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Pergamon 0197-4580(94)00088-3 Neurobtology of Aging, Vol. 15. Suppl. 2, pp. 171-174, 1994 Copyright ~ 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0197-4580/94 $6.00 + .00 Energy, Oxidative Damage, and Alzheimer's Disease: Clues to the Underlying Puzzle M. FLINT BEAL Neurochemistry Laboratory, Warren 408, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 THE ETIOLOGY of cell death in Alzheimer's disease (AD) is unknown. One possibility that has been implicated in several neu- rodegenerative diseases is that a defect in energy metabolism may lead to slow excitotoxic neuronal degeneration. The possibility of a defect in energy metabolism in AD has been strengthened by biochemical studies in both peripheral tissues and in the central nervous system that show reduced cytochrome oxidase activity. A consequence of a defect at the level of the electron transport chain is increased generation of free radicals. Consistent with this, we recently found a threefold increase in oxidative damage to mito- chondrial DNA in AD postmortem brain tissue. These observa- tions raise a number of issues for future investigation. Further- more, they suggest several strategies for therapeutic interventions in AD. EFFECTS OF AGING ON MITOCHONDRIAL ENERGY METABOLISM The most important risk factor for AD is advancing age. The incidence and prevalence of AD increase steeply with age after age 60, increasing to as much as 47% of patients over age 85 in one study (19). One theory to account for age-dependent onset of degenerative diseases such as Alzheimer's disease is that mito- chondrial dysfunction may hasten neuronal death (37,40,69). It has been proposed that the accumulation of mitochondrial genome mutations during life results in a progressive impairment of oxi- dative phosphorylation. The rate of mutations in mitochondrial DNA is about 10 times greater than that in chromosomal DNA (38). A high rate of mutation has been suggested by extensive restriction fragment polymorphism among individual human be- ings (6). Furthermore, mutations in mitochondrial DNA are more likely to have functional consequences because mitochondrial DNA has no noncoding sequences, except for a small segment involved in the replication of mitochondrial DNA. There also ap- pear to be limited repair mechanisms for mitochondrial DNA (9). Mitochondrial DNA may be particularly susceptible to damage due to its lack of protective histories and its close proximity to the inner mitochondrial membrane where reactive oxygen species are generated (37,40,69). Recent studies have demonstrated the presence of an age- dependent deletion between nucleotide positions 8470 and 13459 of the mitochondrial genome (13,37,58). In the heart, the deletion has been detected in individuals starting at age 30 and increases exponentially with advancing age (12,25). A recent quantitative study showed that the deletion was estimated at 3% and 9% in patients of ages 80 and 90, respectively (65). Furthermore, dele- tions are much more frequent in patients with ischemic heart dis- ease (12). We and others have recently shown that there are marked increases in the deletion in human postmortem brain tissue with normal aging (11,61). This finding is consistent with the suggestion that patients with defects in oxidative phosphorylation generate increased amounts of oxygen free radicals, which result in mitochondrial DNA damage (38). Mitochondrial oxidative phosphorylation generates most of the free radicals in the cell, and mitochondrial DNA is particularly susceptible to oxidative damage (51). The respiratory chain components that make the greatest contribution to production of free radicals are ubiquinone and cy- tochrome b566 of complex III (43). An increase in production of superoxide radicals by cytochrome b566 occurs with normal aging (44). Recent studies show that 8-hydroxy-2-deoxyguanosine is a bi- omarker of oxidative DNA damage (56). Of 13 base adducts formed after exposing purified mammalian chromatin to ionizing radiation-generated free radicals, 8-hydroxy-2-deoxyguanosine is the most frequent (16). Several studies indicate that 8-hydroxy-2- deoxyguanosine most frequently codes correctly for cytosine, but also has the monospecific mutagenic ability to pair with adenine about 1% of the time (8,35,55,70). It also results in misreading at adjacent residues (35). lnhibitors of the electron transport chain increase the amount of 8-hydroxy-2-deoxyguanosine in mitochon- drial DNA (29). Concentrations of 8-hydroxy-2-deoxyguanosine increase with normal aging in several rat tissues and in mitochon- drial DNA isolated from human diaphragm and heart muscle (20, 28,30). In heart muscle, the amount of mitochondrial deletions correlates with 8-hydroxy-2-deoxyguanosine concentrations in mi- tochondrial DNA (28). We recently examined 8-hydroxy-2-deoxyguanosine concen- trations in both nuclear and mitochondrial DNA from postmortem brain tissue of 10 normal humans aged 42 to 97 years (39). The amount of 8-hydroxy-2-deoxyguanosine increased progressively with normal aging in both nuclear and mitochondrial DNA; how- ever, the rate of increase with age was much greater in mitochon- drial DNA. There was a significant 10-fold increase in the amount of 8-hydroxy-2-deoxyguanosine in mitochondrial DNA as com- pared with nuclear DNA in the entire group of samples, and a 15-fold significant increase in patients older than 70 years. These results, therefore, show that there is a progressive accumulation of oxidative damage to DNA with normal aging, consistent with free radical theories of aging. Several studies have shown a reduction in capacity for oxida- tive phosphorylation with normal aging in both animal and human tissues. In aged rat brain, variable results have been obtained, but the activity of complex IV appears to reduced (14). In rat brain, there are age-dependent decreases in mitochondrial respiration with complex I substrates but not with complex II substrates (24). S171

