Progress 05/01/04 to 03/19/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations LAB: Neuroscience We have shown that dietary supplementation with fruits and vegetables can forestall and reverse the deleterious effects of aging on neuronal functioning and behavior, and these beneficial effects are the result of plant polyphenols. While fruits and vegetables may have direct effects on oxidative stress(OS) and inflammation in aging, polyphenolic compounds may also enhance protective signaling and neuronal growth. This year we assessed the effects of berryfruits on a type of brain cell, a microglial cell that contributes to the �clean-up� of damaged cells in the brain. However, in aging and neurodegenerative diseases, these cells can become overactivated and begin to clean up healthy cells. We used BV-2 mouse microglial cells and showed that berryfruits and walnuts deactivate the cells and prevent cell death. We showed that these treatments are also effective in shutting down stress signals in hippocampal cells which are important in memory function, and that strawberry/blueberry combinations reduced inflammatory markers and enhanced cellular calcium function in these cell models. To determine the components of walnuts that may be having beneficial effects in cells, we exposed BV-2 cells to inflammatory stressors to determine the protective roles of polyunsaturated fatty acids(PUFAs), specifically the omega-6 fatty acid linoleic acid(LA) and the omega-3 fatty acid, alpha-linolenic acid(ALA). PUFAs are essential components of neuronal cell membranes, maintaining membrane function critical for neurotransmitter communication within neural networks. We found that ALA was a more powerful anti-inflammatory agent than LA. Docosahexanoic acid, a derivative of ALA, was also effective against inflammatory stressors. This suggests that walnut-derived PUFA�s may be effective deterrents of inflammatory effects in aging. We assessed the effects of polyphenolic fruits and nuts on microglial activation in old rats and found that animals consuming a 6% walnut diet exhibited significantly fewer activated microglia in the hippocampus (a part of the brain necessary for learning and memory), suggesting that walnuts reduced inflammation in these rats. We also found in the BV-2 cells a second mechanism that might be operating to account for the beneficial effects of walnuts is the inactivation of a TLR receptor, which is intimately involved in the inflammation process. Also, walnuts decreased an enzyme (acetylcholinesterase)involved in the regulation of age-related declines in memory and increases in neurodegenerative diseases. The effects of walnut extract on BV-2 cells are novel and demonstrate that fruits and nuts can induce dramatic alterations in the stress responses of cells involved in age-related cognitive decline. We also added raspberries to the list of berries that appear to reverse declines in age-related motor function in tests of balance and muscle strength that show declines in aging. LAB: Nutrition & Cognition This project includes the work of a subordinate project at the HNRCA funded through a Specific Cooperative Agreement with Tufts University. For the progress report, see 1950-51000-063-015S (Nutrition & Cognition). Significant Activities that Support Special Target Populations LAB: Neuroscience None. LAB: Nutrition and Cognition None. Technology Transfer Number of New CRADAS: 1 Number of Active CRADAS: 1 Number of New Patent Applications filed: 1
Impacts
(N/A)
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.
- Duffy, K.B., Spangler, E.L., Devan, B.D., Guo, Z., Bowker, J.L., Janas, A. M., Hagapanos, A., Minor, R.K., Decabo, R., Mouton, P.R., Shukitt Hale, B., Joseph, J.A., Ingram, D.K. 2008. A blueberry-enriched diet provides cellular protection against oxidative stress and attenuates a kainate- induced learning impairment in rats. Neurobiology of Aging. 29:1680-1689.
- Shukitt Hale, B., Kalt, W., Carey, A., Vinqvist-Tymchuk, M., Mcdonald, J., Joseph, J.A. 2009. Plum Juice, but Not Dried Plum Powder, is Effective in Mitigating Cognitive Deficits in Aged Rats. Nutrition.25:567-573.
- Joseph, J.A., Fisher, D.R., Cheng, V., Rimando, A.M., Shukitt Hale, B. 2008. Cellular and Behavioral Effects of Stilbene Resveratrol Analogs: Implications for Reducing the Deleterious Effects of Aging. Journal of Agriculture and Food Chemistry.56(22)10544-10551.
- Willis, L., Shukitt Hale, B., Joseph, J.A. Recent Advances in Berry Supplementation and Age-Related Cognitive Decline. Current Opinion in Clinical Nutrition and Metabolic Care. 2009. 12:91-94.
- Shukitt Hale, B., Cheng, V., Joseph, J.A. 2009. Effects of Blackberries on Motor and Cognitive Function in Aged Rats. Nutritional Neuroscience.12:135- 140.
- Rabin, B.M., Joseph, J.A., Shukitt Hale, B. 2008. Effects of Berry Fruits on Neurocognitive Deficits Produced by Exposure to Space Radiation. In: Arora, R. Herbal Radiomodulators Applications in Medicine, Homeland Defence and Space. Wallingford, UK: Institute of Nuclear Medicine and Allied Sciences. p.151-161.
- Willis, L., Shukitt Hale, B., Cheng, V., Joseph, J.A. 2009. Dose-Dependent Effects of Walnuts on Motor and Cognitive Function in Aged Rats. British Journal of Nutrition. 101:1140-1144.
- Rabin, B.M., Carrihill-Knoll, K., Hinchman, M., Shukitt Hale, B., Joseph, J.A., Foster, B.C. 2009. Effects of Heavy Particle Irradiation on Diet on Object Recognition Memory in Rats. Advances in Space Research. 43:1193- 1199.
- Joseph, J.A., Neuman, A., Bielinski, D.F., Fisher, D.R. 2008. Blueberry Antagonism of C-2 Ceramide Disruption of CA2+ Responses and Recovery in MAChR-Transfected COS-7 Cells. Journal of Alzheimer's Disease.15:429-441.
- Willis, L., Shukitt Hale, B., Joseph, J.A. 2009. Modulation of cognition and behavior in aged animals: role of antioxidant and essential fatty acid rich plant foods. American Journal of Clinical Nutrition. 89:1602S-1606S.
- Lau, F., Joseph, J.A., Mcdonald, J.E., Kalt, W. 2009. Attenuation of iNOS and COX2 by blueberry polyphenols is mediated through the suppression of NF-KB activation. Journal of Functional Foods.1(3):274-283.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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. 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. Technology Transfer Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 281 Number of Newspaper Articles,Presentations for NonScience Audiences: 7
Impacts
(N/A)
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.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? By the year 2050, 30 percent of the total population will be over 65 years of age. There is a high probability that this group will exhibit the most common correlative motor and cognitive behavioral changes that occur in aging. These alterations occur even in the absence of specific age-related neurodegenerative diseases, but could interact to exacerbate the behavioral (motor and memory) aberrations exhibited in these conditions. In these cases, and possibly as a function of normal aging as well, it is likely that in cases of severe deficits in memory or motor function hospitalization and/or custodial care would be a likely outcome, with significant increases in health care costs. In both financial and human terms it is extremely important to explore methods to retard or reverse the
age-related neuronal deficits as well as their subsequent, behavioral manifestations. Very little is known about the mechanisms involved in these age-related declines in cognitive and motor behaviors. Attempts to reverse or retard these decrements have been, with very few exceptions, singularly unsuccessful. Even less is known concerning the nutritional modulation that could be employed to retard or reverse these declines. It has been postulated that these behavioral and neuronal declines are the result of an increasing inability to inactivate free radicals or inflammatories that impinge upon the organism, and an increasing vulnerability to these insults, thus creating a fertile environment for aging and the subsequent development of age-related, neurodegenerative diseases. This CRIS encompasses the work of the Nutrition and Neurocognition and the Neuroscience laboratories. The Nutrition and Neurocognition Laboratory aims to gain insight into the interaction between nutritional
factors and age-related cognitive, motor, brain or central nervous system declines in humans. Scientists in this laboratory believe that nutritional modification with fruits and vegetables high in antioxidants, anti-inflammatory activity, and certain B vitamins may be very effective. This program is directed to further specification and identification of these activities and the mechanisms involved in the positive benefits of dietary improvement and nutritional supplementation. Research conducted in the Neuroscience Laboratory has shown that supplementation with fruits and vegetables provide beneficial effects that include both forestalling and reversing the deleterious effects of aging on neuronal functioning and behavior. These effects appear to be the result of compounds (e.g., polyphenolics) that enhance the survivability of the plant, presumably through their antioxidant, and anti-inflammatory properties. While fruits and vegetables may have direct effects on oxidative stress and
inflammation in aging, preliminary data also indicate that polyphenolic compounds may have a plethora of additional effects involving enhanced signaling and neurogenesis in the aged animal that may also contribute to the observed benefits in motor and cognitive function. If this is the case, then it would be important to determine these additional mechanisms with a view toward the establishment of guidelines and dietary recommendations to an aging population concerning the qualitative and quantitative attributes of the fruits and vegetables. The objectives of this research are related to National Program 107 Human program components, 4. Nutrient Requirements; 5. Health Promoting Properties of Plant and Animal Foods; and 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. 2. List by year the currently approved milestones (indicators of research progress) Nutrition and Neurocognition Laboratory Objective 1 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. Objective 2 Characterize the mechanisms by which nutritionally induced hyperhomocysteinemia affects neuronal function and cognitive performance using transgenic mouse models of human cognitive decline. Milestones: 2005 1. Determine the relation between brain volumes, cognitive performance, and homocysteine in Framingham offspring over 10 years. Objective 1 2. Recruit Homebound Elderly for baseline studies. Objective 1 3. Achieve 13% of target enrollment in the FAVORIT Cognitive Ancillary Study. Objective 1 4. Determine the effect of aging in rats on one carbon metabolism with emphasis on difference among various tissues. Objective 2 5. Determine the impact of dietary imbalances in folate and/or methionine and their resultant homocysteinemias on Morris water maze performance young rats. Objective 2 6. Determine the impact of
chronic (43 week) B12-deficiency and its resultant homocysteinemia on Morris water maze performance and on the radiant-heat tail flick test in rats. Objective 2 7. Characterize the effect of chronic induced adult-onset homocysteinemia on several behaviors in a pilot study of transgenic mice with an inducible mutation in a homocysteine metabolizing gene (human cystathionine beta synthetase). Objective 2 2006 1. Further characterization of the effect of diet on vascular impairment in rodent models. Objective 2 2. Further characterization of cognitive dysfunction in rodent models for discerning between effect of homocysteine vs vitamin deficiency. Objective 2 2007 1. Complete and analyze cross-sectional studies of Homebound Elderly MRIs cognition, and nutritional biochemistries. Objective 1 2008 1. Complete and analyze cross-sectional baseline findings of cognition, and nutritional biochemistries in the FAVORIT Cognitive Ancillary Trial. Objective 1 2009 1. Complete data collection of
cognitive effect of homocysteine lowering. Objective 1 Neuroscience Laboratory Objective 1: (a) 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. (b) Assess the protective capability of berry fruit 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. Muscarinic receptors are involved in regulating both memory and motor function and show change with age. (c) 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 polyphenolic and berry fruit extracts. Objective 2: (a)
Establish the effects of dietary berry fruit extracts and the most effective component polyphenolics on neuronal function in aging by determining the effects on motor and cognitive behaviors as a function of age, (b) Identify brain regional localization of berry fruit compounds and correlating the amounts seen with the behavioral performance, and (c) Determine their effects on signaling and the generation of new neurons in aging. 2005 1 Develop and test procedures to construct chimerics and point mutations in M1 and M3 muscarinic receptors (AChR) and determine sensitivity to oxidative stress and inflammation. Objective 1a. 2. Identify blueberry and strawberry polyphenolics that offer protection against oxidative stress and inflammation in M1AChR-transfected COS 7 cells. Objective 1b. 3. Determine motor and cognitive behaviors in young and old rats following blueberry or strawberry supplementation. Objective 2a. 4. Begin to determine the extent of new neuron growth (neurogenesis) in
