0733-8619 /98 $8.00 THE NEUROLOGY OF AGING + .OO AGE-RELATED NEURODEGENERATION AND OXIDATIVE STRESS Putative Nutritional Intervention James A. Jose...

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AGE-RELATED NEURODEGENERATION AND OXIDATIVE STRESS Putative Nutritional Intervention James A. Joseph, PhD, Natalie Denisova, PhD, Derek Fisher, BS, Barbara Shukitt-Hale, PhD, Paula Bickford, PhD, Ronald Prior, PhD, and Giohua Cao, PhD

It is rare to see a day pass in which we are not told through some popular medium that the US population is becoming older. Along with this information comes the revelation that as we enter the next millennium there will be increases in age-associated diseases (e.g., cancer and cardiovascular disease) including the most devastating of these which involve the nervous system (i.e.,Alzheimer’s and Parkinson’s diseases). By the year 2050, fully 30% of the total population will be over 65 years of age and there is a high probability that they will be exhibiting the most common correlative motor and cognitive behavioral changes that occur in aging. Notably, these changes occur even in the absence of specific age-related neurodegenerative diseases but could interact to exacerbate the behavioral aberrations exhibited in these conditions. The alterations in motor function may include decreases in balance, muscle strength, and ~oordination,3~,~~ whereas memory deficits appear to occur primarily in secondary memory systems and are reflected in the retrieval of newly acquired information. Indeed, these characterizations have been supported by a great deal of research both in animal^^,^*,^ and human^.^,^^ It should be evident that in cases of severe deficits in memory or motor function during aging or in age-related diseases, hospitalization and/or custodial care would be a likely outcome. This means that unless some means is found to reduce these age-related decrementsin neuronal function, health care costs will continue to rise exponentiallyand today‘s costs will pale by comparison as this ”gerofactor”becomes increasingly important. Unfortunately, very little is known about the mechanisms involved in these agerelated declines in cognitive and motor behaviors, and attempts to reverse or reFrom the USDA Human Nutrition Research Center on Aging at Tufts University Boston, Massachusetts (JAJ, ND, DF, BSH, RP, GC); and the Veterans Medical Center, Denver, Colorado (PB) NEUROLOGIC CLINICS OF NORTH AMERICA VOLUME 16 * NUMBER 3 * AUGUST 1998



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tard these decrements have been, with very few exceptions, singularly unsuccessful. Even less is known concerning the nutritional modulation that could be used to retard or reverse these declines. Among the prime candidates responsible for producing the neuronal changes mediating these behavioral deficits, however, appear ,to be free radicals and the oxidative stress (0s)they generate. The free radical hypothesis of aging has been used to suggest that age-related changes occur as a result of an inability to cope with 0s that occurs throughout the life span. If this is the case, then it becomes very important to determine if these neuronal changes may be prevented or retarded by antioxidants, especially dietary antioxidants contained in fruits and vegetables. Extrapolating from the results seen with respect to the age-related diseases previously mentioned (cancer and cardiovascular disease) it appears that diets high in fruits and vegetables may reduce their incidence. For the former, the consumption of fruits and vegetables has been found to reduce both the incidence and mortality rates of cancer in several human cohort and case-control studies for all common cancer site~.'9,58,5~ Similarly, in animal experiments, diets containing fruits and vegetables that are common in human diets have been found to have antitumorigenic effect^.^,^,^^ For many years a strong association also has been found between fresh fruit and vegetable intake and reductions in ischemic heart disea~e.,~,~, Vegetarians and nonvegetarians with a high intake of fruits and vegetables also have reduced blood p r e s s ~ r eand ~ , ~lower ~ cerebrovascular disease mortality.' The important question here is whether diets high in fruit and vegetable intake will be effective in reducing the effects of neuronal aging. Attempts to reduce neuronal "biomarkers of aging" with nitrone trapping agents or antioxidants are only now beginning to be addressed. In this regard, it might be said that the majority of studies concerned with examining the effects of dietary antioxidants as a means to reduce the rather ubiquitous effects of aging have been done without a great deal of knowledge concerning their sites of action, whether the deficits that are being observed are sensitive to OS, or even if the correct combinations of antioxidants are being used. It is clear that this shotgun approach has not been particularly successful. More information could be gained if tests were used that were sensitive to both the effects of age and 0s. This review describes the nature and effects of 0s in aging and outlines some initial experiments that describe their reversal or prevention through nutritional intervention. OXIDATIVE STRESS

