2007 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 Subordinate projects for this project include: 1950-51000-063-04T – Trust Fund Cooperative Agreement with California Dried Plum Board 1950-51000-063-05T – Trust Fund Cooperative Agreement with Welch Foods Inc. 1950-51000-063-08R – Reimbursable Agreement with University of Maryland (UMBC) 1950-51000-063-09T – Trust Fund Cooperative Agreement with The Stewart and Linda Resnick Revocable Trust (POM Wonderful) 1950-51000-063-10T – Trust Fund Cooperative Agreement with California Strawberry Commission 1950-51000-063-11T – Trust Fund Cooperative Agreement with California Walnut Commission 1950-51000-063-12T – Trust Fund Cooperative Agreement with U.S Highbush Blueberry Council 1950-51000-063-13T – Trust Fund Cooperative Agreement with Wild Blueberry Association of North America (WBANA) 1950-51000-063-14T – Trust Fund Cooperative Agreement with Sunnyridge Farm 1950-51000-063-16R – Reimbursable Agreement with University of Maryland at Baltimore (UMBC)-NASA Subaward For a complete report on the progress of these subordinate projects, see the corresponding annual report. 4.Accomplishments LAB:NEUROSCIENCE 1) Differential protection among fractionated blueberry polyphenolic families against oxidative stress. Hippocampal cells in the brain are very important in memory function, decline in function during aging and are sensitive to oxidative and inflammatory stressors. The most significant accomplishment during FY2007 addressed the problem of identifying the effects of the polyphenolic compounds derived from blueberries in lowering the deleterious effects of oxidative stress and the products of oxidative stress (e.g., cytokines) in hippocampal cells. Overall these results suggested that, except for a few instances, the more refined (e.g., pure) the polyphenolic component the less the effectiveness in protecting the cells against oxidative stressors such as dopamine. They also suggest that one of the major mechanisms involved in the effects of blueberries is the specific reductions in the numerous signals that are generated by oxidative stress which results in the generation of products such as cytokines that are deleterious to the brain. The results make a good case for eating the whole berry, rather than attempting to define the most efficacious polyphenolic compound or family within the blueberry. 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 NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 2) Blueberry (BB) extract antagonizes combined lipid and oxidative/inflammatory stressor effects. One of the significant accomplishments during FY2007 addressed the problem of determining how changes in lipid membranes, such as the accumulation of a lipid called sphingomyelin and one of its metbolites, C2 ceramide, may alter a cell’s response to oxidative stress. C2 ceramide has been shown to have several negative cellular effects, including oxidant formation and cytokine formation. We used COS-7 cells as a model in these experiments, since we can place a certain type of receptor called a muscarinic receptor in them and look at the response of this receptor and the membrane in relative isolation. This allows us also to look at the function of the cell by assessing its ability to clear calcium (CAR) that has moved inside the cell. CAR that has not been cleared can damage the cell. The results indicated that the effects of several stressors (e.g., dopamine) on CAR deficits were prevented by pretreatment with the BB extract in both ceramide- and non-ceramide- treated cells. BBs appear to accomplish these benefits by reducing several negative effects on the cells such as oxidant and cytokine formation. As above, 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 NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 3) Blueberry (BB) and strawberry (SB) effects on cognition and motor function in aging. Determine the effects of BB and SB supplementation on motor and cognitive function. Results showed that SB-fed rats had improved performance compared to the BB-fed rats on the large plank, BB rats were better than SB rats on rod walking, while both diets improved motor function on the rotarod compared to control. Both berryfruit diets enhanced working memory in the Morris water maze. 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 NP107 Human Nutrition program Component 5-Health Promoting Properties of Plant and Animal Foods. 4) Blueberry antagonism of DA-induced disruption of calcium clearance (CAR) in COS-7 cells involves reductions in stress signaling at several sites. One of the major changes that occurs in the brain in aging is an increasing inability of the cells to remove calcium after calcium moves into them when they are activated (i.e., depolarized). This is especially seen if the cell is exposed to an oxidative stressor. We wanted to determine whether berries would increase a cell’s ability to remove calcium when it was exposed to an oxidative stressor such as dopamine. CAR that has not been cleared can damage the cell. We found that dopamine induced decrements in CAR were antagonized by BB and this appeared to have been accomplished by blocking stress signals such as nuclear factor kappa B. When we used specific inhibitors of these signals we found that none were as effective as the BB, suggesting that BB may be blocking these signals at several points in the stress pathway. 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 NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. 