strawberry - or blueberry-fed animals Objective 2b. 2006 1. Determine the effects of lipid raft modification on oxidative stress and inflammation sensitivity. Objective 1a 2. Develop and test procedures to induce point mutations in M1 and M3AChR. Objective 1a 3. Determine the signaling (complex proteins that control cell communication and function) mechanisms involved in berry fruit protection in the cell models (e.g., COS-7 cells (Objective #1b; BV-2) and hippocampal cells. Objective 1c 4. Determine brain regions for signaling, neurogenesis and oxidative stress and inflammation markers, after behavior is determined in the rats. Objective 2a 5. Determine the alterations in complex proteins that affect communication between neurons and correlate these with the behavioral changes. Objectives 2a, 2c 2007 1. Determine oxidative stress and inflammation sensitivity in M1 and M3AChR transfected COS-7cells with point mutations in the transmembrane loops. Objective 1a 2. Determine the effect
of these mutations on the lipid raft microenvironment and determine if the berry fruits affect the microenvironment. Objective 1b 3. Begin to determine the protective effects of polyphenolic extracts derived from blueberries in our cell models. Objectives 1b, 1c. 4. Determine protective capacity against oxidative stress and inflammation insults in tissue obtained from young and senescent control animals or those supplemented with blueberries or strawberries. Objectives 2a 5. Begin to determine the localization the various blueberry or strawberry polyphenolics in the brain following blueberry or strawberry supplementation in the senescent or young rodents Objective 2b 6. Confirm neurogenesis effects in blueberry or strawberry fed animals using double and triple labels and their controls. Objective 2c 2008 1. Determine the effects of oxidative stress and inflammation lipid raft modification on sensitivity of COS-7 cells. Objective 1a 2. Begin gene array analyses on the effects of the
various alterations in mAChR structure on oxidative stress- and inflammation -induced gene expression. Utilize immunocytochemistry to validate the gene array analyses. Objective 1a 3. Determine the effects of blueberry or strawberry-derived polyphenolics identified in the cell work as showing oxidative stress and inflammation protection Objective 2a 2009 1. Complete gene array analyses on the effects of the various alterations in mAChR structure on oxidative stress- and inflammation -induced gene expression. Utilize immunocytochemistry to validate the gene array analyses. Objective 1a 2. Determine signaling effects of oxidative stress, inflammation, blueberry or strawberry effects in the BV and hippocampal cell models and relate to changes in gene markers Objectives 2a and 2c. 3. Determine the localization of the various blueberry or strawberry polyphenolics in the striatal and cortical areas following blueberry or strawberry supplementation in the senescent or young rodents Objective
2b 4a List the single most significant research accomplishment during FY 2006. Animal Models of Homocysteine-Related Cognitive Dysfunction: The Nutrition and Neurocognition Laboratory developed rodent models to test homocysteine-related cognitive function. Cognitive decline and dementia afflicts up to one-third of elders over the age of 75. With the aging of the US population it is vital to develop means of preventing or slowing these cognitive impairments. Nutritional factors such as B- vitamin deficiency and its associated elevations of blood homocysteine concentrations (homocysteinemia) might modify the course of brain aging and provide an opportunity to intervene in age-related cognitive impairments. The development of physiologically relevant animal models of these processes is necessary in order to determine the relation of nutrition to brain aging in the human population and to develop effective dietary and other interventions. We demonstrated that feeding diets that raised
homocysteine levels in genetically engineered mice prone to vascular disease and brain degeneration causes poor performance on a specific test of memory and learning and subtle chemical changes in brain without causing apparent degeneration to brain cells, or increasing the deposition of Alzheimers disease type brain protein deposits. The diets that caused these changes were different than those that enhanced damage to aorta. Our findings suggest that different nutritional imbalances that raise homocysteine levels may lead to different end organ dysfunctions and diseases. They do not support the idea that homocysteinemia accelerates Alzheimers type pathology directly, but rather that it may contribute to cognitive dysfunction in Alzheimers disease and through a separate mechanism of brain damage. This work is aligned to National Program 107 Human Nutrition component 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. 4b
List other significant research accomplishment(s), if any. Nutrition and Neurocognition Laboratory Homocysteine and cognitive performance A diet deficient in folate and vitamin B12 raised blood homocysteine levels and impaired cognitive performance in mice transgenic for the human mutant amyloid precursor protein. These animals showed increased sensitivity to high homocysteine in behavioral measures. These findings have a potential impact on determining nutrient requirements for the maintenance of brain health and cognitive function in Alzheimers disease. This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. . Homocysteine and vascular toxicity Prepared brain tissue from aged transgenic homocysteinemic and control mice for measurement of brain vascular density. The brains of B vitamin- deficient hyperhomocysteinenemic mice had smaller capillary vascular beds. This method is used to identify nutrient requirements for maintaining brain
vascular health and cognitive function as humans age (objective 2). This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. Homocysteine and amino acid metabolism Developed methodology for determining amino acid imbalances which could effect neuronal function in brain tissue from homocysteinemic rodents (objective 2). These results have the potential impact on determining nutrient requirements for the maintenance of brain health and cognitive function in old age). This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. Brain aging and insulin Determined brain insulin like-growth factor type 1 and glucose transporter content as markers of microvascular damage in homocysteinemic rat brain (objective 2). These results have the potential impact on determining nutrient requirements for the maintenance of brain health and cognitive function in old age. This work is aligned to National Program 107 Human
Nutrition component 4 Nutrient Requirements. Homocysteine lowering trial with B vitamins Enrolled 35% of target population in the FAVORIT cognitive ancillary study and analyzed preliminary cognitive data from subjects enrolled during first year of the trial. Trial will determine effect homocysteine lowering on cognitive decline after 5 years. (objective 1). This work has the potential impact on determining nutrient requirements for the maintenance of brain health and cognitive function in old age. This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. Vitamin K and cognition Prepared tissue samples from Fisher rats (F344) to study the effect of dietary vitamin K on sulfatide levels which might mediate function in different organs and in brain regions associated with behavior of males and female Fisher rats in aging (collaborative study with Vitamin K Laboratory). This work has a potential impact on determining nutrient requirements for the
maintenance of brain health and cognitive function in old age (objective 2). This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. Animal models of cognitive behavior: Designed and implemented methods to measure cognitive behavior in rodent models of aging and hyperhomocystenemia These experiments are designed to examine the impact of nutritive status and aging on cognitive performance. The use of animal models allows for precise manipulations of diet and nutritive status in the examination of cognition in a manner that is not possible in human studies (Objective 2) This work is aligned to National Program 107 Human Nutrition component 4 Nutrient Requirements. Neuroscience Laboratory Oxidant Signaling Reductions by Blueberry Extract in COS-7 cells Milestone 3, 2006: Determine the signaling (complex proteins that control cell communication and function) mechanisms involved in berry fruit protection in COS-7 cells (Objective 1c). The
most significant accomplishment during FY2006 addressed the problem of identifying the role of blueberry supplementation in lowering oxidative stress signaling in COS-7 cells. COS-7 cells are used as a model in these experiments, since they normally do not contain muscarinic receptors and this enables us to transfect them with one of five subtypes of muscarinic receptors so that we can study the response of these receptors to the various polyphenolic compounds in berry fruit in isolationthat is without the interference of other muscarinic receptors. The polyphenolic compounds contained in blueberries and other berry fruits can act as potent antioxidants. Muscarinic receptors (MAChRs) are intimately involved in various aspects of both neuronal and vascular functioning, and there is selective oxidative stress sensitivity (OSS) among MAChR subtypes with M1, M2, and M4 showing > OSS than the COS-7 cells transfected with M3 or M5 receptor subtypes. We assessed OSS by examining the
inability of the cell to extrude or sequester calcium following stimulation of the cell with a compound called oxotremorine, which causes calcium to move into the cell from outside the cell. Some cells are exposed to dopamine (DA) an oxidative stressor or amyloid beta, (a peptide found in the brains of Alzheimer disease patients that is associated with neurotoxicity). Some of the COS-7 muscarinic receptor-transfected COS-7 cells (e.g., M1) are pretreated with BB or other berry fruit extracts (e.g., strawberry) to determine if the changes in calcium can be prevented. We made changes in the structure of both M1 and M3 receptors by altering a particular loop in these receptors or switching the i3 loop from the M1 or M3 receptors to determine if these changes would affect receptor sensitivity. The study was carried out to determine if: a) BB treatment of the cells transfected with wild type (no changes in the structure of the receptor), truncated (I3 loop removed) or chimeric [where
the i3 loop of one receptor was switched with i3 loop of the other; i.e., M1(M3i3) and M3(M1i3)] receptors would alter DA-induced changes in calcium buffering and would confer protection through alterations in phospho (p) mitogen activated protein kinase (MAPK), p cyclic AMP response element binding protein (CREB) or protein kinase C (PKC) signaling. These are important molecules that serve as signaling agents to oxidative stress. Thus, they are called stress signals. The findings suggested that M1/M3 OSS differences may involve differential signaling in pMAPK and pCREB, under OS-treatment conditions, with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggested that BB may antagonize OS effects by lowering activation of pCREB and possibly PKC? induced by DA. In the truncated (with the i3 loop removed) and chimeric (the i3 loop of an M1 switched with and M3 and the reverse) receptors, findings indicated that BB reduced OSS to DA in M1-transfected
cells. However, BBs were also effective in preventing these Ca2+ buffering deficits in cells transfected with truncated M1 receptors but only partially enhanced the protective effects of the M3 i3 loop in the M1(M3i3) chimerics. A similar partial effect of BBs was seen in the M3(M1i3) chimerics which showed increased OSS to DA. It appeared that antioxidants found in BBs might be targeting additional sites on these chimerics to decrease OSS. These initial findings have already made some impact on the scientific community when presented at recent national and international meetings, and the work concerned with reduction of stress signals has been cited in the popular press. The paper reporting on these studies has been submitted to the Journal of Alzheimer Disease. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. A. Milestone 1, 2006: Determine the effects of lipid raft modification on oxidative
stress and inflammation sensitivity (Objective 1a). A plethora of research suggests that the naturally occurring sphingolipid, ceramide, may have several negative cellular effects, including: oxidant formation, mitogenic stimuli, and cytokine formation. It also appears that negative responses to ceramide increase as a function of age. The present study was carried out to determine whether: a) C2 ceramide would differentially affect M1 and M3 AChR- transfected COS-7 cells and b) BB treatment would prevent any deleterious effects of C2 ceramide on oxo- induced calcium buffering. Results, thus far, have suggested that, unlike previous findings with H202 and DA, C2 ceramide disrupted oxo-induced responses in both M1 and M3 transfected cells. However, BB-treatment of the cells offered protection against the effects of the C2 ceramide in M1 but not M3- transfected cells. Although previous findings showed that the M3 i3 loop offered protection against DA induced deficits in Ca2+ buffering,
this protection does not extend to ceramide. We are assessing possible differences in PKC isoforms (e.g., epsilon), CREB, and caspases that could account for these differences, but previous findings indicate that BB induction of protective ERK activity is higher in M1-transfected COS-7 cells than those transfected with M3AChR, suggesting that magnitude of ERK signaling may be important in this protection. The potential impact of this work is that for the first time we will be able to begin to discern the mechanisms of action of berry fruit at the molecular level. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. B. Milestone 4, 2006: Determine brain regions for signaling, neurogenesis and oxidative stress and inflammation markers, after behavior is determined in the rats (Objective 2a). Previously, we have shown that whole, crude blueberry (BB) extracts are able to reverse several parameters of