Although there have been many hypotheses concerning the rather ubiquitous declines that occur in numerous systems in aging, the determination of the one overriding mechanism has been elusive. As indicated, one implication of a theory that suggests 0s involvement in the deleterious effects of aging is that 0s sensitivity shows increases throughout the life span. As is well known, (see Halliwel125,26 and Yu6*for reviews), this insult can arise from both extrametabolic (e.g., pollution, radiation, toxins, etc.) and metabolic sources. Concerning the latter of these, among the most significant biologic sources of free radicals are those that lead to oxygen-derived superoxide (0;) from electron transport associated with mitochondria1membranes. In this case, the conversion of oxygen to water requires electron transfer. Among the products formed by these reactions are the hydroperoxyl radical (HO,.), hydrogen peroxide (H,O,), and the hydroxyl radical (.OH), which is potentially the most damaging pro-oxidant in cellular systems. Other cellular sources of free radical generation are those of microsomal and nuclear



membranes. These membranes also contain electron transport systems, cytochromes P-450 and b,, which may produce free radicals. One other free radical known to be produced metabolically that has received considerable attention in recent years is nitric oxide. It has been demonstrated, for example, that the excessive production of nitric oxide may be an important factor in N-methyl-D-aspartate (NMDA) neurotoxicity in strokes and neurodegenerative disease (Paakkari and Lindsbergso for review). Fortunately, there are a number of antioxidant defenses that remove excess superoxides and H,O,. These include superoxide dismutase (SOD), catalase, and various peroxidases. These enzymes participate in retarding membrane lipid peroxidation. In addition, there also are low molecular mass antioxidants such as glutathione, vitamin E, and ascorbic acid. Because organisms do not scavenge free radicals with 100% efficiency, the repair of oxidative damage in DNA, proteins, and lipids is extremely important. Thus, a wide variety of enzymes, proteases, and chain-breaking antioxidants exist to aid in this repair (see reviews by Halliwellz and YU"). In aging there are indications of decreases in repair (Adams et aP) following 0s damage. Moreover, in aging there are accumulations of advanced glycation end that may interact to further increase the deleterious effects on cell function. It is believed that these deficits and alterations act in concert with declines in antioxidant defenses to serve as the primary factors involved in the ubiquitous functional declines observed in aging. As examples consider 1. There is a great deal of evidence suggesting that 0s is of primary impor-

tance in cellular aging3,46,51 and the aforementioned age-associated diseases such as cancer and cardiovascular however, there is less evidence demonstrating a role for 0s in normal aging, especially in the brain. There have been few studies directed toward demonstrating agerelated increases in sensitivity to 0s on age-sensitiveneuronal/behavioral indices. The central nervous system (CNS) may show heightened vulnerability to 0s relative to other areas, since it exhibits reduced free radical scavenging ability and uses high amounts of oxygen.47 2. In age-related neurodegenerative diseases, evidence continues to mount that suggests that 0s may be a primary factor contributing to the pathophysiologic and subsequent behavioral alterations that are observed (e.g., Alzheimer's and Parkinson's d i ~ e a s e ) . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 3. Research also indicates that several indices of antioxidant protection appear to be altered in aging including (1)decreased glutathione in hippo(2)~ increased levels of a-tocopherol, poscampus, cortex, and ~ t r i a t u m ~ ; sibly as a compensatory attempt to respond to age-related increases in OS63;(3) increased lipofuscin accumulation6zin lipofuscin-containingvacuoles of neurones, glia, and vascular cells43;and (4) reduced glutamine synthetase.10,12 4. One other factor that may be of extreme importance in increased 0s sensitivity in aging, especially brain aging, may be reduced calcium homeostasis such that there are increases in intracellular calcium and reduction in the ability of the cell to extrude or sequester calcium, especially after depolarization. It is believed that long-lasting increases in cytosolic calcium may contribute to cell death by several mechanisms, including free radical activation through xanthine oxidase activation40as well as the generation of nitric oxide synthase and phosph~lipases.~~ In addition, there may be a rather "vicious circle" created in aging since 0s insult may enhance calcium dysfunction (eg., Cheng et all3) in cells that are al-


