition of respiratory chain complexes, followed by a potentializing effect of Ab on mitochondrial H2O2 production. The fourth and last mode of action of Ab on mitochondrial function observed by their study is a release of cytochrome c, at high Ab concentrations (Aleardi et al., 2005). Caspersen et al. recently confirmed these observations with the in vivo analysis of a

transgenic mice model of AD (Caspersen et al., 2005), showing for instance that Ab progressively accumulates in mitochondria and that Ab is associated with diminished enzymatic activity of respiratory chain complexes. Studies in cell culture demonstrated a role for proapoptotic Bax in Ab-induced apoptosis, mediated via Bax-induced increased permeability of the mitochondrial outer membrane, followed by caspase activation and cell death (Zhang et al., 2002). This indicates that mitochondria remain important suspects as pathogenic contributors to Ab-mediated cellular dysfunction. In our own studies, we could clearly demonstrate that Ab causes oxidative stress and mitochondrial malfunction in a cell culture model (Keil et al., 2004). In particular, Ab has been shown to generate free radicals in vitro (Henlsey et al., 1994) and to reduce mitochondrial respiration through inhibition of cytochrome c oxidase activity (Keil et al., 2004). To further elucidate the biochemical pathways affected by Ab, we carried out studies on PC12 cells and HEK cells bearing the Swedish APP double mutation. This mutation results in increased Ab production compared to wild-type APP (APPwt) bearing cells. Our studies evidenced significantly increased NO levels, diminished cytochrome c oxidase activity and reduced ATP levels compared to APPwt and control cells. Expression of APPsw rendered PC12 cells vulnerable to the induction of cell death after exposure to oxidative stress (Eckert et al., 2001; Leutz et al., 2002). In addition, in PC12 cells bearing APPsw, caspase 3 activity was significantly elevated compared to APPwt and vector-transfected control cells after oxidative stress (Eckert et al., 2001). Another important fact are our observations that the JNK pathway is enhanced in APPsw cells as indicated by an attenuation of apoptosis through protection of mitochondrial function by SP600125, a JNK inhibitor (Marques et al., 2003). Concluding, Ab seems to cause defects in mitochondrial energy metabolism, associated with increased production of ROS. This might lead to release of apoptogenic factors from mitochondria, such as cytochrome c and SMAC followed by the activation of caspases and apoptotic cell death. Nevertheless it has to be mentioned, that PC12 cells are tumorderived cell lines, which have a modified energy metabolism. Therefore, the results obtained by using PC12-cells have to be considered carefully. Therefore, we continued our studies with 3-month-old APP transgenic mice, based on amyloid protein precursor mutations similar to those found in human familial AD (APP; Swedish and London mutation), and non-tg littermate control animals (non-tg). In accordance with the findings in PC12 cells, we observed a reduction in ATP levels and a drop of mitochondrial membrane potential in brain cells of these mice (Keil et al., 2004). In addition, 3-month-old APP transgenic mice show increased levels of 4-hydroxynonenal (HNE), which is a marker for lipid peroxidation. These increased HNE levels were accompanied by reduced activity of Cu/Zn-superoxide dismutase. This suggests that impaired antioxidants defence is causally responsible for increased formation of HNE (Schuessel et al., 2005).

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Using isolated mitochondria from 3-month-old APP transgenic (tg) mice and non-tg littermate control animals (non-tg) we could detect decreased basal levels of mitochondrial membrane potential in tgAPP mice compared to littermate non-tg control mice (unpublished data), similar to the decrease in dissociated cells. Our data suppose that the increased Ab production by APP transgenic mice might trigger the dysfunction of the mitochondrial respiratory chain. In addition, hydrogen peroxide, the nitric oxide donor sodium nitroprusside and extracellular Ab damaged the isolated mitochondria significantly by decreasing the mitochondrial transmembrane potential in littermate non-tg control mice but not in tgAPP mice (unpublished data). Most probably, this is due to the preliminary insult caused by the chronic Ab exposure. Moreover, we observed a significant decrease in state 3 respiration and again a significant decrease of respiration after uncoupling with FCCP in non-tg control mice after treatment with extracellular Ab. Most interestingly, we did not detect any differences between the mitochondrial respiratory chain using 6-month-old littermate compared to tgAPP transgenic mice (unpublished data). We suppose that this subtle phenotype is probably due to compensatory effects mediated by the other respiratory chain enzymes and maybe effects appear later. Our findings are in good agreement with a recent study by Caspersen et al., using isolated mitochondria of Tg mAPP mice. This group observed comparable oxygen consumption at 4 months in Tg mAPP and non-Tg littermates, a trend toward lower levels in Tg mAPP mice at 8 months that achieved statistical significance at an age of 12 months (Caspersen et al., 2005). Furthermore beside the reduction in the rate of oxygen consumption, they observed diminished enzymatic activity of respiratory chain complexes III and IV from Tg mAPP and non-Tg littermates at 8 months of age. Taken together, our data facilitate the intracellular hypothesis since we demonstrated that mitochondrial defects such as decreased mitochondrial potential and ATP levels in 3 months old transgenic mice, which have increased intracellular Ab load but no Ab plaques (Blanchard et al., 2003) Our observations are also in accordance with a recent study performed with isolated rat mitochondria showing an Ab concentration dependent inhibition of respiratory chain complexes. In addition, the authors could demonstrate an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O (Aleardi et al., 2005). Cytochrome c oxidase, a-ketoglutarate dehydrogenase and pyruvate activities were also diminished in isolated mitochondria incubated with Ab (Casley et al., 2002). Increasing evidence suggests that Ab accumulates in mitochondria. Lustbader et al. (2004) demonstrated that Abbinding alcohol dehydrogenase (ABAD) is a direct molecular link between Ab and mitochondrial toxicity. They found that Ab interacts with ABAD in the mitochondria of AD patients and APP transgenic mice. In the context of mitochondria, there are several mechanisms through which Ab might accumulate. One possibility is that severely damaged mitochondria passively accumulate Ab. Another possibility is that APP is processed in mitochondria