Energy, oxidative damage, and Alzheimer's disease: Clues to the underlying puzzle

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0197-4580(94)00088-3

Neurobtology of Aging, Vol. 15. Suppl. 2, pp. 171-174, 1994 Copyright ~ 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

0197-4580/94 $6.00 + .00

Energy, Oxidative Damage, and Alzheimer's Disease: Clues to the Underlying Puzzle

M. FLINT BEAL

Neurochemistry Laboratory, Warren 408, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

THE ETIOLOGY of cell death in Alzheimer's disease (AD) is unknown. One possibility that has been implicated in several neu- rodegenerative diseases is that a defect in energy metabolism may lead to slow excitotoxic neuronal degeneration. The possibility of a defect in energy metabolism in AD has been strengthened by biochemical studies in both peripheral tissues and in the central nervous system that show reduced cytochrome oxidase activity. A consequence of a defect at the level of the electron transport chain is increased generation of free radicals. Consistent with this, we recently found a threefold increase in oxidative damage to mito- chondrial DNA in AD postmortem brain tissue. These observa- tions raise a number of issues for future investigation. Further- more, they suggest several strategies for therapeutic interventions in AD.

EFFECTS OF AGING ON MITOCHONDRIAL ENERGY METABOLISM

The most important risk factor for AD is advancing age. The incidence and prevalence of AD increase steeply with age after age 60, increasing to as much as 47% of patients over age 85 in one study (19). One theory to account for age-dependent onset of degenerative diseases such as Alzheimer's disease is that mito- chondrial dysfunction may hasten neuronal death (37,40,69). It has been proposed that the accumulation of mitochondrial genome mutations during life results in a progressive impairment of oxi- dative phosphorylation. The rate of mutations in mitochondrial DNA is about 10 times greater than that in chromosomal DNA (38). A high rate of mutation has been suggested by extensive restriction fragment polymorphism among individual human be- ings (6). Furthermore, mutations in mitochondrial DNA are more likely to have functional consequences because mitochondrial DNA has no noncoding sequences, except for a small segment involved in the replication of mitochondrial DNA. There also ap- pear to be limited repair mechanisms for mitochondrial DNA (9). Mitochondrial DNA may be particularly susceptible to damage due to its lack of protective histories and its close proximity to the inner mitochondrial membrane where reactive oxygen species are generated (37,40,69).

Recent studies have demonstrated the presence of an age- dependent deletion between nucleotide positions 8470 and 13459 of the mitochondrial genome (13,37,58). In the heart, the deletion has been detected in individuals starting at age 30 and increases exponentially with advancing age (12,25). A recent quantitative study showed that the deletion was estimated at 3% and 9% in patients of ages 80 and 90, respectively (65). Furthermore, dele- tions are much more frequent in patients with ischemic heart dis- ease (12). We and others have recently shown that there are

marked increases in the deletion in human postmortem brain tissue with normal aging (11,61). This finding is consistent with the suggestion that patients with defects in oxidative phosphorylation generate increased amounts of oxygen free radicals, which result in mitochondrial DNA damage (38). Mitochondrial oxidative phosphorylation generates most of the free radicals in the cell, and mitochondrial DNA is particularly susceptible to oxidative damage (51). The respiratory chain components that make the greatest contribution to production of free radicals are ubiquinone and cy- tochrome b566 of complex III (43). An increase in production of superoxide radicals by cytochrome b566 occurs with normal aging (44).

Recent studies show that 8-hydroxy-2-deoxyguanosine is a bi- omarker of oxidative DNA damage (56). Of 13 base adducts formed after exposing purified mammalian chromatin to ionizing radiation-generated free radicals, 8-hydroxy-2-deoxyguanosine is the most frequent (16). Several studies indicate that 8-hydroxy-2- deoxyguanosine most frequently codes correctly for cytosine, but also has the monospecific mutagenic ability to pair with adenine about 1% of the time (8,35,55,70). It also results in misreading at adjacent residues (35). lnhibitors of the electron transport chain increase the amount of 8-hydroxy-2-deoxyguanosine in mitochon- drial DNA (29). Concentrations of 8-hydroxy-2-deoxyguanosine increase with normal aging in several rat tissues and in mitochon- drial DNA isolated from human diaphragm and heart muscle (20, 28,30). In heart muscle, the amount of mitochondrial deletions correlates with 8-hydroxy-2-deoxyguanosine concentrations in mi- tochondrial DNA (28).