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ready compromised, further increasing intracellular calcium levels, thus resulting in enhanced pro-oxidant generation, subsequent loss of functional capacity of the cell, and cell death. Hence, there are several factors that may combine to enhance the deleterious effects of 0s in aging. Although the above discussion provides evidence that increased sensitivity to 0 s may be of considerable importance in aging, it is only recently that their putative role in neuronal aging using. "age sensitive" tests has begun to be characterized. By using such tests, more information can be obtained concerning such factors as (1) locus and mechanisms of oxidative damage in neuronal cell death; (2) the putative sites involved in membrane damage and subsequent alterations in neurotransmitter receptor mediated signal transduction; (3) interactions between membrane lipids such as cholesterol (CHL), sphingomyelin (SPM) (which increase as a function of and 0s; and (4) responsiveness of the alterations to various antioxidants. In this regard, one test that we have used that has both age-sensitivity and 0s ~ensitivity~~ involves the determination of oxotremorine-enhancement of K+evoked dopamine release from striatal slices. This test assesses the loss in sensitivity expressed by muscarinic receptors as a function of age in the striatum. This brain area is involved in motor control and shows significant functional and morphologic declines as a function of age. The results from several experiments have indicated that the oxotremorine (0x0) enhancement of dopamine (DA) release (K+ERDA) (in perfused striatal slices) shows significant declines with aging.29,30,33 In addition, 0 s generated in the whole animal (via 100% oxygen exposure or whole body i r r a d i a t i ~ n induces ~~) changes similar to those seen in aging. Moreover, exposure of the striatal slices to hydrogen peroxide or dopamine in vitro to generate 0s can also produce these deficits in oxotremorine enhancement of K+-ERDA, and the tissue obtained from the old animals appears to be more sensitive to the effects of exposure to hydrogen Additional studies have shown that these 0 s and age deficits in oxotremorine enhancement of K+-ERDAare primarily the result of alterations in signal transduction early in the transduction p r o c e ~ s ~ ~ , ~ ~ probably at in the membrane where the receptor interfaces with its respective regulatory G protein. Importantly, subsequent experiments have indicated that the age- and OS-induced deficits could be prevented by the in vivo or in vitro administration of antioxidant^^^ (e.g., Trolox, a Vitamin E analog, cx-phenyln-butyl nitrone [PBN]). To analyze further the possible role of 0s on membrane lipids, we examined the interaction of these two factors on calcium flux. Calcium flux is the movement of calcium between extracellular and intracellular compartments. Of specific interest was the determination of the effects of 0s and alterations of membrane lipids on the ability of PC-12 cells to extrude or sequester calcium after stimulation (i.e., depolarization). We had found previously that oxidative stress can significantly lower the ability of these cells to extrude or sequester calcium after depol a r i ~ a t i o nAs . ~ ~pointed out above, high levels of intracellular calcium can lead to increased cell death, and it is known that calcium homeostasis is altered in aging.39 Thus, 0 s was examined in control PC-12 cells and in those treated with CHL, SPM, or SPM/CHL. After CHL and SPM treatment, the levels of these lipids in the cell membranes were equivalent to those seen in aging. The results indicated that 0 s decreased the ability of the cell to extrude or sequester calcium, and the 0s effects were increased to a greater extent in SPM- and SPM/CHL-treated cells.17 Subsequent experiments have indicated that an SPM metabolite, sphingosine-lphosphate (SP-1-P) significantly increased 0s vulnerability. Thus, these findings indicate that one factor that is important in determining 0s vulnerability is membrane lipid content, especially those of SPM and its metabolite SP-1-P. Therefore, as can be seen from this study, and those cited herein, there is



strong evidence to suggest that the neuronal deficits seen in aging are the result of age-related increases in sensitivity to 0s that arise from several factors: most notably, decreased antioxidant protection and repair as well as increased membrane SPM and altered calcium homeostasis. Given these considerations it is especially important to determine the role of dietary antioxidants in reducing the deleterious effects of this increased 0s sensitivity on neuronal function in aging. NUTRITIONAL INTERVENTION