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leading to mitochondrial accumulation of Ab. Recently, Anandatheerthavarada et al. (2003) studied the relationship between mutant APP and mitochondrial dysfunction in neuronal cells of Tg2576 mice. They demonstrated that APP can accumulate in mitochondrial membranes. Another potential mechanism for mitochondrial accumulation of Ab might be the import of Ab into mitochondria. 3. Effects of tau on mitochondria The appearance of neurofibrillary tangles, primarily composed of aggregated hyperphosphorylated tau protein within specific neuronal populations, is a known neuropathological feature in several diseases known as tauopathies, such as Alzheimer’s disease. Tau proteins are a group of neuronal microtubule-associated proteins that are formed by alternative mRNA splicing. Tau is a phosphoprotein and its phosphorylation negatively regulates its ability to stimulate microtubule assembly. Further on, tau is the major component of the paired helical filaments (PHFs) that make up the neurofibrillary tangles (NFTs) in AD brains. Because the pathological diagnosis of AD is dependent upon NFTs and the brain areas affected by NFTs correlate with disease progression, it is widely assumed that NFTs are central mediators of AD pathogenesis. Subsequently, it was reported that microtubule assembly in brain extracts from AD cases is impaired and that the hyperphosphorylation of tau may contribute to this deficit (Iqbal et al., 1986). The fact, that some mouse models of AD show impairment of cognitive functions prior to observation of NFTs, leads to the assumption, that NFTs might only play a role in the late stage of the disease. Little is known about the distinct intracellular mechanism underlying the consequences of tau pathology. Increasing evidence highlights a connection between AD and mitochondrial dysfunction together with a deregulation of energy metabolism and oxidative stress. Furthermore some datas suggest, that tau plays a key role in the pathogenic cascades. For example, neurons from tau-knockout mice are resistant to Ab-induced neurotoxicity (Rapoport et al., 2002) and the expression of pseudophosphorylated tau constructs in cells is toxic (Fath et al., 2002). In addition PHF-tau is usually assumed to be a neurotoxic agent and several mechanisms have been suggested for its role in neurodegeneration. It has been demonstrated, that phosphorylated tau inhibits microtubule assembly and causes the disassembly of microtubules (Alonso et al., 2001). Furthermore PHF-tau is also thought to compromise microtubule stability and function, resulting in a loss or decline in axonal or dendritic transport in AD disease (Salehi et al., 2003). Using transgenic mice overexpressing the P301L mutant human tau protein, we could demonstrate mitochondrial dysfunction by proteomic and functional analyses in these mice (David et al., 2005). The P301L transgenic mice express the human pathogenic mutation P301L of tau together with the longest human brain tau isoform under control of the neuronspecific mThy1.2 promoter (Gotz et al., 2001a). This isoform