brain aging (e.g., deficits in cell communication) as well as age-related motor and cognitive deficits when fed to rats 19-21 months of age. Last year we assessed 3 different BB- derived diets: control diet or one with 5.4% crude BB extract a 2% pre- C18 column BB extract, or a 0.1% post-C18 column semi-purified BB extract (a mixture of only BB phenolics with the sugars and organic acids removed) , all equated on phenolic level, to determine whether the effects observed with the whole, crude blueberry extract are indeed due to polyphenolics or whether other compounds were contributing to the age- related improvements in behavior. Results showed that only the crude blueberry extract diet improved rotarod (a slowly turning rod in which we assess the length time that the rat can remain on the rod-it is a measure of motor function which, includes coordination an balance). performance, while all three blueberry-derived diets improved working memory in the Morris water maze. This year we
extended these findings as per Milestone 4 where new neuronal growth (neurogenesis) in these groups was assessed by using cell counts of bromodeoxyuridine (BrdU). BrDU assesses the rate the rate of incorporation of DNA into a cell. It assessed the amount of new cells formation, since DNA is primarily incorporated into newly formed cells in the hippocampus. This is an important memory control area that shows considerable decline in function, as well as reductions in neurogenesis in aging. The results of these analyses indicated that there was a trend for the animals fed the crude BB extract to show increases in BrdU incorporation into the hippocampus, while the animals fed the pre or post C-18 BB extract diets showed significantly more neurogenesis than either the control or crude BB extract fed diets. There was a greater number of animals that were fed the supplemented diets that showed enhanced performance on Morris water maze (MWM) performance than seen in the control diet fed
animals. Many of the non-supplemented diets showed negative performance between trial 1 and trial 2 in the MWM (61.5%) , while in the supplemented groups negative performance was 21% or less, suggesting a significant percentage of each supplemented group showed enhanced trial 2 performance on the task. Therefore, one mechanism involved in this enhanced increased cognitive performance may be related to increased neurogenesis by BB polyphenols. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. C. Milestone 4, 2006: Determine brain regions for signaling, neurogenesis and oxidative stress and inflammation markers, after behavior is determined in the rats. Objective 2a. Colleagues from the ARS Natural Products Utilization Research Unit in Mississippi showed that pterostilbene, a natural methoxylated analog of resveratrol which is found in blueberries, showed antioxidant activity that was as active as
resveratrol and Trolox (a soluble form of Vitamin E) in antagonizing herbicide-induced oxidative damage assessed as electrolyte leakage from cucumber plant tissue. Pterostilbene also showed some inhibition against COX-1, and weak inhibition of COX-2, suggesting some anti-inflammatory activity, as well as some cholesterol lowering activity. Given these findings, we carried out studies to determine whether resveratrol analogs, including pterostilbene, would be effective in protecting against oxidative stress in our COS-7 cells transfected with muscarinic receptors that increased the vulnerability of these cells to such stressors. Pterostilbene pretreatment, in particular, was effective in protecting against an oxidative stressor regarding the ability of the cell to clear calcium. Therefore, we conducted another study to determine whether pterostilbene-supplemented senescent rodents would show reversals in cognitive deficits as compared to unsupplemented animals. The high dose of
pterostilbene, 0.016%, equivalent to 10mg/kg body weight, was effective in improving working memory performance in the Morris water maze test, which measures spatial learning and memory, compared to the control group. Therefore, pterostilbene has been shown to protect against oxidative stress and inflammation, the two stressors seen in aging, in several different models. This is an extremely important finding which shows the efficacy of resveratrol-like compounds on aging. We are applying for a patent on this compound. If these studies are supported by additional research, it could mean that we have important compound to prevent or reduce the deleterious effects of brain aging on behavior. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. D. Milestones 3 and 5, 2006: Determine the signaling (complex proteins that control cell communication and function) and gene activation mechanisms
involved in berry fruit protection in the cell models and animals (Objectives 1c, 2a, 2c). D1) We are constructing a rat brain mini-cDNA library containing 24 genes that are involved in inflammation, oxidative stress and cell death signaling pathways along with three house-keeping genes. These genes formed a panel of indicators for the study of the beneficial effects of blueberries on gene regulation in the rat brain or in cell-culture paradigms. We used a subset of these to determine gene alteration in blueberry-fed animals and controls exposed to central administration of the neurotoxin, kainic acid (see D2). Using a murine microglial cell line (BV-2), that has been used previously as an in vitro model for the study of pathogenesis in Alzheimers disease (AD), we found that treatment with blueberry (BB) extracts significantly and dose-dependently reduced the lipopolysaccharide (LPS)-induced NO production in conditioned media from BV2 murine microglial cells. Reactive oxygen species
(ROS) release was also reduced in BB-treated LPS-activated BV2 cells., BB extracts significantly attenuated the protein expression of the inducible NO synthase (iNOS), cyclo-oxygenases 2 (COX-2), and the pre-processed form of IL1-beta in the LPS-activated BV2 cells. Furthermore, the secretion of the inflammatory cytokines IL-1 beta and TNF-alpha into the conditioned media from the LPS-activated BV2 cells was inhibited by BB treatment. The results from this study suggest that BB polyphenols attenuate inflammatory responses of the brain microglial cells and could be used to modulate inflammatory conditions in the central nervous system. D2) We investigated whether the polyphenols in blueberries (BBs) can reduce the deleterious effects of inflammation induced by central administration of kainic acid (KA), an inflammatory agent by altering the expression of genes associated with inflammation. Four- month old male Fischer 344 (F344) rats were fed a control, 0.015% piroxicam (PX, an
NSAID) or 2% BB diet for 8 weeks before either Ringers (R) or KA was bilaterally microinfused into the hippocampus. Two weeks later, following behavioral evaluations, the rats were sacrificed and total RNA from the hippocampus was extracted and used in real-time quantitative RT-PCR (qRT- PCR) to analyze the expression of inflammation-related genes. Behavioral studies showed that KA had deleterious effects on cognitive behavior as KA-injected rats on the control diet exhibited increased latencies to find the hidden platform in the Morris water maze (MWM) compared to R- injected rats and utilized non-spatial strategies during probe trials. The BB diet, and to a lesser degree the PX diet, was able to improve cognitive performance. Immunohistochemical analyses of OX-6 expression revealed that KA produced an inflammatory response by increasing the OX-6 positive areas in the hippocampus of KA-injected rats. Further analyses showed that KA up-regulated the expression of inflammatory
cytokines IL-1 beta and TNF-alpha, the neurotrophic factor IGF-1 and the transcription factor NF-KB, which are all important molecules in stress and stress signaling. BB and PX supplementations were found to reduce the expression of IL-1 beta, TNF-alpha, and NF-KB, while only BB was able to increase IGF-1 expression. These results indicate that BB polyphenols exert anti- inflammatory actions, perhaps via alteration of gene expression. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. 4d Progress report. The Effects of California Dried Plums on Cognitive and Motor Function in Aging 1950-51000-063-03S This report serves to document research conducted under a Specific Cooperative Agreement between Agri-Canada and ARS. In a 2005 study conducted in conjunction with 51000-063-4T, a Trust Fund Cooperative Agreement between the California Dried Plum Board and ARS, we tested dried plum powder for its
efficacy in reversing cognitive and motor deficits in aged rats. We supplied plasma collected from the animals tested in 51000-063- 4T to scientists from Agri-Canada for Oxygen Radical Absorbance Capacity (ORAC) testing. No significant differences were seen in plasma ORAC between the dried plum supplemented and control animals, suggesting that the supplementation did not increase antioxidant activity. The study was completed and terminated this year. We are preparing a manuscript that reports on these results. The Effects of California Dried Plums on Cognitive and Motor Function in Aging 1950-51000-063-04T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the California Dried Plum Board. In fiscal year 2004 we tested the effects on cognitive and neuronal function of dried plum powder that was incorporated into an NIH-31 diet. The results indicated that there were no beneficial effects observed in either motor or cognitive
behavior in the plum powder. We also saw no differences between control and supplemented groups on oxotremorine regulation of striatal dopamine release. After having the powder further tested (see report for 51000-063-03S) for polyphenol levels, we concluded that many of the polyphenols, particularly anthocyanins, were not present. Therefore, in 2005 we repeated the experiments and gave the rats plum juice from which the dried plum extract was made. We found significant reversals in deficits in cognitive function in the rats as measured by Morris water maze performance. This year, we have finished analyzing the results and are preparing a manuscript that reports on these findings. We have just received a short extension from the California Dried Plum Board to look at the anti-inflammatory effects of the dried plum juice in our BV-2 cells. THE EFFECTS OF PURPLE GRAPE JUICE ON COGNITIVE AND MOTOR DEFICITS IN AGING 1950-51000-063-05T This report serves to document research conducted
under a Trust Fund Cooperative Agreement between ARS and the Welchs Foods. Animals and humans show increased motor and cognitive declines with aging, which are thought to be due to increased susceptibility to the long-term effects of oxidative stress and inflammation. Previous findings have suggested that improvements in these age-related declines might be accomplished by increasing the dietary intake of polyphenolics found in fruits and vegetables, especially those identified as being high in antioxidant and anti-inflammatory activity. Therefore, we investigated the beneficial effects of two concentrations (10% and 50%) of Welch's Concord grape juice compared to a calorically-matched placebo for their effectiveness in reversing age-related deficits in behavioral and neuronal function in aged Fisher 344 rats. Rats that drank the 10% grape juice from age 19 to 21 months had improvements in oxotremorine-enhancement of K+-evoked release of dopamine from striatal slices as well as
cognitive performance on the Morris water maze, while the 50% grape juice produced improvements in motor function. These findings suggest that, in addition to their known beneficial effects on cancer and heart disease, polyphenolics present in foods may be beneficial in reversing the course of neuronal and behavioral aging, possibly through a multiplicity of direct and indirect effects that can affect a variety of neuronal parameters. The manuscript written from this study has been published, and is cited below. This year we continued the study of grape juice by assessing juices of varying anthocyanin or proanthocyanin levels on these behaviors in aged animals. The behavioral assays have been carried out, as well as assessments of dopamine release, and we are now analyzing the results. LOCALIZATION OF FRUIT POLYPHENOLICS IN VITRO: BENEFICIAL BIOLOGICAL ACTIONS 1950-51000-063-06T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS
and the Wild Blueberry Association of North America. This trust has been terminated and work is continuing under 51000-063-12T Mechanisms Involved in the Beneficial Effects of Blueberries on Neuronal Aging and Behavior. Additional details of this research can be found in the report for the parent CRIS 1950-51000-063- 00D Nutritional Modulation of Brain Aging and Cognitive Decline. THE LOCALIZATION OF FRUIT POLYPHENOLOCS IN VIVO: BENEFICIAL BIOLOGICAL ACTION 1950-51000-063-07T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the US Highbush Blueberry Council. This trust has been terminated and work is continuing under 51000-063-13T Mechanisms Involved in the Beneficial Effects of Blueberries on Neuronal Aging and Behavior THE ROLE OF DIETARY ANTIOXIDANTS AND AGING IN THE DELETERIOUS EFFECTS OF OXIDATIVE AND INFLAMMATORY INSULTS VIA HEAVY PARTICLE IIRRADIATION 1950-51000-063-08R This report serves to document research conducted
under a Reimbursable Cooperative Agreement between ARS and the University of Maryland Baltimore County (UMBC). Previous work has shown that exposure to heavy particle irradiation produces neurochemical and cognitive deficits in young animals that are characteristic of much older animals. As a result, it has been suggested that exposure to heavy particles can produce accelerated aging. In collaboration with scientist from UMBC, we found that the doses of 56Fe particles needed to affect neurobehavioral endpoints decreases as a function of the age of the rat. For both measurements of anxiety using the elevated plus maze and responsiveness to environmental contingencies using operant responding on an ascending fixed-ratio schedule, the older animals are more susceptible to the effects of irradiation than are the young animals. It appears from our findings last year that age may be a risk factor for the behavioral effects of exposure to heavy particles. The effects are not necessarily
