2008 Annual Report 1a.Objectives (from AD-416) LAB:Nutrition and Cognition Determine whether and how nutritional factors, especially B vitamins, can be employed in the understanding and prevention of age-related cognitive impairment in humans and in human populations. Charecterize the mechanisms by which nutritionally induced hyperhomocysteinemia affects neuronal function and cognitive performance using transgenic mouse models of human cognitive decline. LAB:Neuroscience Identify the structural and compositional difference among muscarinic receptor subtypes and the lipid microenvironment (lipid rafts) or their combination that contribute to increased vulnerability to oxidative stress and inflammation in aging in the COS-7 cell model. Assess the protective capability of berryfruit polyphenolic extracts and determine the most effective component polyphenol(s) against oxidative stress and inflammatory agents in a muscarinic receptor transfected COS-7 cell model. Assess the vulnerability to oxidative and inflammatory stressors in microglia cells (which may affect loss of neuronal function in aging), or hippocampal cells (which may be involved in memory function) and determine the effects of polyphonolic and berryfruit extracts. Establish the effects of dietary berryfruit extracts and the most effective component polyphenolics on neuronal function in aging by determining the effects on motor cognitive behaviors as a function of age. (a) Identify brain regional localization of berryfruit compounds and correlating the amounts seen with the behavioral performance. (b) Determine their effects on signaling and the generation of new neurons in aging. 1b.Approach (from AD-416) LAB:Nutrition and Cognition Collaboration will continue with the Framingham Heart Study cohorts to examine prospective relationships among nutritional status, cognitive performance, homocysteine, and brain morphology by imaging. Using the homebound elderly population, we will study these relationships first cross-sectionally, and then prospectively. We will add other factors, including antioxidant vitamin status and its relation to inflammatory markers and adhesion molecules in the vasculature. We have designed a trial as an adjunct to the large homocysteine lowering intervention on subjects who have undergone renal transplantation to measure the impact of homocysteine lowering on cognitive decline and performance. The animal models we intend to use are the apoE knockout, the APP/PSI and an APP/London, in which we can modify nutritional status with respect to B vitamins and homocysteine levels and examine the relationships between increased sensitivity to behavioral decline. We will compare age-related status of membrane sphingolipids and vitamins K & A in brain regions controlling behavior in aged Fisher rats. We will also compare the status of membrane sphingolipids, one carbon metabolism, and neuronal viability in a human neuronal cell culture model. LAB:Neuroscience COS-7 cells transfected with various muscarinic receptor subtypes and their chimerics will be used to identify the structural and membrane lipid raft differences contributing to increased oxidative stress vulnerability by exposing them to dopamine and assessing alterations in calcium flux and viability. Motor and cognitive assessments will be undertaken in senescent rats given diets supplemented with whole berryfruits or extracts derived from them. Correlative determinations of neurogenesis, neuronal signaling and gene activation or inhibition will also be undertaken in various brain regions of these control and supplemented animals. 3.Progress Report The following is a progress report a laboratory led by an ARS scientist. This project includes the work of one subordinate project at the HNRCA funded through a SCA with Tufts University. See 1950-51000-063-15S for more info. We have shown that supplementation with fruits and vegetables can both forestall and reverse the deleterious effects of aging on neuronal functioning and behavior, and these beneficial effects are the result of polyphenolics in the plants. While fruits and vegetables may have direct effects on oxidative stress (OS) and INF in aging, polyphenolic compounds may also enhance signaling and neurogenesis in aging. Discovery of these additional mechanisms might lead to guidelines and dietary recommendations for an aging population concerning fruits and vegetables. This year, we determined how berryfruits affected the plasma membrane microenvironment. The plasma membranes of a cell line in which receptors are placed (muscarinic receptors) are altered by lipids and receptor functioning is observed. C2C is known to be increased in the cell membranes in aging