We recently examined 8-hydroxy-2-deoxyguanosine concen- trations in both nuclear and mitochondrial DNA from postmortem brain tissue of 10 normal humans aged 42 to 97 years (39). The amount of 8-hydroxy-2-deoxyguanosine increased progressively with normal aging in both nuclear and mitochondrial DNA; how- ever, the rate of increase with age was much greater in mitochon- drial DNA. There was a significant 10-fold increase in the amount of 8-hydroxy-2-deoxyguanosine in mitochondrial DNA as com- pared with nuclear DNA in the entire group of samples, and a 15-fold significant increase in patients older than 70 years. These results, therefore, show that there is a progressive accumulation of oxidative damage to DNA with normal aging, consistent with free radical theories of aging.

Several studies have shown a reduction in capacity for oxida- tive phosphorylation with normal aging in both animal and human tissues. In aged rat brain, variable results have been obtained, but the activity of complex IV appears to reduced (14). In rat brain, there are age-dependent decreases in mitochondrial respiration with complex I substrates but not with complex II substrates (24).

S171

S172 BEAL

A study of aged rat muscle showed the activities of complex I and complex IV were reduced, but the activity of complex II-III was unchanged (66). The numbers of cytochrome-c oxidase-deficient cardiomyocytes in the human heart increases with aging (41). A study of muscle biopsies in 29 subjects between ages 16 and 92 showed a significant negative correlation between activated mito- chondrial respiration rates and age with all substrates tested (67). Similar observations were made in human liver biopsy samples (71). In a recent study of human muscle biopsies, the activities of complex I and complex IV were decreased, while complex II-III activity was unaffected (10). The decrease in activity in skeletal muscle is not associated with decreases in respiratory chain protein content, suggesting that it is not due to impaired mitochondrial DNA transcription or translation (7). Metabolic studies of oxygen utilization in human skeletal muscle and brain have also confirmed a decline with normal aging (2,45). The results of prior studies indicate that complex I and complex IV in brain and muscle may be particularly vulnerable to age-dependent decreases in activity, while complex II-III activity is relatively preserved. In our stud- ies, we examined the effects of aging on activity of the electron transport chain in cerebral cortex of primates across the entire age span of the species (4). We found that there was a progressive decrease in complex I and complex IV activity in cerebral cortex with normal aging; however, complex II and complex V activities were spared. The finding of an age-related decline in complex I and complex IV activities is consistent with damage to mitochon- drial DNA because it encodes seven subunits of complex I and three subunits of complex IV, whereas complex II is encoded exclusively by nuclear DNA (69).

EVIDENCE FOR IMPAIRED ENERGY METABOLISM IN ALZHEIMER'S DISEASE

The largest body of evidence suggesting an impairment of en- ergy metabolism in AD has come from studies of glucose metab- olism using positron emission tomography. The major difficulty with these studies is determining whether alterations play a role in the disease process or are merely secondary to neuronal loss. Stud- ies of cerebral blood flow, oxygen utilization, and glucose metab- olism show consistent decreases in AD in a temporo-parietal pat- tern (17,26,27). Comparing patients with early and more advanced dementia, it was shown that a substantial decrease in cerebral glucose metabolism may precede cognitive impairment. Several reports show reduced glucose transport in microvessels of AD patients (33); however, this does not appear to be sufficient to account for the decrease in glucose metabolism (33).

Neurochemical studies have also suggested that energy metab- olism may be impaired in AD. Sims and colleagues studied cere- bral biopsies and found that the adenylate energy charge was un- changed, but oxygen uptake was significantly increased under conditions of submaximal metabolic activity, consistent with un- coupling of mitochondrial energy metabolism (59).

Electronmicroscopic studies of cortical biopsies in AD show abnormal mitochondria with increased matrix density and paracrystalline inclusions in the intercristal space (53). This find- ing is of interest due to the observation of paracrystalline inclu- sions in the mitochondria of patients with known mitochondrial diseases. Mitochondrial dysfunction is also suggested by studies showing 70%-100% reductions in activity of the thiamine- dependent mitochondrial enzymes pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase (22,62). Parker and colleagues re- ported a deficiency in cytochrome oxidase (complex IV) activity in mitochondria isolated from AD platelets (46). This was disputed in a study using platelet mitochondria obtained with less rigorous purification procedures, but has been confirmed recently by Parker in a second study (47,68). A recent study showed reduced cy- tochrome oxidase activity in several cortical regions in AD post-

mortem brain tissue (34). We and others have confirmed this result (42.48). This finding appears to be relatively specific because no abnormalities in activity of the other electron transport enzymes were found (42). Furthermore, one study found that cytochrome aa 3 levels were normal, while cytochrome oxidase activity was reduced (48). Cytochrome aa 3 is an integral part of cytochrome oxidase. This argues that the reduced activity is likely to represent an abnormality in catalytic activity rather than a decrease in the amount of enzyme. Paradoxically, however, an increase in ex- pression of cytochrome oxidase subunit 3 mRNA was reported in AD brain (1). An increase in expression could occur as a com- pensatory response to an electron transport defect.