linear (i.e., middle aged rats seem more susceptible to the deleterious effects of exposure to 56Fe particles than young or very old animals). This year we showed that there are a series of age-related decrements in performance following exposure to 56Fe particles. Operant responding on an ascending fixed-ratio schedule (pressing the bar a fixed number times to receive a reward) has been used to evaluate age-related decrements in performance because the same animals can be repeatedly tested on that task over intervals of up to 12 to 18 months. This test has revealed an age-related deterioration in performance as a function of age and time. However, this research also revealed that rats that had been maintained on 4% blueberry- or strawberry-supplemented diets and tested one year after exposure to the radiation showed no deficit in response rate when compared to the non-irradiated rats maintained on the control diet. Additional findings emerging from the blueberry and strawberry
supplementation studies indicated that maintaining organisms on diets containing antioxidant phytochemicals (blueberry and strawberry extract) can provide a significant degree of radiation protection, depending upon the specific diets and neurobehavioral endpoints. In this respect, findings indicate that maintaining rats on diets containing blueberry or strawberry extract at the time of radiation can prevent the occurrence of both the acute and late degenerative changes that result from exposure to 56Fe particles. Both strawberries and blueberries rank high in their ability to scavenge free radicals and the observation that they are capable of ameliorating the effects of exposure to 56Fe particles is consistent with the observation that exposure to heavy particles produces oxidative stress and that oxidative stress in the central nervous system may play a role in mediating the neurobehavioral consequences of irradiation. Data cited last year indicated that the effects of antioxidant
diets on the neurobehavioral effects of exposure to 56Fe particles vary as a function of diet and endpoint. In cognitive tasks the 2% strawberry diet prevented the late degenerative changes in paradigms testing place memory, while 2% blueberry supplementation was very effective in preventing deterioration in spatial memory. This year we showed that at least 8 weeks of feeding of the antioxidant diets may be necessary to achieve this protection, since short-term supplementations (2 weeks) did not significantly protect against the loss of radiation-induced memory performance. The results also suggest that a dietary cocktail may be necessary to provide the maximum protection against radiation. These findings have important implications for aging, since it may be that a similar berry cocktail may be important in forestalling or reversing the deleterious effects of aging. The results of the study regarding radiation and diet, and effects on spatial memory, have been accepted for
publication in Neurobiology of Aging THE EFFECTS OF POMEGRANATE JUICE ON COGNITIVE AND MOTOR DEFICITS IN AGING 1950-51000-063-09T This report serves to document research conducted under a Cooperative Agreement Trust between ARS and Pom-Wonderful/the Stewart and Linda Resnick Revocable Trust of Los Angeles, CA. In the first study funded by this organization the results that we obtained indicated only minimal beneficial effects of pomegranate juice (POM) on behavior in the aged animals. There were no differences between the groups on any of the motor tests. When examining Morris water maze (cognitive) performance, we performed separate t-tests between the two trial latencies for each group, to determine if the different juice groups significantly improved their performance from Trial 1 to Trial 2, showing improved working memory. Trial 1 measures reference memory while Trial 2 is a measure of working memory. No effects were seen in the high POM group. The low POM juice showed improved
working memory (i.e., there was a significant difference between Trial 1 and Trial 2 performance in this group), however, Trial 1 performance in the low POM juice group was worse than the group given water to drink, i.e., the low POM juice group took longer to find the hidden platform on the reference memory trial. Working memory performance was not different between the groups. Therefore, one cannot conclude that the low POM juice had improved performance compared to the water group. There were indications that the muscarinic receptor sensitivity was increased in the striata of the aged animals as we have seen previously with the blueberries. For these reasons we believed that the concentrations used in these studies were too low (.33 % and 3.3%) to see an effect on behavior. However, it was encouraging that both concentrations had an effect on muscarinic receptor sensitivity. We have received additional funding from Pom-Wonderful to carry out the behavior and neuronal
evaluations again using higher doses of a concentrated extract of the pomegranates. THE EFFECTS OF STRAWBERRIES ON COGNITION AND NEURONAL COMMUNICATION IN AGING: MECHANISTIC CONSIDERATIONS. All studies with pomegranate juice were completed in 2005. 1950-51000-063-10T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the California Strawberry Commission. Previously, we have shown that whole, crude berry extracts are able to reverse several parameters of brain aging, as well as age- related motor and cognitive deficits when fed to rats from 19-21 months of age. These effects may be the result of direct effects on brain signaling or indirect effects through antioxidant and anti-inflammatory properties of the polyphenols. The study conducted this year examined two different berry fruit diets (blueberry, BB and strawberry, SB) to determine whether the effects observed are indeed the result of differential effects of the polyphenols
on the brain. Old (19 mo) F344 rats were fed a control, 2% BB, or 2% SB diet for 8 weeks prior to motor and cognitive testing. 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 berry fruit diets enhanced working memory in the Morris water maze. We are currently assessing regional localization of the BB and SB polyphenols and their putative differential effects on signaling parameters in these rats, with a view toward determining selective bioavailability and mechanism(s) of action. THE EFFECTS OF WALNUTS ON COGNITIVE AND NEURONAL COMMUNICATION IN AGING: POSSIBLE SYNERGISTIC EFFECTS WITH BLUEBERRIES 1950-51000-063-11T This report serves to document research conducted under a Cooperative Trust Agreement between ARS and the California Walnut Commission. The initial findings have been analyzed from our
first study in which we assessed Morris water maze and motor performance in senescent rats maintained for 8 weeks on a control diet or one containing 2% blueberry extract (BB), 2% walnut powder, or 1% BB and 1% walnut powder. Results indicated that, overall, either BB or walnut supplementation produced increases in the mean difference between Trial 1 and Trial 2 performance in the Morris water maze, indicating a greater degree of memory for the platform location in the supplemented groups than in the control group. Initial analysis of the motor behavioral data indicated that there was some increase in performance in all of the supplemented groups in rotorod performance. However, we are still analyzing these and other results and will also be correlating these with our measurements of neuronal function. MECHANISMS INVOLVED IN THE BENEFICIAL EFFECTS OF BLUEBERRIES ON NEURONAL AGING AND BEHAVIOR 1950-51000-063-12T This report serves to document research conducted under a Trust Fund
Cooperative Agreement between ARS and the Wild Blueberry Association of North America. Previously, we found that one of the major effects of blueberry (BB) application to cells may be to alter oxidative stress/inflammatory signaling. Our findings suggest that BB may antagonize oxidative stress effects by enhancing phospo (p) mitogen activated protein kinase (MAPK) and lowering activation of p cyclic AMP response element binding protein (CREB). pCREB is important in acetylating Forkhead proteins, which prevents their interaction with sirtuins. Sirtuins are very important in mediating cellular health and functioning. Since BB supplementation appears to lower pCREB activation the Forkhead proteins are not deactivated. We also showed that in M1muscarinic receptor chimerics (M1 receptors containing an M3 muscarinic receptor i3 loop) the dopamine- induced increases in pMAPK were reduced, as were the increases in pCREB, suggesting that at least part of the protection against oxidative
stress- induced decrements in calcium buffering seen in the chimerics may be mediated through the i3 loop of the receptor and enhancement of pCREB. Although it appears that oxidative stress induced deficits in calcium buffering in the M3 truncated - and chimeric-transfected COS 7 cells, it is clear that these changes are less dependent upon alterations MAPK or CREB. It appears that antioxidants might be targeting additional sites on these chimerics to decrease oxidative stress sensitivity. This year we found that BBs also interact with genes that regulate oxidative and inflammatory stressors (e.g., IL-1beta). These genes are downregulated in BV-2 mouse microglial cells treated with the inflammatory agent lipopolysaccharide (LPS). Treatment with BB extracts significantly and dose-dependently reduced the lipopolysaccharide (LPS)- induced NO production in conditioned media from these cells. Reactive oxygen species (ROS) release was also reduced in BB-treated LPS-activated BV2 cells. In
addition, BB extracts significantly attenuated the protein expression of the inducible NO synthase (iNOS), cyclo-oxygenases 2 (COX-2) , and the pre-processed form of IL-1beta in the LPS-activated BV2 cells. The secretion of the inflammatory cytokines IL-1beta and TNF-alpha into the conditioned media from the LPS-activated BV2 cells was inhibited by BB treatment. The results from this study suggest that BB polyphenols attenuate inflammatory responses of the brain microglial cells and could be used to modulate inflammatory conditions in the central nervous system. . MECHANISMS INVOLVED IN THE BENEFICIAL EFFECTS OF BLUEBERRIES ON NEURONAL AGING AND BEHAVIOR 1950-51000-063-13T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the Wild Blueberry Association of North America. Last year we assessed three different BB-derived diets, all equated on phenolic levels, to determine whether the effects observed with the whole, crude BB
extract are due to polyphenolics or if the other compounds were contributing to the age-related improvements in behavior. Old (19 mo) F344 rats were fed a control diet or one with 5.4% crude BB extract (as before), a 2% pre-C18 column BB extract, or a 0.1% post-C18 column semi- purified BB extract (a mixture of only BB phenolics with the sugars and organic acids removed) for 8 weeks prior to motor and cognitive testing. Results showed that only the crude BB extract diet improved rotarod performance, while all three BB-derived diets improved working memory in the Morris water maze. In work recently completed, we carried out analyses of new neuronal growth (neurogenesis) in these groups by using cell counts of bromodeoxyuridine (BrdU) incorporation in dentate gyrus of the hippocampus. This is an important memory control area that shows considerable decline in function, as well as reductions in neurogenesis in aging. The results of these analyses indicated that there was a trend for the
animals fed the crude BB extract to show increases in BrdU incorporation into the dentate gyrus, while the animals fed the pre or post C-18 BB extract diets showed significantly more neurogenesis than either the control or crude BB extract fed diets. There was a greater number of animals that showed enhanced performance on the Morris water maze performance task among those fed the supplemented diets than seen in the control diet fed animals. Many of the non-supplemented diets showed negative performance between trial 1 and trial 2 in the Morris water maze (61.5%), while in the supplemented groups negative performance was 21% or less, suggesting a significant percentage of each supplemented group showed enhanced trial 2 performance on the task, indicating improved working memory. Therefore, one mechanism involved in this enhanced increased in cognitive performance may be related to increased neurogenesis by BB polyphenols. 5. Describe the major accomplishments to date and their
predicted or actual impact. Over the life of the project the Nutrition and Neurocognition Laboratory has observed in human population studies that common vitamin B12 and folate deficiencies and high blood homocysteine levels are often associated with cognitive decline and dementia. It is not clear if and how these conditions contribute to the observed cognitive impairments. In animal studies scientists fed rodents experimental diets that increased blood homocysteine levels from either folate or vitamin B12 deficiency or by high intake of methionine without vitamin deficiency. These models pave the way for elucidating the relation of dietary imbalances in these factors and cognitive decline in human populations. These findings are related to Objective 2, Milestone 6 for 2005, and to National Program 107 - Human Nutrition program components: 4. Nutrient Requirements; and 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. Neuroscience Laboratory