As indicated above, there is an abundance of literature to suggest that diets high in fruits and vegetables are important in preventing or moderating such major disorders as cancer and heart disease; however, as was also indicated, there is less research examining the effects of these diets in the case of neuronal functioning. Indirect evidence that nutritional intervention might be effective in reducing or retarding the effects of neuronal aging can be derived from a large number of studies indicating that improper or persistent inadequate nutrition can lead to neurologic dysfunction even in adulthood. To cite just a few instances, a variety of nutritional diseases of the nervous system have been documented, such as nutritional neuropathy (e.g., from thiamin or B,, deficiencies), cerebellar degeneration (e.g., from chronic alcoholism),retinal degeneration (vitamin E deficiency), and paresthesia (B12deficiency) to name just a few. Psychologic aberrations from B,, deficiency include apathy, irritability, and even in some cases, dementia (eg., reviewed in Dreyfus and SeyalZ0and Swain55;see also Crystal et all6).It seems clear that inadequate nutrition may play a role in neuronal dysfunction. Deficits in brain function leading to behavioral aberrations are also observed in aging even in situations that appear to be of nonnutritional origins or when nutrition seems adequate. This is especially seen in the case of Parkinson's disease in which the central absorption (and consequently the efficacy) of L-dopa treatment has been reported to be enhanced by dietary manipulation^.^^ It also appears that deficits in ferritin may increase iron levels in the substantia nigra, leading to enhanced oxidative damage and subsequent neurodegeneration and cell loss.6,15,1s Therefore, it may be possible to reduce the progressive deterioration (which may be increased by L-dopa administration) resulting from this disease by altering ferritin or iron levels. In the case of aging, there is evidence from studies in both rats and humans indicating that Ginkgo biloba has positive effects on several age-related neuronal functional indices including memory impairment;, difficulties in concentraand decreases in the Ca +,-induced increases in the oxidative metabolism of brain n e ~ r o n s .Considering ~,~~ the above discussion concerning alterations of calcium homeostasis in aging, a dietary supplement that can alter its deleterious effects could have profound effects in aging. In direct tests of the effectiveness of dietary intervention on neuronal dysfunction, the Oxygen Radical Absorbance Capacity (ORAC) assay was used to identify the total antioxidant capacity of fruits and vegetables that could be used in conjunction with the models previously described herein. The ORAC assay uses 0-phycoerythrin (p-PE) or R-PE as an indicator protein, 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and Trolox (as soluble form of vitamin E) as a standard." Thus far, the results from the assay have shown that among foods with the highest ORAC activity are spinach, blueberries, and strawberries. Therefore, we used these foods to assess the effects of dietary intervention in our age and OS-sensitive models. In these experiments Fischer 344 rats (6- to 8-months old) were maintained for 8 weeks prior to 0, exposure on control diets or those that contained one of the following: vitamin E


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(500 IU/kg), strawberry extract (9.4 g/kg dried aqueous extract, DAE), spinach (6.7g/kg DAE), or blueberry extracts (10 g/kg DAE). Results showed that all of the diets were effective in preventing striatal oxotremorine enhancement of K+ERDA as well as two other age- and OS-sensitive parameters: decreases in basal forebrain nerve growth factor (NGF) and P-adrenergic receptor functioning in the cerebellum. In an effort to validate further the efficacy of the diets in preventing these age-associated decrements in signal transduction (e.g., striatal 0x0-enhanced K+ERDA, cerebellar P-adrenergic receptor sensitivity), we maintained rats on the control, strawberry, or spinach diets from the age of 6 months to 15 months. The preliminary results indicated that the each of the diets prevented the age-related loss in striatal oxotremorine enhancement of K+-ERDA.The animals maintained on the spinach diet showed almost 100% higher values in this parameter than control rats. We are presently attempting to determine if these diets also will be effective in reversing the deleterious effects of aging on the above parameters as well as on motor and cognitive behaviors. These findings, however, thus far suggest that nutritional intervention with fruits and vegetables may play an important role in preventing or perhaps even reversing the effects of 0 s in aging on brain function. We are presently attempting to delineate the sites of actions of the dietary antioxidants contained in these foods and particular antioxidant classes (e.g., flavonoids) that are the most effective in preventing these age-related deficits.

SUMMARY This review described some of the age-associated changes that occur in neuronal function and cites evidence to show that these alterations may be the result of increased sensitivity to 0s. Evidence is presented to show that the abilities to mitigate the 0 s effects and to repair the damage from 0 s show decline as a function of age. Results are given, using age- and OS-sensitive tests, which indicate that one of the major sites of action of 0 s is the membrane, especially membranes compromised by high amounts of sphingomyelin, and one of the major effects of 0 s is to alter further the calcium disregulation in aging. It is suggested that attempts to increase antioxidant protection through diets high in fruits and vegetables identified as being high in total antioxidant activity might prevent or reverse the deleterious 0s effects on neuronal aging.

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Address reprint requests to J.A. Joseph, PhD USDA Human Nutrition Research Center on Aging at Tufts University 711 Washington St. Boston, MA 02111