and to alter the functioning of the membrane and receptors. When a cell sends a signal, calcium is transported in then out of the membrane. Cells from aged animals and cells exposed to C2C and/or OS lose this ability, but pretreatment of the cells with blueberry (BB) extract restored this functioning, although the effects blunted. We also showed that the mechanisms found in the BB protection of the cells from C2C and OS involved the suppression of signals that enhance OS/INF. Thus, BBs are actually shutting down OS signals and protecting the cells. Similar findings were seen in hippocampal cells (important in memory control) with walnut based polyunsaturated fatty acids. Similar to BB, the results indicated that the walnut oils protected by not allowing the stressor to affect calcium movement out of the cell. Research in old animals to determine the most effective polyphenolics that would enhance cognitive and motor function showed that stilbene compounds (which are resveratrol analogs found in BB, concord grapes and red wine) produced these effects. The most efficacious stilbene compound in a cell study was pterostilbene (PTS) which was fed to aged (19 mo) rats. PTS was effective in reversing cognitive behavioral deficits (e.g., working memory), and performance was correlated with PTS levels in the hippocampus. In a mechanistic study with BB or strawberry (SB) supplementation in senescent rats we showed that there was a significant increase in the rate of hippocampal neurogenesis in SB-fed animals and a trend toward increased neurogenesis in the BB-fed rats. This is an important finding since neurogenesis (the growth and survival of new neurons) normally shows some significant decline in aging that is related to increases in cognitive decline. The finding that there were new neurons in the supplemented areas and that these neurons survived as well as proliferated may be an important mechanism in the positive benefits of these supplementations.This work is aligned with National Program 107 – Human Nutrition Component: 5. Health Promoting Properties of Plant and Animal Models. 4.Accomplishments 1. LAB: Nutrition and Cognition Vitamin K in Brain Aging Since the human brain is richly endowed with vitamin K, we set out to examine the function of this vitamin in brain and its possible protection against brain aging. Rats were fed diets to create different levels of vitamin K in brain and then important fatty substances in brain were measured to assess the effect of different levels of vitamin K. There was a relationship between the amount of vitamin K in brain tissue and substances which coat and protect brain fibers, suggesting important functions of vitamin K in brain metabolism. These findings may be potentially important for the design of clinical studies in prevention of cognitive decline and brain aging. This accomplishment is aligned with NP107 Human Nutrition Program component 5 (Health promoting properties of plant and animal foods). 2. Dietary folate and choline affect metabolism in brain. Folate and choline have been suggested to affect brain metabolism. However, the mechanism is not known. Rats were placed on diets sufficient or deficient in folate and choline to judge effects on brain metabolism. Dietary folate deficiency resulted in severe depletion of folate in blood and liver, and mild deficiency in brain. This resulted in depletion of one of the most important fat substances in brain which is involved in many aspects of brain function. Supplemental choline alleviated these effects. This study adds to understanding of how vitamins and related subjects may be involved in the protection of brain against age-related cognitive decline and could lead to further studies of age related cognitive decline in humans. This accomplishment is aligned with NP107 Human Nutrition Program Component 5 (Health Promoting Properties of Plant and Animal Foods). 3. B-vitamin deficiency causes an impairment of small blood vessel supply to brain in mice. The mechanism by which B vitamin deficiency impairs cognitive brain function is not well understood, we created B vitamin deficiency in mice. Deficient animals had an elevation of the blood levels of homocysteine, which has been previously shown to be related to blood vessel impairment. B vitamin deficiency caused a decrease in the small blood vessels in brain, which are necessary to deliver blood and nutrients to brain tissue. These findings are consistent with human studies of vascular impairment in brain aging and cognitive decline and provide evidence for the importance of the maintenance of blood vessels in brain aging, and the potential benefit of dietary or nutritional factors. This accomplishment is aligned with NP107 Human Nutrition Program Component 4 Nutrient Requirements. 