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contains exons 2 and 3 as well as four microtubule-binding repeats. P301L tau transgenic mice show tau hyperphosphorylation already at 3 months and NFT formation starts at 6 months of age (Gotz et al., 2001a,b). Our functional analysis demonstrated reduced NADHubiquinone oxidoreductase (complex I) activity and, with age, impaired mitochondrial respiration and ATP synthesis in P301L tau mice. In particular, the reduction in state 3 respiration reflects a reduced capacity of mitochondria to metabolize oxygen and the complex I substrates in the presence of a limited quantity of ADP (David et al., 2005). Mitochondrial dysfunction was also associated with higher levels of reactive oxygen species in aged transgenic mice. Furthermore, P301L tau mitochondria displayed increased vulnerability towards Ab peptide insult, suggesting a synergistic action of tau and Ab pathology on the mitochondria. Together, this evidence supports a role of tau pathology in mitochondrial and metabolic dysfunction. However, it remains unclear how tau accumulation mediates these changes. Although, it is uncertain exactly how tau mechanistically affects mitochondrial function, we can postulate that the mutant tau acts either indirect by modifying microtubule stability, axonal transport and mitochondrial network or direct by inhibiting energy production through complex I of the respiratory chain. 4. Summary Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer’s disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein and tau alterations in this type of dementia remains controversial. Although, it remains inconclusive whether Ab or tau initiates AD pathology, a vast array of evidence has demonstrated that pathologically altered Ab metabolism plays an essential role in the aging-AD continuum. In the ‘intracellular hypothesis’ of Ab, a large amount of evidence suggests that mitochondria could intervene in the mechanism by which intraneuronal Ab triggers neuronal dysfunction and degeneration. Several studies raised the possibility that intraneuronal accumulation of Ab in mitochondrial membranes could impair organellar functions and participate in the physiopathology of AD. Besides the amyloid-cascade hypothesis suggests that the accumulation of Ab in specific brain regions (hippocampus, and cerebral cortex) is the primary pathogenic process, which triggers a cascade of various physiological events such as microglial and astrocytic activation, oxidative damage, formation of tau pathology, synaptic loss and progressive cognitive decline (Hardy and Selkoe, 2002). The most compelling observation providing a direct link between AD pathogenesis and Ab abnormalities is the discovery of the genetic mutations that are causative of familial AD. In addition, the gene, which encodes tau is not genetically linked to AD, but tau mutations cause FTDP-17 and neuropathological investigations of AD brains have indicated

that filamtous tau aggregates are more closely related to neuronal loss than Ab plaques (Cummings and Cotman, 1995). The identification of disease-causing mutations in tau establishes that tau dysfunction suffices to cause neurodegeneration. The lack of genetic association to AD, however, further corroborates the evidence that tau lies downstream of Ab in the neurodegenerative cascade. This does not imply that tau pathology is irrelevant or innocuous in the pathogenesis of AD, because neurodegeneration induced by tau dysfunction might have a pivotal role in AD. The role of tau and Ab together in the pathogenesis has proven difficult to unravel, in part because of unanticipated challenges of reproducing both pathologic hallmarks in transgenic mice. One very interesting approach used by Lewis et al., (2001) demonstrated that double transgenic mice, transgenic for mutant APP and mutant tau, developed enhanced neurofibrillary pathology compared with singlemutant tau mice. In addition it was demonstrated that the intracranial administration of Ab into mutant tau mice led to the generation of tangles within the amygdala (Gotz et al., 2001b). These observations suggest an interaction between Ab and tau, although the mechanism underlying this effect is unknown. Moreover, it might be important to know, in which concentrations Ab and tau are toxic to mitochondria. This is still a matter of debate, because it is difficult to measure. Caspersen et al. gave an indication of how much Ab accumulates in mitochondria of a transgenic mice model of AD. In 12-month-old mice levels of Ab1–42 had reached w2800 pg/mg of mitochondrial protein. This accumulation of Ab in the mitochondrion was accompanied by a decrease of the respiratory control ratio. Thus, accumulation of Ab within mitochondria correlates with changes in mitochondrial function. On the basis of these findings together with our own observations we suppose a hypothetical sequence of events linking AD, Ab production and tau pathology with mitochondrial dysfunction. Ab as well as tau pathology lead to reduced mitochondrial membrane potential, including significantly reduced complex IV activity in APP transgenic mice and significantly reduced complex I activity in P301L tau transgenic mice. Nevertheless, both reduced activities lead to reduced ATP levels, followed by enhanced ROS production. When the inhibition of mitochondrial function has reached a phenotypic threshold and severe energy deprivation appears, mitochondrial and synaptic dysfunction could appear. A similar explanation was already mentioned in the article by Aleardi et al. They proposed that when the inhibition of mitochondrial respiratory chain has reached a phenotypic threshold, the sudden drop in energy production could lead to neuronal dysfunction and impairment of cognitive capacities (Aleardi et al., 2005). Mitochondria are essential for neuronal function, because the limited glycolytic capacity of these cells makes them highly dependent on aerobic oxidative phosphorylation for their energy needs. In the end, this whole vicious cycle lead to enhanced apoptosis (unpublished data; David et al., 2005).

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