2005: A. Previous experiments under Milestone 1 have revealed that deletions of the entire i3 loop increased dopamine (DA) sensitivity (where a lower percentage of cells showing recovery following depolarization) in both the M1 and M3 subtypes. Additionally, chimerics are created by switching the i3 loop of the M3AChR with the i3 loop of the M1AChR (M1M3i3) and the reverse) of M1 (showed that the DA sensitivity was reduced (percent of cells showing increases in calcium clearance) following depolarization. In the M3 chimerics containing M1i3 (M3M1i3), the i3 loop offered no protection against DA-induced decrements in calcium buffering. We found that M1/M3 differences in oxidative stress vulnerability may involve differential signaling in pMAPK (phospho mitogen activated protein kinase) and pCREB (phospho cyclic AMP response element binding protein) under oxidative stress treatment conditions, with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggest
that blueberries may antagonize oxidative stress effects by enhancing pMAPK and lowering activation of pCREB. Interestingly, in the M1 chimerics the DA induced increases in pMAPK were reduced, as were the increases in pCREB, suggesting that at least part of the protection against oxidative stress calcium buffering seen in the chimerics may be mediated through the i3 loop and the alterations in pCREB and pMAPK transcription factors. This is the first time that the possible actual locus of the antioxidant effect of blueberries in receptor structures has been identified. This should impact the design of new agents to increase their specificity and efficacy against stress signaling. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. B. Determine motor and cognitive behaviors in young and old rats following strawberry or blueberry supplementation. Milestone 3 for 2005. Previously, we had shown that
whole, crude blueberry (BB) extracts are able to reverse several parameters of brain aging (e.g., deficits in cell communication) as well as age-related motor and cognitive deficits when fed to rats from 19-21 months of age. These effects appear to be the result of compounds (polyphenolics) that enhance the survivability of the plant, possibly through direct effects on brain signaling or indirectly through their antioxidant and anti-inflammatory properties. We assessed 3 different BB-derived diets, all equated on phenolic level, to determine whether the effects observed with the whole, crude blueberry extract are indeed due to polyphenolics or whether other compounds were contributing to the age-related improvements in behavior. Old (19 month) F344 rats were fed a control diet or one with 5.4% crude blueberry extract (as before), a 2% pre-C18 column BB extract, or a 0.1% post-C18 column semi- purified blueberry extract (a mixture of only BB phenolics with the sugars and organic acids
removed) for 8 weeks prior to motor and cognitive testing. Results showed that only the crude blueberry extract diet improved rotarod performance, while all three blueberry-derived diets improved working memory in the Morris water maze. Therefore, phenolics are important components in the beneficial effects of blueberries on age-related improvements in cognition, but other compounds may play a role in motor improvements. This is the first attempt at extraction to identify the active components of the blueberries that are responsible for the beneficial effects on aging. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. C. Oxidant Signaling Reductions by Blueberry Extract in COS-7 cells The most significant accomplishment during FY2006 addressed the problem of identifying the role of blueberry supplementation in lowering oxidative stress signaling in COS-7 cells. COS-7 cells are used as a model
in these experiments, since they normally do not contain muscarinic receptors and this enables us to transfect them with one of five subtypes of muscarinic receptors so that we can study the response of these receptors to the various polyphenolic compounds in berry fruit in isolationthat is without the interference of other muscarinic receptors. The polyphenolic compounds contained in blueberries and other berry fruits can act as potent antioxidants. Muscarinic receptors (MAChRs) are intimately involved in various aspects of both neuronal and vascular functioning, and there is selective oxidative stress sensitivity (OSS) among MAChR subtypes with M1, M2, and M4 showing > OSS than the COS-7 cells transfected with M3 or M5 receptor subtypes. We assessed OSS by examining the inability of the cell to extrude or sequester calcium following stimulation of the cell with a compound called oxotremorine, which causes calcium to move into the cell from outside the cell. Some cells are exposed
to dopamine (DA) an oxidative stressor or amyloid beta (A?), (a peptide found in the brains of Alzheimer disease patients that is associated with neurotoxicity). Some of the COS-7 muscarinic receptor- transfected COS-7 cells (e.g., M1) are pretreated with BB or other berry fruit extracts (e.g., strawberry) to determine if the changes in calcium can be prevented. We made changes in the structure of both M1 and M3 receptors by altering a particular loop in these receptors or switching the i3 loop from the M1 or M3 receptors to determine if these changes would affect receptor sensitivity. The study was carried out to determine if: a) BB treatment of the cells transfected with wild type (no changes in the structure of the receptor), truncated (I3 loop removed) or chimeric [where the i3 loop of one receptor was switched with i3 loop of the other; i.e., M1(M3i3) and M3(M1i3)] receptors would alter DA-induced changes in calcium buffering and would confer protection through alterations in
phospho (p) mitogen activated protein kinase (MAPK), p cyclic AMP response element binding protein (CREB) or protein kinase C (PKC) signaling. These are important molecules that serve as signaling agents to oxidative stress. Thus, they are called stress signals. The findings suggested that M1/M3 OSS differences may involve differential signaling in pMAPK and pCREB, under OS-treatment conditions, with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggested that BB may antagonize OS effects by lowering activation of pCREB and possibly PKC? induced by DA. In the truncated (with the i3 loop removed) and chimeric (the i3 loop of an M1 switched with and M3 and the reverse) receptors, findings indicated that BB reduced OSS to DA in M1-transfected cells. However, BBs were also effective in preventing these Ca2+ buffering deficits in cells transfected with truncated M1 receptors but only partially enhanced the protective effects of the M3 i3 loop in the
M1(M3i3) chimerics. A similar partial effect of BBs was seen in the M3(M1i3) chimerics, which showed increased OSS to DA. It appeared that antioxidants found in BBs might be targeting additional sites on these chimerics to decrease OSS. These initial findings have already made some impact on the scientific community when presented at recent national and international meetings, and the work concerned with reduction of stress signals has been cited in the popular press. The paper reporting on these studies has been submitted to the Journal of Alzheimer Disease. This work is aligned with National Program 107 Human Nutrition program component: 5. Health Promoting Properties of Plant and Animal Foods. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The
Nutrition and Neurocognition Laboratorys research on B vitamin nutrition and age related cognitive function has been reported in peer- reviewed journals and has been presented to industry groups and scientists at professional meetings. As addressed in our progress reports for subordinate projects, there are several organizations to whom the findings of the Neuroscience Laboratory concerned with the effects of fruits and vegetables on brain aging have been transferred through research agreements. Additionally, the findings from our previous CRIS concerned with blueberry effects in the aged animals have been disseminated throughout the blueberry industry. The per capita U.S consumption of blueberries has increased significantly since 1999 when our first paper in this area was published. Since then several other commodity groups (e.g., strawberry, walnut, avocado, etc.) have expressed interest in supporting investigations involved with possible beneficial effects of their fruits etc.,
on brain/behavioral function in aging. Members of this laboratory have also given talks on healthy eating to various nutrition groups who are concerned about the quality of nutrition in the US and other countries. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Rosenberg, IH : Soaring sales of supplements have scientists asking questions combining fortified food & large doses of vitamin tablets could be too much of a good thing, cautions a group of scientists. American Medical News. June 19, 2006. Tailored vitamins better than multivitamins. NPR Morning Edition. May 18, 2006. Dementia off the Menu: Mediterranean diet tied to low Alzheimers risk. Science News. April 22, 2006; Vol. 169, No.16. Muscle loss threatens health of the elderly. The Hartford Courant. April 20, 2006. Older, Wiser, Fitter: As they add on years, theyre also adding muscle.
Looking whos crowding gyms in record numbers. The Boston Globe. April 16, 2006. Movement, Sports to help defeat diseases. Der Spiegel (Cover Story). January 30, 2006 Presentations Rosenberg, IH. Folic Acid From Preconception to Predementia. Invited talk. Friedman School of Nutrition Science & Policy, Tufts University, Boston, MA. February 2006. Rosenberg, IH. Vitamins: Who needs them? Invited talk. Friedman School of Nutrition Science & Policy, Tufts University, Boston MA. March 8, 2006. Rosenberg, IH. Research Challenges and Opportunities. Invited talk. NIH State-of-the-Science Conference on Multivitamin/Mineral Supplements and Chronic Disease Prevention, Bethesda, MD. May 2006. Rosenberg, IH. New/Underutilized Research Techniques and the DRIs. Invited talk. Dietary Reference Intake Research Synthesis Workshop, IOM, Washington, DC. June 7, 2006. Troen, AM. Evaluating Cognitive Function in Renal Transplant Recipients: An Ancillary Study of the FAVORIT Trial. Invited talk. Kidney
Interagency Coordinating Subcommittee Meeting, NIH, Bethesda MD. March 24, 2006. Troen, AM. Neurotoxicity or metabolic insult? : Modeling homocysteine- related brain dysfunction. Invited talk. FASEB Summer Conference, Indian Wells, CA. August 5-10, 2006 Troen, AM, French Emily E, Roberts Jessica F, Selhub Jacob, Ordovas Jose M, Parnell Laurence D, Lai Chao-Qiang. Lifespan modification by glucose and methionine in Drosophila melanogaster fed a chemically defined diet. Poster presentation at the 35th Annual Meeting of the American Aging Association, Boston MA. June 2-4, 2006. Abstract Published in Age 28(1):67. Neuroscience Laboratory 1) Psychology Today, Brain Food Stroke Protection, October 1, 2005 2) Kenly News, Remember Where You Put Your Blueberries? October 5, 2005 3) Cooking Light, Raise a Glass to Your Health, November 1, 2005 4) Union Democrat, New Book Extols Virtues of Blueberries, November 1, 2005 5) LC:GC North America, Determination of Stilbenes in Blueberries, November
1, 2005 6) Newsday, How to Maximize Meals With Needed Nutrients, November 15, 2005 7) CBS (NY), Drinking to Good Health, November 18, 2005 8) Metro Silicon Valley, 5 Foods That Make You Smart, November 23, 2005 9) Indianapolis Star, Nutrition Needs in the Golden Years, November 27, 2005 10) Modern Baking, Blueberries: That Healthy Glow Means More Than Meets the Eye, December 1, 2005 11) Union County Leader, How To Protect Your Brain From The Damage Of Aging, December 2, 2005. 12) Third Age, Antioxidants in Fruits and Veg May Fight the Effects of Aging, December 14, 2005 13) Star Tribune, Eating Smart, December 18, 2005 14) Emedia Wire Orchard of Health, Natural Benefits From Everyday Fruit, December 19, 2005 15) Life Extension, Blueberries - The World's Healthiest Food, January 15, 2006 16) Patriot-News, Boost Your Brain Power, January 17, 2006 17) Star Phoenix, Blueberries & Cranberries Benefit Baby Boomers, January 26, 2006 18) Life Extension, Blueberries - One of Nature's Most
Potent Antioxidants Offers Powerful Neuroprotective and Other Benefits, February 1, 2006 19) AARP (The Magazine), Healing Foods, March 1, 2006 20) Shape Magazine, Eat Right News, March 1, 2006 21) Natural Health, The Longevity Care Package, March 1, 2006 22) Men's Fitness, The 20 Fittest Foods, April 1, 2006 23) Patriot-News, Super Foods, Harrisburg, PA, May 2, 2006 24) Los Angeles Times, An Orchard of Good Health, May 9, 2006 25) Health & Healing, Blueberries for Memory, June 1, 2006 26) CS Christian Single, Health Flash - 9 Foods You Gotta Eat, July 1, 2006
Impacts
(N/A)
Publications
- Joseph, J.A., Bielinski, D., Fisher, D.R. 2004. Blueberry extract inhibits DA-induced increases in MAPK signaling in Muscarinic M1 and M3-transfected COS-7 cells. Soc.Neurosci. Abs. 2004, 30, 1017.17.
- Shukitt Hale, B., Carey, A.N., Belinski, D., Lau, F.C., Galli, R.L., Spangler, E.L., Ingram, D.K., Joseph, J.A. 2004. Fruit polyphenols prevent inflammatory mediated decrements in cognition. Soc. Neurosci. Abs. 2004, 30,565.5.
- Smith, M.A., Atwood, C.S., Joseph, J.A., Perry, G. 2002. Predicting the failure of amyloid-beta vaccine. Lancet. 359:1864-1865.
- Casadesus, G., Stellwagen, H.M., Shukitt Hale, B., Rabin, B.M., Joseph, J. A. 2005. Hippocampal neurogenesis and psa-ncam expression following exposure to 56fe particles mimics that seen during aging. Gerontology Journal. 40 249-254
- Joseph, J.A., Shukitt Hale, B., Casadesus, G. 2005. Reversing the deleterious effects of aging on neuronal communication and behavior: the beneficial properties of fruit polyphenolics. American Journal of Clinical Nutrition 2005, 81 (Suppl.) 313S-316S
- Andres-Lacueva, C., Shukitt Hale, B., Galli, R.L., Jaurengui, O., Lamuela- Raventos, R.M., Joseph, J.A. 2005. Anthocyanins in aged blueberry-fed rats are found centrally and may enhance memory. Nutritional Neuroscience, April 2005:8(2): 111-120.