4. Cognitive impairment in folate deficient rats corresponds to depleted brain phosphatidylcholine. Folate deficient rats were studied for the impact of the deficiency on brain chemistry. These studies gave further evidence that the interplay between vitamin and choline may be important for the maintenance of normal brain function, especially in aging, and provide a foundation for further studies to understand the means of possible prevention of cognitive decline with age. This addresses one of the most important changes in function with an aging population. This accomplishment is aligned with NP107 Human Nutrition Program Component 4 Nutrient Requirements. 5. LAB: Neuroscience Blueberry (BB) extract antagonizes the effect of combined lipid and oxidative/inflammatory stressor. Lipids, such as ceramide can alter a cell’s response to oxidative (oxidative stress) and inflammatory insults. C2 ceramide has been shown to have several negative cellular effects, including oxidant (oxidative stress) and cytokine (inflammatory agents) formation causing loss of cell functioning, neuronal transmission and ultimately declines in motor and cognitive function. This year we showed that several of the deleterious inflammatory and oxidative stress signals (e.g., a very complex one called cyclic AMP response element binding protein, CREB) that result in enhancement of negative inflammatory stressors such as cytokines that produce inflammation in tissue were reduced by blueberry supplementation in these cells. Importantly, however, blueberry treatment also increased protective signals. However, when we exposed these cells to ceramide, we found that the effects of the blueberry supplementation were blunted, and the cells showed some loss of function. The ceramide that was added to the cells was similar in amount to that seen in the cells of aged animals, suggesting that age can have an effect on the ability of the blueberries to exert their positive effects and those alterations in membranes via ceramide may artificially “age” the cells. This work has been discussed in lay terms in the lay press, and has provided for the first time mechanisms other than the inactivation of free radicals (molecules producing oxidative stress) as being involved in the beneficial effects of the blueberries. These findings are important in both industry and health areas, since they point once again to the importance of including berries in the diet for promoting healthy aging. This work is aligned with National Program 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models. 6. Walnut-based PUFAs protect against stress-induced decrements in Ca2+ buffering in primary hippocampal cells via differential effects on stress signals. Alterations in plasma membranes are known to occur as a function of age, and it became of interest to see if dietary lipids from walnuts could alter membrane function to maintain calcium levels in cells. If high calcium levels remain in a cell after it “fires” or sends a message it could be deleterious. We used a cell model to see if walnut oil could prevent stress-induced alterations on the ability of these cells to clear calcium. Two concentrations of the oil did prevent these stress-induced alterations, and the best protection against the stressors was seen when oils were used from the whole walnut rather than isolated oils such as linoleic acid. Being able to prevent changes in membrane lipids with walnut oils is important since these changes occur as we age and are deleterious to the function of the cell. These findings are important in both industry and health areas, since they point once again to the importance of including whole berries and nuts in the diet for promoting healthy aging especially with changes in membrane lipids. This work is aligned with National Program 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models. 7. Pterostilbene, a resveratrol analog found in blueberries, can improve cognitive function in aged rats. As we age, we are more likely to develop age-related decreases in cognition; therefore, we must find strategies to forestall or reverse these changes. Research from our laboratory suggests that the combinations of antioxidant/anti-inflammatory polyphenolics found in fruits and vegetables may show efficacy in reducing age-related decline in cognition. However, it is difficult to discern from a crude blueberry (BB) extract the contribution of individual polyphenols or even non-polyphenols (sugars, organic acids, etc.) to their positive effects in aged rats. One particular group of compounds that may be responsible for these effects and that