5) Blueberry (BB) and strawberry (SB) effects on neurogenesis. Determine the effects of BB and SB supplementation on both proliferation and survival of new neurons, and confirm that they are neurons. One mechanism for the improvement in age-related deficits in behavior with BB and SB supplementation is increased neurogenesis. In preliminary analyses, we have found that the berryfruit diets increase both proliferation and survival of new neurons compared to control. In double-labeling experiments, it was confirmed that the bromodeoxyuridine (BrdU)-positive cells did not co-localize with the glial fibrillary acidic protein (GFAP)-positive cells, showing that new neuronal cells and not glial cells were increased. 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 NP107 Human Nutrition Component 5-Health Promoting Properties of Plant and Animal Foods. LAB: NUTRITION AND COGNITION 1)Cognitive Impairment in an animal model was demonstrated to be caused by nutritional changes in brain membranes Homocysteinemia is associated with age-related cognitive decline and dementia but it is uncertain whether these cognitive dysfunctions are due to homocysteine toxicity or to underlying metabolic impairments. Homocysteinemia was induced in Sprague Dawley rats fed control or folate deficient diets, with or without supplemental methionine. After 9 weeks of feeding, rats underwent behavioral testing followed by tissue collection for hematological and biochemical analysis. Folate deficiency alone with normal methionine resulted in impaired Morris Water Maze performance, a test of memory and learning in rodents; however, this impairment was mitigated by the addition of supplemental methionine to the folate deficient diet. Plasma homocysteine was moderately elevated during folate deficiency, irrespective of dietary methionine. Similarly, brain SAM, S-adenosylmethionine, a derivative of methionine, and SAH, S-adenosylhomocysteine, a product of the utilization of SAM, were not significantly altered by the different diets. However, the ratio of phospatidylcholine to phosphatidylethanolamine in brain membranes was specifically and significantly reduced by folate deficiency alone, but was restored by supplemental methionine. In contrast, acid phospholipids (phosphatidylserine and phosphatidylinositol) were not affected by diet. These observations move us in direction of understanding how nutritional factors may affect brain function, memory and cognition. This accomplishment is aligned with NP107 Human Nutrition Component 5-Health Promoting Properties of PLant and Animal Foods. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 281 Number of newspaper articles and other presentations for non-science audiences 7 Review Publications Joseph, J.A., Shukitt Hale, B., Lau, F.C. 2007. Fruit Polyphenols and their effects on Neuronal Signaling and Behavior in Senescence. Annals of the New York Academy of Sciences. 1100:470-485. Lau, F.C., Bielinski, D., Joseph, J.A. 2007. Inhibitory effects of Blueberry Extract on the Production of Inflammatory Mediators in LPS-activated BV2 Microglia. Journal of Neuroscience Research. 85:1010-1017. Lau, F.C., Shukitt Hale, B., Joseph, J.A. 2006. Beneficial effects of berryfruit polyphenols on neuronal behavioral aging. Journal of the Science of Food and Agriculture.86:2251-2255. Mcguire, S.O., Sortwell, C.E., Shukitt Hale, B., Joseph, J.A., Hejna, M.J., Collier, T.J. 2006. Dietary supplementation with blueberry extract improves survival of transplanted dopamine neurons. Nutritional Neuroscience. 9(5/6): 251-258. Carey, A.N., Shukitt Hale, B., Rabin, B.M., Joseph, J.A. 2007. Interaction between age and exposure to 56Fe particles on behavior and neurochemistry. Advances in Space Research. 39:987-993. Shukitt Hale, B., Casadesus, G., Carey, A.N., Rabin, B.M., Joseph, J.A. 2007. Exposure to 56Fe irradiation accelerates normal brain aging and produces deficits in spatial learning and memory. Advances in Space Research. 39:1087-1092. Shukitt Hale, B., Carey, A.N., Jenkins, D., Rabin, B.M., Joseph, J.A. 2007. Beneficial effects of fruit extracts on neuronal function and behavior in a rodent model of accelerated aging. Neurobiology of Aging.28:1187-1194. Joseph, J.A., Fisher, D.R., Carey, A.N., Bielinski, D.F. 2006. Dopamine-Induced Stress Signaling in COS-7 Cells Transfected With Selectively Vulnerable Muscarinic Receptor Subtypes is Partially Mediated Via the i3 Loop and Antagonized By Blueberry Extract. Journal of Alzheimer's Disease. 10:423-437. Crews, F.T., Nixon, K., Kim, D., Joseph, J.A., Shukitt Hale, B., Qin, L., Zou, J. 2006. Bht blocks nfkb activation and ethanol-induced brain damage. Alcoholism: Clinical and Experimental.30(11):1938-1949. Lau, F.C., Shukitt Hale, B., Joseph, J.A. 2007. Nutritional Intervention in Brain Aging: Reducing the Effects of Inflammation and Oxidative Stress. Inflammation in the Pathogenesis of Chronic Diseases:The COX-2 controversy. Subcellular Biochemistry. Volume 42. New York, NY:Springer Publication. p.299-318. Crivello, N.A., Rosenberg, I.H., Dallal, G.E., Bielinski, D., Joseph, J.A. 2005. Age-related changes in neutral sphingomyelin-specific phospholipase c activity in striatum, hippocampus, and frontal cortex: implication for sensitivity to stress and inflammation. Neurochemistry International. 47(8): 573-579. D'Anci, K.E., Rosenberg, I.H. 2005. B vitamins and the brain: depression. Nutrition in Clinical Care. 8(4):143-8. D'Anci, K.E., Rosenberg, I. 2004. Folate and brain function in the elderly. Current Opinion in Clinical Nutrition and Metabolic Care. 7(6):569-64. D'Anci, K.E., Constant, F., Rosenberg, I. 2006. Hydration and cognitive function in children. Nutrition Reviews. 64(10 Pt 1):457-64. Balk, E.M., Raman, G., Tatsioni, A., Chung, M., Lau, J., Rosenberg, I. 2007. Vitamin B6, B12 and folic acid supplementation and cognitive function: a systematic review of randomized trials. Archives of Internal Medicine. 167(1):21-30. Raman, G., Chung, M., Tatsioni, A., Rosenberg, I., Lichtenstein, A.H., Lau, J., Balk, E.M. 2007. Heterogeneity and lack of good quality studies limit association between folate, vitamin b-6 and b-12, and cognitive function. Journal of Nutrition. 137(7): 1789-1794.