- Smith, M.A., Zhu, X., Casadesus, G., Aliev, G., Ogawa, O., Nunomura, A., Takeda, A., Joseph, J.A., Peterson, R.B., Perry, G., 2003. Alzheimer disease: causes, consequences and surprises. Biogerontology 2003, 4 (Suppl 1): 88-89.
- Shukitt Hale, B., Meterko, V., Carey, A.N., Bielinski, D., Mcguie, T., Galli, R., Joseph, J.A. 2005. Dietary supplementation with fruit polyphenolics ameliorates age-related deficits in behavior and neuronal markers of inflammation and oxidative stress. Age. 2005, 27, 49-57.
- Rabin, B.M., Carrihill-Knoll, K.L., Carey, A.N., Shukitt Hale, B., Joseph, J.A. 2005. Effect of diet on the disruption of operant responding at different ages following exposure to fe56 particles. Age. 205, 27, 69-73.
- Joseph, J.A., Shukitt Hale, B., Smith, M. 2004. Nutrition and communication in the aged brain: can you hear us now? Acarology International Congress Proceedings.
- Lee, H., Casadesus, G., Zhu, X., Tabaton, M., Joseph, J.A., Perry, G., Smith, M. 2004. Different perspectives on the amyloid-b cascade hypothesis. Journal of Alzheimer's Disease.
- Joseph, J.A., Fisher, D.R., Carey, A.N. 2004. Fruit extracts antagonize ab- or da-induced deficits in ca2+ flux in m1-transfected cos-7 cells. Journal of Alzheimer's Disease 6(2004) 403-411.
- Youdim, K.A., Mcdonald, J., Kalt, W., Joseph, J.A. 2002. Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults. Journal of Nutritional Biochemistry. 13:282-288.
- Obrenovich, M.E., Joseph, J.A., Atwood, C.S., Perry, G., Smith, M.A. 2002. Amyloid-2: a (life) preserver for the brain. Neurobiology of Aging.
- Rottkamp, C.A., Atwood, C.S., Joseph, J.A., Akihiko, N., Perry, G., Smith, M.A. 2002. The state versus amyloid-beta: the trail of the most wanted criminal in alzheimer's disease. Peptides 23 (2002) 1333-1341.
- Youdim, K.A., Joseph, J.A. 2003. Phytochemicals and brain aging: a multiplicity of effects. Book Chapter.
- Youdim, K.A., Shukitt Hale, B., Joseph, J.A. 2004. Flavonoids and the brain: interactions at the blood brain barrier and their physiological effect on the cns. Free Radical Biology and Medicine, VOL 37, NO11 PP. 1683-1693.
- Joseph, J.A., Fisher, D.R., Carey, A.N., Szprengiel, A. 2004. The m3 muscarinic receptor i3 domain confers oxidative stress protection on calcium regulation in transfected cos-7 cells. Aging Cell (2004)3, pp263- 271.
- Martin, A., Smith, M.A., Perry, G., Joseph, J.A. 2004. Nutritional antioxidants, vitamins, cognition and neurodegenerative disease. Principles of Gender Specific Medicine, Volume 2. Elsevier: San Diego, 2004, 813-823.
- Casadesus, G., Shukitt Hale, B., Stellwagen, H.M., Zhu, X., Lee, H., Smith, M.A., Joseph, J.A. 2004. Modulation of hippocampal plasticity and cognitive behavior by short-term blueberry supplementation in aged rats. Nutritional Neuroscience, Volume 7 Number 5/6 (October/December 2004), pp. 309-316
- Rabin, B.M., Joseph, J.A., Shukitt Hale, B., 2005. Effects of age and diet on the heavy particle-induced disruptions of operant responding produced by a ground-based model for exposure to cosmic rays. Brain Research 1036(2005) 122-129.
- Shukitt Hale, B., Carey, A.N., Joseph, J.A. Phytochemicals in foods and everages: Effects on the Central Nervous System. In lieberman, H.R., Kanarek, R.B., and Prasad, C.eds. Nutritional Neuroscience. CRC Press, Taylor & Francis Group, LLC Boca Raton, FL, 2005, 393-404.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? By the year 2050, 30 percent of the total population will be over 65 years of age. There is a high probability that this group will exhibit the most common correlative motor and cognitive behavioral changes that occur in aging. These alterations occur even in the absence of specific age-related neurodegenerative diseases, but could interact to exacerbate the behavioral (motor and memory) aberrations exhibited in these conditions. In these cases, and possibly as a function of normal aging as well, it is likely that in cases of severe deficits in memory or motor function hospitalization and/or custodial care would be a likely outcome, with significant increases in health care costs. In both financial and human terms it is extremely important to explore methods to retard or reverse the age-related
neuronal deficits as well as their subsequent, behavioral manifestations. Very little is known about the mechanisms involved in these age-related declines in cognitive and motor behaviors. Attempts to reverse or retard these decrements have been, with very few exceptions, singularly unsuccessful. Even less is known concerning the nutritional modulation that could be employed to retard or reverse these declines. It has been postulated that these behavioral and neuronal declines are the result of an increasing inability to inactivate free radicals or inflammatories that impinge upon the organism, and an increasing vulnerability to these insults, thus creating a "fertile environment" for aging and the subsequent development of age-related, neurodegenerative diseases. The Nutrition and Neurocognition Laboratory aims to gain insight into the interaction between nutritional factors and age-related cognitive, motor, brain or central nervous system declines in humans. Scientists in this
laboratory believe that nutritional modification with fruits and vegetables high in antioxidants, anti-inflammatory activity, and certain B vitamins may be very effective. This program is directed to further specification and identification of these activities and the mechanisms involved in the positive benefits of dietary improvement and nutritional supplementation. Research conducted in the Neuroscience Laboratory has shown that supplementation with fruits and vegetables provide beneficial effects that include both forestalling and reversing the deleterious effects of aging on neuronal functioning and behavior. These effects appear to be the result of compounds (e.g., polyphenolics) that enhance the survivability of the plant, presumably through their antioxidant, and anti-inflammatory properties. While fruits and vegetables may have direct effects on oxidative stress and inflammation in aging, preliminary data also indicate that polyphenolic compounds may have a plethora of
additional effects involving enhanced signaling and neurogenesis in the aged animal that may also contribute to the observed benefits in motor and cognitive function. If this is the case, then it would be important to determine these additional mechanisms with a view toward the establishment of guidelines and dietary recommendations to an aging population concerning the qualitative and quantitative attributes of the fruits and vegetables. The objectives of this research are related to National Program 107 Human program components, 5. Health Promoting Properties of Plant and Animal Foods; and 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. 2. List the milestones (indicators of progress) from your Project Plan. Nutrition and Neurocognition Laboratory Objective 1 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. Objective 2 Characterize the mechanisms by which nutritionally induced hyperhomocysteinemia affects neuronal function and cognitive performance using transgenic mouse models of human cognitive decline. Milestones: 2005 1. Determine the relation between brain volumes, cognitive performance, and homocysteine in the Framingham offspring over 10 years. Objective 1 2. Recruit homebound elderly for baseline studies. Objective 1 3. Achieve 13 percent of target enrollment in the FAVORIT Cognitive Ancillary Study. Objective 1 4. Determine the effect of aging in rats on one-carbon metabolism with emphasis on difference among various tissues. Objective 2 5. Determine the impact of dietary imbalances in folate and/or methionine and their resultant homocysteinemias on Morris water maze performance young rats. Objective 2 6. Determine the impact of chronic (43 week) B12-deficiency and its resultant homocysteinemia on Morris water maze performance and on the radiant-heat tail flick
test in rats. Objective 2 7. Characterize the effect of chronic induced adult-onset homocysteinemia on several behaviors in a pilot study of rodents with an inducible mutation in a homocysteine metabolizing gene (human cystathionine beta-synthase). Objective 2 2006 1. Further characterization of the effect of diet on vascular impairment in rodent models. Objective 2 2. Further characterization of cognitive dysfunction in rodent models for discerning between the effects of homocysteine vs. vitamin deficiency. Objective 2 2007 1. Complete and analyze cross-sectional studies of Homebound Elderly MRIs cognition, and nutritional biochemistries. Objective 1 2008 1. Complete and analyze cross-sectional baseline findings of cognition, and nutritional biochemistries in the FAVORIT Cognitive Ancillary Trial. Objective 1 2009 1. Complete data collection of cognitive effect of homocysteine lowering. Objective 1 Neuroscience Laboratory Objective 1: (a) 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. (b) 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. Muscarinic receptors are involved in regulating both memory and motor function and show change with age. (c) Assess the vulnerability to oxidative and inflammatory stressors in microglial 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 polyphenolic and berryfruit extracts. Objective 2: (a) Establish the effects of dietary berryfruit extracts and the most effective component polyphenolics on neuronal function in
aging by determining the effects on motor and cognitive behaviors as a function of age. (b) Identify brain regional localization of berryfruit compounds and correlating the amounts seen with the behavioral performance. (c) Determine their effects on signaling and the generation of new neurons in aging. MILESTONES 2005 1. Develop and test procedures to construct chimerics and point mutations in M1 and M3 muscarinic receptors (AChR) and determine sensitivity to oxidative stress and inflammation. Objective 1a 2. Identify blueberry and strawberry polyphenolics that offer protection against oxidative stress and inflammation in M1AChR-transfected COS-7 cells and begin to identify the signaling mechanisms involved. Objective 1b 3. Determine motor and cognitive behaviors in young and old rats following blueberry or strawberry supplementation. Objective 2a 4. Begin to determine the extent of new neuron growth (neurogenesis) in strawberry- or blueberry-fed animals. Objective 2b 2006 1.