The role of point mutations in mitochondrial DNA in Alzhei- mer's disease has recently been examined. It was reported that point mutations in subunit 2 of NADH dehydrogenase may be associated with AD (36). This point mutation, however, was sub- sequently reported to be a normal polymorphism (50,57). Shoffner and colleagues, however, found three point mutations in mito- chondrial DNA of patients with either AD, Parkinson's disease (PD), or AD with PD (57). The most frequent was a tRNA Gtn gene mutation found in 5.2% of patients but only 0.7% of Caucasian controls. An NDI point mutation in an evolutionarily highly con- served region was found in two unrelated patients who had AD with PD. A third mutation was an insertion in the 12s rRNA gene. The significance of these mutations remains to be clarified, but they may be analogous to the situation in Leber's disease in which some mutations are high risk factors for the illness while others increase one's risk but are not sufficent by themselves to cause the illness (5).

EVIDENCE FOR OXIDATIVE DAMAGE IN All)

A consequence of impaired mitochondrial function may be the generation of free radicals. Increases in protein oxidation products (carbonyl groups) occur in aged controls and AD brain samples as compared with young controls (60). The activity of glutamine synthetase is particularly susceptible to free radical damage. There is a loss of enzyme activity with normal aging in gerbil brain. In AD, there is a significant decrease in activity in the frontal pole but not in the occipital pole as compared with age-matched controls (60). This finding has been interpreted as supporting increased oxidative damage in AD. There is also evidence that lipid perox- idation is increased in AD cerebral cortex (23,64).

FUTURE DIRECTIONS

We and others have shown that an impairment of energy me- tabolism can lead to neuronal cell death by secondary excitotoxic mechanisms (3). In addition, a consequence of impaired mito- chondrial function is the generation of free radicals. Consistent with this we recently found a threefold significant increase in oxidative damage to mitochondrial DNA in AD postmortem brain tissue as compared with age-matched controls (Mecoeci et al., unpublished data). Oxidative damage may play a role in the patho- genesis of AD by leading to aggregation of amyloid and by dam- aging cytoskeletal proteins such as tau (18,67). Amyloid itself may be capable of generating free radicals (31) and of impairing mitochondrial function (54). Interest in the role of oxidative dam- age in neurodegenerative diseases has recently increased following the finding of point mutations in superoxide dismutase in patients with familial ALS (15,52). An impairment of energy metabolism may also lead to increased production by I3-amyloid. Treatment of cultured neurons with sodium azide leads to the generation of amyloidogenic fragments (21). Ischemic lesions are also associ- ated with increased expression of the amyloid precursor protein (63).

Based on these observations, there are several directions for future research in AD. Further work is needed to determine the

ENERGY, OXIDATIVE DAMAGE, AND AD S173

relationship of amyloid production to impairments of mitochon- drial function in vivo. Further studies are also needed to determine whether there is other evidence of increased oxidative damage to cellular constituents in AD postmortem brain tissue. It would be of interest to determine whether oxidative stress can accelerate amy- loid deposition in transgenic mice overexpressing the amyloid pre- cursor protein. A major area for research will be molecular genetic studies of mitochondrial DNA in AD. The most convincing evi- dence would be the finding of an association of AD with a mito- chondrial point mutation that was maternally transmitted through a large pedigree. If mitochondrial point mutations are found in AD, they will then need to be screened for in large populations of patients and controls.

If a defect in energy metabolism is determined to be either a risk factor or a direct pathogenetic factor in AD, this will have major implications for therapy. It would suggest several therapeu-

tic strategies that may be useful either alone or in combination. These would be to (a) attempt to improve mitochondrial function, (b) to block either glutamate release or excitatory amino acid re- ceptors in an attempt to ameliorate secondary excitotoxic neuronal injury, and (c) to use antioxidants to treat oxidative damage that may be a consequence of mitochondrial dysfunction. Future re- search will, therefore, be needed to assess the efficacy of these strategies in experimental animals and to develop improved phar- macological agents. Another focus for future research will be to develop improved methods for assessing oxidative stress and mon- itoring antioxidative therapies in man.

ACKNOWLEDGEMENT

This work was supported by NIH Grants POIAGII337 and P50AG05134.

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