has received a great deal of attention are the stilbenes (e.g., resveratrol), which have potent antioxidant activity and are found in berries and grapes. Experiment I, we utilized resveratrol, a stilbene, and six other similar compounds and examined their efficacies in preventing oxidative stress in cells. In Experiment II, we utilized one of more effective analogs (pterostilbene) from Experiment I and added a low or a high concentration of pterostilbene to a diet which was fed to the aged rats. Pterostilbene was effective in reversing cognitive behavioral deficits and short-term memory was correlated with pterostilbene levels in the brain. These findings show that pterostilbene might be one of the active components in blueberries and grapes that impart the protective effects against aging and cognition. Based on these findings, we have submitted a use-patent for pterostilbene. These findings are also important in both industry and health areas, since they point once again to the importance of including berries and grapes in the diet for promoting healthy aging. This work is aligned with National Program 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models. 8. Blueberry (BB) and strawberry (SB) effects on neurogenesis. Up until a few years ago, it was believed that once an individual was born, neurons were only lost throughout the lifespan and no new neurons were made. We now know that new neurons (neurogenesis) are made throughout the lifespan, but the rate slows down as a function of age. A few years ago we showed that BB can increase neurogenesis in senescent animals; however, we studied the role of strawberry or blueberry supplementation on both the growth of new neurons and their survival. This year we expanded upon our initial studies on neurogenesis by quantifying cells in an area of the brain called the dentate gyrus which is critically involved in memory and cognition and shows declines in aging. We fed 24 month old rats strawberry- or blueberry- supplemented diets and then studied their cognitive behavior. There was a significant increase in the rate of dentate gyrus neurogenesis in strawberry-fed animals and a trend for increased neurogenesis in the blueberry-fed rats. This work is aligned with National Program 107 – Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Models. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 1 Number of Non-Peer Reviewed Presentations and Proceedings 3 Number of Newspaper Articles and Other Presentations for Non-Science Audiences 1 Review Publications Rabin, B.M., Joseph, J.A., Shukitt Hale, B., Carey, A.N. 2007. Dietary modulation of the effects of exposure to 56FE particles. Advances in Space Research. 40(4):576-580. Ghosh, D., Mcghie, T.K., Fisher, D.R., Joseph, J.A. 2007. Cytoprotective effects of anthocyanins and other phenolic fractions of Boysenberry and blackcurrant on dopamine and amyloid Beta induced oxidative stress in transfected COS-7 cells. Journal of the Science of Food and Agriculture. 87:2061-2067. Kalt, W., Joseph, J.A., Shukitt Hale, B. 2007. Blueberries and Human Health: A Review of Current Research. Journal of American Pomological Society. 61:151-160. Shukitt Hale, B., Lau, F.C., Joseph, J.A. 2008. Berry Fruit Supplementation in the Aging Brain. Journal of Agriculture and Food Chemistry. 56:636-641. Denissova, N., Rosenberg, I., Shukitt Hale, B., Bielinski, D., Dallal, G., Joseph, J.A. 2007. Aging modifies brain region-specific vulnerability to experimental oxidative stress induced by low dose hydrogen peroxide. American Aging Association. 29:191-203. Joseph, J.A., Smith, M.A., Perry, G., Shukitt Hale, B. 2008. Polyphenols and Polyunsaturate Fatty Acids: The Pollyanna's of Age-Related Cognitive Decline, Neurodegenerative Disease. In Coulston, A.M. and Boushey, C.J., editors. Nutrition in the Prevention and Treatment of Disease. 2nd edition. San Diego: Elsevier. p. 269-287. Joseph, J.A., and Gibson, G.E. 2007. Coupling of neuronal function to oxygen and glucose metabolism through changes in neurotransmitter dynamics as revealed with aging, hypoglycemia and hypoxia. In Gibson, G.E. and Dienel, G., editors. Handbook of Neurochemistry and Molecular Biology. 3rd edition. Volume 5. New York, NY: Springer. p.297-320. Shukitt Hale, B., Lau, F.C., Carey, A.N., Galli, R.L., Spangler, E.L., Ingram, D.K., Joseph, J.A. 2008. Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus. Nutritional Neuroscience. 11(4):172-182. Joseph, J.A., Carey, A.N., Brewer, G.J., Lau, F.C., Fisher, D.R. 2007. Dopamine and ABeta-induced Stress Signaling and Decrements in CA2+ Buffering in Primary Neonatal Hippocampal Cells are Antagonized by Blueberry Extract. Journal of Alzheimer's Disease. 11:433-446.