Determine the effects of lipid raft modification on oxidative stress and sensitivity to inflammation. Objective 1a 2. Develop and test procedures to induce point mutations in M1 and M3AChR. Objective 1a 3. Determine the signaling (complex proteins that control cell communication and function) and gene activation mechanisms involved in berryfruit protection in the cell models (e.g., COS-7, BV-2 and hippocampal cells). Objectives 1b, 1c 4. Determine brain regions for signaling, neurogenesis and oxidative stress and inflammation markers, after behavior is determined in the rats. Objective 2a 5. Begin to determine the alterations in complex proteins and genes that affect communication between neurons and correlate these with the behavioral changes. Objectives 2a, 2c 2007 1. Determine oxidative stress and inflammation sensitivity in M1 and M3AChR transfected COS-7 cells with point mutations in the transmembrane loops. Objective 1a 2. Determine the effect of these mutations on the lipid
raft microenvironment and determine if the berryfruits affect the microenvironment. Objective 1b 3. Begin to determine the protective effects of polyphenolic extracts derived from blueberries in our cell models. Objectives 1b, 1c 4. Determine protective capacity against oxidative stress and inflammation insults in tissue obtained from young and senescent control animals or those supplemented with blueberries or strawberries. Objectives 2a 5. Begin to determine the localization the various blueberry or strawberry polyphenolics in the brain following blueberry or strawberry supplementation in the senescent or young rodents. Objective 2b 6. Confirm neurogenesis effects in blueberry or strawberry fed animals using double and triple labels and their controls. Objective 2c 2008 1. Determine the effects of oxidative stress and inflammation of lipid raft modification on sensitivity. Objective 1a 2. Begin gene array analyses on the effects of the various alterations in mAChR structure on
oxidative stress- and inflammation-induced gene expression. Utilize immunocytochemistry to validate the gene array analyses. Objective 1a 3. Determine the effects of blueberry or strawberry-derived polyphenolics identified in the cell work as showing oxidative stress and inflammation protection. Objective 2a 2009 1. Complete gene array analyses on the effects of the various alterations in mAChR structure on oxidative stress- and inflammation-induced gene expression. Utilize immunocytochemistry to validate the gene array analyses. Objective 1a 2. Determine signaling effects of oxidative stress, inflammation, blueberry or strawberry effects in the BV-2 and hippocampal cell models and relate to changes in gene markers. Objectives 2a and 2c 3. Determine the localization of the various blueberry or strawberry polyphenolics in the striatal and cortical areas following blueberry or strawberry supplementation in the senescent or young rodents. Objective 2b 3a List the milestones that were
scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Nutrition and Neurocognition Laboratory Milestone # 1 Determine the relation between brain volumes, cognitive performance, and homocysteine in the Framingham offspring over 10 years. Objective 1 Milestone Substantially Met 2. Nutrition and Neurocognition Laboratory Milestone # 2 Recruit homebound elderly for baseline studies. Objective 1 Milestone Substantially Met 3. Nutrition and Neurocognition Laboratory Milestone # 3 Achieve 13 percent of target enrollment in the FAVORIT Cognitive Ancillary Study. Objective 1 Milestone Substantially Met 4. Nutrition and Neurocognition Laboratory Milestone # 4 Determine the effect of aging in rats on one-carbon metabolism with emphasis on difference among various tissues. Objective 2 Milestone Substantially Met 5. Nutrition and Neurocognition Laboratory Milestone # 5 Determine the impact of dietary
imbalances in folate and/or methionine and their resultant homocysteinemias on Morris water maze performance young rats. Objective 2 Milestone Substantially Met 6. Nutrition and Neurocognition Laboratory Milestone # 6 Determine the impact of chronic (43 week) B12-deficiency and its resultant homocysteinemia on Morris water maze performance and on the radiant-heat tail flick test in rats. Objective 2 Milestone Substantially Met 7. Nutrition and Neurocognition Laboratory Milestone # 7 Characterize the effect of chronic induced adult-onset homocysteinemia on several behaviors in a pilot study of rodent with an inducible mutation in a homocysteine metabolizing gene (human cystathionine beta-synthase). Objective 2 Milestone Substantially Met 8. Neuroscience Laboratory Milestone # 1 Develop and test procedures to construct chimerics and point mutations in M1 and M3 muscarinic receptors (AChR) and assess sensitivity to oxidative stress and inflammation. Objective 1a Milestone Substantially
Met 9. Neuroscience Laboratory Milestone # 2 Identify blueberry and strawberry polyphenolics that offer protection against oxidative stress and inflammation in M1AChR-transfected COS 7 cells and begin to identify the signaling mechanisms involved. Objective 1b Milestone Substantially Met 10. Neuroscience Laboratory Milestone # 3 Determine motor and cognitive behaviors in young and old rats following strawberry or blueberry supplementation. Objective 2a Milestone Substantially Met 11. Neuroscience Laboratory Milestone # 4 Begin to determine the extent of new neuron growth (neurogenesis) in strawberry- or blueberry - fed animals. Objective 2b Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Nutrition and Neurocognition Laboratory 2006 1. Further characterization of the effect of diet on vascular impairment in rodent
models. Objective 2 Expected results: measurements of brain vascular densities in rodent models of different types of dietary and genetic homocysteinemia. 2. Further characterization of cognitive dysfunction in rodent models for discerning between the effects of homocysteine vs. vitamin deficiency. Objective 2 Expected Results: measurements of the relation between diet and genetically induced behavioral and brain biochemical and pathological changes in rodent models of homocysteinemia. 2007 1. Complete and analyze cross-sectional studies of Homebound Elderly MRIs cognition, and nutritional biochemistries. Objective 1 Expected results: Determination of the association of blood vitamins and homocysteine in the brain function and anatomy. 2008 1. Determination of the cross-sectional relationship of baseline homocysteine and B-vitamins in renal transplant recipients. Expected Results: High baseline blood homocysteine and low vitamin concentrations are expected to correlate with poor
baseline performance on cognitive tests in renal transplant recipients. Neuroscience Laboratory 2006 1. Determine the effects of lipid raft modification on oxidative stress and inflammation sensitivity. Objective 1a Anticipated outcome: We anticipate that C2 ceramide, a metabolite of sphingomyelin, would differentially affect M1 and M3AChR-transfected COS- 7 cells and that blueberry treatment would prevent any deleterious effects of C2 ceramide on oxotremorine-induced calcium buffering (i.e., the ability of the cells to clear or buffer calcium when stimulated). 2. Develop and test procedures to induce point mutations in M1 and M3AChR. Objective 1a Anticipated outcome: We anticipate that point mutations in the i3 loop (the internal loop of the third transmembrane segment) will have the most effect on oxidative stress vulnerability in the M1 and M3 transfected cells. Point mutations in other transmembrane segments will have less effect on this vulnerability 3. Determine the
signaling (complex proteins that control cell communication and function) and gene activation mechanisms involved in berryfruit protection in the cell models (e.g., COS-7, BV-2 and hippocampal cells). Objectives 1b, 1c Anticipated outcome: We predict that berryfruit (e.g., blueberries) will have important effects on complex signaling pathways in all of these cell models. As examples: As an important interface between cell and animal models, primary hippocampal cells offer a valuable model to examine the effects of various oxidative stress (OS) insults (e.g., dopamine or amyloid beta). We predict that berryfruit extracts will be effective in protecting against oxidative stress induced by dopamine on calcium buffering in these cells. Similar effects should be seen against inflammatory stimulants in the BV-2 cells, as well as the mAChR- transfected COS-7 cells. We also expect that there will be alterations in gene regulation in these cell models. 4. Determine brain regions for
signaling, neurogenesis and oxidative stress and inflammation markers, after behavior is assessed in the rats. Objective 2a Anticipated outcome: Since we have some preliminary data showing that berryfruit-supplementation increases bromodeoxyuridine incorporation (a marker for DNA incorporation and thus, cell proliferation) in the hippocampus we anticipate that the berryfruit will increase hippocampal neurogenesis. The slides for the confirmatory neurogenesis determinations from one study have been cut and prepared with bromodeoxyuridine antibody in order to assess the number of dividing cells. These will then need to be probed with additional markers to ensure that the cells showing division are localized among neurons. 5. Begin to assess alterations in complex proteins and genes that affect communication between neurons and correlate these with the behavioral changes. Objectives 2a, 2c Anticipated outcome: Preliminary data has indicated that blueberry supplementation affects
signaling parameters in aged rats and that these changes are correlated with behavioral performance. Therefore, we predict that signaling parameters such as mitogen activated protein kinases (MAPk), cyclic AMP response element binding proteins (CREB) and others will be affected by blueberry and other berryfruit supplementations. 2007 1. Determine oxidative stress and sensitivity to inflammation in M1 and M3AChR transfected COS-7 cells with point mutations in the transmembrane loops. Objective 1a Anticipated outcome: Based on previous research we anticipate that removal of lysine and histidine residues will decrease the sensitivity of M1AChR in the transfected COS-7 cells. Conversely, increasing the number of these amino acids in the M3AChR will increase their sensitivity to oxidative stress and inflammation. 2. Determine the effect of these mutations on the lipid raft microenvironment and determine if the berryfruits affect the microenvironment. Objective 1b Anticipated outcome:
We anticipate that C-2 ceramide will affect the responses of these cells to stimulation and will negatively affect the ability of the cells to clear or buffer calcium. These responses may have a synergistic affect with the point mutations that also alter calcium clearance (see milestone 1 for 2007). 3. Begin to determine the protective effects of polyphenolic extracts derived from blueberries in our cell models. Objectives 1b, 1c Anticipated outcome: We anticipate using anthocyanins, reseveratrol, proanthocyanidins and elagitannins in the cell models, and we predict that there will be differential levels of protection offered by the various polyphenolic families, with the whole blueberry extract having the greatest effects. 4. Determine protective capacity against oxidative stress and inflammation insults in tissue obtained from young and senescent control animals or those supplemented with blueberries or strawberries. Objectives 2a Anticipated outcome: We anticipate that the
various brain regions obtained from the supplemented animals exposed to hydrogen peroxide or lipopolysaccharide will show greater protection than those from the non- supplemented animals, as assessed via stimulated neurotransmitter (e.g., dopamine release). 5. Begin to determine the localization the various blueberry or strawberry polyphenolics in the brain following blueberry or strawberry supplementation in the senescent or young rodents. Objective 2b Anticipated outcome: Based upon a previous study we anticipate that there will be differential localization of the berryfruit polyphenolics in the various brain regions. As examples, the proanthocyanidins may show greater localization in the striatum, while the elagitannins may show greater affinity for the hippocampus an important memory control area. 6. Confirm neurogenesis effects in blueberry or strawberry fed animals using double and triple labels and their controls. Objective 2c Anticipated outcome: Based upon our previous
research we anticipate that the berryfruit increases in cells will primarily involve neuronal cells. Therefore, markers of increased neuronal cell number (e.g., NeuN) will increase, while those involving glial cells will not show increases following these supplementations. 2008 1. Determine the effects of oxidative stress and inflammation of lipid raft modification on sensitivity. Objective 1a Anticipated outcome: We anticipate that the application of C-2 ceramide or other sphingolipids to the COS-7 cells will increase their sensitivity to oxidative stress induced by dopamine or amyloid beta. Similar effects should be seen with lipopolysaccharide, an inflammatory mediator. 2. Begin gene array analyses on the effects of the various alterations in mAChR structure on oxidative stress- and inflammation-induced gene expression. Utilize immunocytochemistry to validate the gene array analyses. Objective 1a Anticipated outcome: We have limited data from our previous research that suggests
that the berryfruit decreases expression of genes showing increased activity during conditions of oxidative stress (nuclear factor kappaB) or inflammation (cyclooxygenase II). An expansion of this work through microarray analyses should tremendously increase our ability to determine the activities of a large number of the genes associated with these insults. 3. Determine the effects of blueberry or strawberry-derived polyphenolics identified in the cell work as showing oxidative stress and inflammation protection. Objective 2a Anticipated outcome: As with the cells, we anticipate that none of the single polyphenolic families (e.g., anthocyanins) will be as effective in preventing or reversing cognitive and motor function as the combinations of polyphenolics found in the whole fruit. 4a What was the single most significant accomplishment this past year? Animal models of homocysteine-related cognitive dysfunction: The Nutrition and Neurocognition Laboratory developed rodent models to
test homocysteine-related cognitive function. Cognitive decline and dementia afflicts up to one-third of elders over the age of 75. With the aging of the US population it is vital to develop means of preventing or slowing these cognitive impairments. Nutritional factors such as B- vitamin deficiency and its associated elevations of blood homocysteine concentrations (homocysteinemia) might modify the course of "brain aging" and provide an opportunity to intervene in age-related cognitive impairments. The development of physiologically relevant animal models of these processes is necessary in order to determine the relation of nutrition to brain aging in the human population and to develop effective dietary and other interventions. 4b List other significant accomplishments, if any. 1. Oxidant Signaling Reductions by Blueberry Extract in COS-7 cells: Milestone 2, Objective 1b The most significant accomplishment by the Neuroscience Laboratory during FY 2005 addressed the problem of
identifying the role of blueberry supplementation in lowering oxidative stress signaling in COS-7 cells. It has long been known that the polyphenolic compounds contained in blueberries can act as potent antioxidants. However, our research shows that the blueberries may actually reduce signals such as calcium response element binding protein (CREB) and protein kinase C gamma, which are potent transcription factors for oxidative stress and inflammation. These studies should provide a whole new approach for elucidating the mechanisms involved in the antioxidant activity of berryfruits, and the initial findings have already made some impact on the scientific community when presented at recent national and international meetings. 2. The Effects of Manipulation of the i3 Loop on Sensitivity to Oxidative Stress and Signaling. Milestones 1 (Objective 1a) and 2 (Objective 1b) for 2005. Previous experiments related to this milestone have revealed that deletions of the entire i3 loop increased
dopamine (DA) sensitivity (a lower percentage of cells showing recovery following depolarization) in both the M1 and M3 subtypes. Additionally, chimerics of M1 where the i3 loop of the M3AChR was switched with the i3 loop of the M1AChR (M1M3i3) showed that the DA sensitivity was reduced (percent of cells showing increases in calcium clearance) following depolarization. In the M3 chimerics containing M1i3 (M3M1i3), the i3 loop offered no protection against DA-induced decrements in calcium buffering. In FY2005 we found that M1/M3 differences in oxidative stress vulnerability may involve differential signaling in pMAPK (phospho- mitogen activated protein kinase) and pCREB (phosphor cyclic AMP response element binding protein) under oxidative stress treatment conditions, with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggest that blueberries may antagonize oxidative stress effects by enhancing pMAPK and lowering activation of pCREB. In the M1 chimerics
the DA induced increases in pMAPK were reduced, as were the increases in pCREB, suggesting that at least part of the protection against oxidative stress calcium buffering seen in the chimerics may be mediated through the i3 loop and the alterations in pCREB and pMAPK transcription factors. Although it appears that oxidative stress induced deficits in calcium buffering in the M3 truncated - and chimeric -transfected COS-7 cells, it is clear that these changes are less dependent upon alterations in these transcription factors. It appears that antioxidants might be targeting additional sites on these chimerics to decrease oxidative stress sensitivity. 3. Identify blueberry and strawberry polyphenolics that offer protection against oxidative stress and inflammation in M1AChR-transfected COS-7 cells and begin to identify the signaling mechanisms involved. Milestone 2 (Objective 1b) for 2005. Muscarinic receptors (MAChRs) are intimately involved in various aspects of both neuronal and
vascular functioning, show loss of sensitivity in aging and AD, and are selectively sensitive to oxidative stress with MAChR subtypes M1, M2, and M4 showing greater oxidative stress sensitivity [the ability of the cell to extrude or sequester Ca2+ following depolarization by 750 M oxotremorine and exposure to dopamine DA) or A 25-35 than M3 or M5 subtypes in transfected (tn) COS-7 cells. However, blueberry (BB) extract pretreatment prevented the DA or A induced deficits in Ca2+ buffering. Although previous findings showed that the M3 receptor i3 loop offered protection against DA induced deficits in Ca2+ buffering, this protection does not extend to ceramide. We are assessing possible differences in protein kinase C (PKC) isoforms (e.g., epsilon), CREB, and caspases that could account for these differences, but previous findings indicate that BB induction of protective ERK activity is higher in M1-tn COS-7 cells than those in M3AChR, suggesting that magnitude of ERK signaling may be
important in this protection. 4. Determine motor and cognitive behaviors in young and old rats following strawberry or blueberry supplementation. Milestone 3 (Objective 2a) for 2005. Previously, we had shown that whole, crude blueberry (BB) extracts are able to reverse several parameters of brain aging (e.g., deficits in cell communication) as well as age-related motor and cognitive deficits when fed to rats from 19-21 months of age. These effects appear to be the result of compounds (polyphenolics) that enhance the survivability of the plant, possibly through direct effects on brain signaling or indirectly through their antioxidant and anti-inflammatory properties. This year we assessed 3 different BB-derived diets, all equated on phenolic level, to determine whether the effects observed with the whole, crude blueberrry extract are indeed due to polyphenolics or whether other compounds were contributing to the age-related improvements in behavior. Old (19 mo) F344 rats were fed a
control diet or one with 5. 4% crude blueberry extract (as before), a 2% pre-C18 column BB extract, or a 0.1% post-C18 column semi-purified blueberry extract (a mixture of only BB phenolics with the sugars and organic acids removed) for 8 weeks prior to motor and cognitive testing. Results showed that only the crude blueberry extract diet improved rotarod performance, while all three blueberry-derived diets improved working memory in the Morris water maze. Therefore, phenolics are important components in the beneficial effects of blueberries on age-related improvements in cognition, but other compounds may play a role in motor improvements. 5. Begin to determine the extent of new neuron growth (neurogenesis) in strawberry- or blueberry - fed animals. Milestone 4 (Objective 2b) for 2005. The slides for the neurogenesis determinations from two studies in old animals that were given control or blueberry diets have been cut and prepared with bromodeoxyuridine antibody in order to assess
the number of cells dividing. Early results suggest both age and blueberry effects. We will probe these with additional markers to determine the neuronal localization of these changes. 6. Determine the signaling (complex proteins that control cell communication and function) and gene activation mechanisms involved in berryfruit protection in the cell models and animals. Milestone 3 (Objectives 1b, 1c) and Milestone 5 (Objectives 2a, 2c) for 2006. a) We are constructing a rat brain mini-cDNA library containing 24 genes that are involved in inflammation, oxidative stress and cell death signaling pathways along with three housekeeping genes. These genes formed a panel of indicators for the study of the beneficial effects of blueberries on gene regulation in the rat brain or in cell-culture paradigms. We utilized a subset of these to determine gene alteration in blueberry-fed animals and controls exposed to central administration of the neurotoxin, kainic acid. We showed that in the
animals fed the control diet there was up-regulation in cytokines as well as nuclear factor kappa B, which are markers of inflammation, following kainic acid administration. However, these cytokines and NF B were downregulated in the BB-supplemented animals. b) Using a murine microglial cell line (BV-2), that has been used previously as an in vitro model for the study of pathogenesis in Alzheimer's disease (AD), we found that treatment with blueberry (BB) extracts significantly and dose-dependently reduced the lipopolysaccharide (LPS)-induced NO production in conditioned media from BV2 murine microglial cells. Reactive oxygen species (ROS) release was also reduced in BB-treated LPS-activated BV2 cells. In addition, BB extracts significantly attenuated the protein expression of the inducible NO synthase (iNOS), cyclo-oxygenases 2 (COX-2), and the pre-processed form of IL1 beta in the LPS-activated BV2 cells. Furthermore, the secretion of the inflammatory cytokines IL-1 beta and
TNF-alpha into the conditioned media from the LPS-activated BV2 cells was inhibited by BB treatment. The results from this study suggest that BB polyphenols attenuate inflammatory responses of the brain microglial cells and could be used to modulate inflammatory conditions in the central nervous system. 4d Progress report. 1950-51000-063-03S This report serves to document research conducted under a Specific Cooperative Agreement between Agri-Canada and ARS. This study was carried out in conjunction with 1950-51000-063-04T, which is a Trust Fund Cooperative Agreement between the California Dried Plum Board and ARS. In this project HNRCA ARS scientists tested dried plum powder for its efficacy in reversing cognitive and motor deficits in aged rats. HNRCA scientists supplied plasma collected from rats to Dr. Wilhelmina Kalt from Agri Canada for testing of the Oxygen Radical Absorbance Capacity (ORAC). The results indicated that no significant differences were seen in plasma ORAC between
the dried plum-supplemented and control animals, suggesting that supplementation did not increase antioxidant activity in the supplemented animals. Results are being written at present. 1950-51000-063-04T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the California Dried Plum Board. In fiscal year 2004, HNRCA scientists tested the effects of a dried plum powder on motor and memory performance in 21-month-old Fischer 344 rats. There were no beneficial effects observed in either motor or cognitive behavior among rats consuming an NIH-31 diet containing the plum powder. There were no differences between control and supplemented groups on dopamine release, a measure of cell signaling. After having the powder tested for polyphenol content, scientists concluded that many of the polyphenols, particularly anthocyanins, were not present. Therefore, they repeated the experiments this year and fed the rats plum juice from which the
dried plums were made. Significant reversals in deficits in cognitive function were found in these rats as indicated by a Morris water maze performance test and dopamine release. 1950-51000-063-05T This report serves to document research conducted under a Trust Fund Cooperative Agreement between the ARS and Welchs Foods. Animals and humans show increased motor and cognitive declines with aging that are thought to be due to increased susceptibility to the long-term effects of oxidative stress and inflammation. Previous findings have suggested that improvements in these age-related declines might be accomplished by increasing the dietary intake of polyphenolics found in fruits and vegetables, especially those identified as being high in antioxidant and anti-inflammatory activity. We investigated the beneficial effects of two concentrations of Welch's Concord grape juice (10% and 50%) compared to a calorically-matched placebo for their effectiveness in reversing age- related deficits in
behavioral and neuronal function in aged Fischer 344 rats. Rats which drank the 10% grape juice from age 19 to 21 months had improvements in oxotremorine-enhancement of potassium-evoked release of dopamine from striatal slices, as well as cognitive performance on the Morris water maze, while the 50% grape juice produced improvements in motor function. These findings suggest that, in addition to their known beneficial effects on cancer and heart disease, polyphenolics present in foods may be beneficial in reversing the course of neuronal and behavioral aging, possibly through a multiplicity of direct and indirect effects that can affect a variety of neuronal parameters. The manuscript on this work has been submitted for publication and reviewed in the journal Nutrition - The International Journal of Applied and Basic Nutritional Sciences. It is now being revised according to the reviewers suggestions. 1950-51000-063-06T This report serves to document research conducted under a Trust
Fund Cooperative Agreement between ARS and Wild Blueberry Association of North America. Scientists have found that one of the major effects of blueberry (BB) application to cells may be to alter oxidative stress induced signaling (OSS). Our findings suggest that BB may antagonize oxidative stress effects by altering cell signaling. In this way, cells are able to better communicate with one another, which enhances behavioral functions. We also showed that, in M1 muscarinic receptor hybrids, the dopamine-induced increases in the signaling marker pMAPK were reduced, as were the increases in another signaling marker pCREB, suggesting that at least part of the protection against oxidative stress calcium buffering seen in the hybrids may be mediated through the i3 loop (the variable section of this receptor) and the alterations in pCREB and pMAPK signaling markers. These experiments also showed that antioxidants might be targeting additional sites on these hybirds to decrease OSS. It
also appears that BBs interact with genes that regulate oxidative and inflammatory stressors, (e.g., IL-1beta). Our laboratory has shown that these genes are down regulated in BV-2 mouse microglial cells treated with the inflammatory agent lipopolysaccharide. 1950-51000-063-07T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the U.S. Highbush Blueberry Council. Scientists assessed three different blueberry (BB)-derived diets, all equated on phenolic level, to determine whether the effects observed with the whole, crude BB extract are due to polyphenolics or whether other compounds were contributing to the age-related improvements in behavior. Old (19 months) F344 rats were fed a control diet or one with 5.4% crude BB extract (as before), a 2% pre-C18 column BB extract, or a 0.1% post- C18 column semi-purified BB extract (a mixture of only BB phenolics with the sugars and organic acids removed) for 8 weeks prior to motor and
cognitive testing. Results showed that only the crude BB extract diet improved rotarod performance, while all three BB-derived diets improved working memory in the Morris water maze a device used to test special learning and memory. Therefore, phenolics are important components in the beneficial effects of BBs on age-related improvements in cognition, but other compounds may play a role in motor improvements. We also assessed the anti-inflammatory effects of blueberries since age-related cognitive deficits in neurodegenerative diseases such as Alzheimers disease (AD) and Parkinsons disease (PD) may be attributable to chronic exposure and/or increased susceptibility to inflammation. We investigated if the polyphenols in blueberries (BBs) can reduce the deleterious effects of inflammation induced by kainic acid (KA). Central administration of KA induces a characteristic behavioral syndrome and neurodegeneration in several brain areas including the hippocampus. Four-month old male F344
rats were fed a control or 2% BB diet for 8 weeks before either Ringers (R) or KA was bilaterally microinfused into the hippocampus. Two weeks later, following behavioral evaluations, the rats were euthanized, their brains removed and dissected. Total RNA from the hippocampus was extracted and used in real-time quantitative RT-PCR (qRT-PCR) to analyze the expression of genes that are involved in inflammatory response. Behavioral studies showed that KA had deleterious effects on cognitive behavior. KA-injected rats on the control diet exhibited increased latencies to find the hidden platform in the Morris water maze on days 1- 3 and on day 4 during reversal learning, compared to R-injected rats. These deficits were improved by BB supplementation: latencies to find the platform on days 2-4 in the BB-KA group were not different than the control diet-R group. Immunohistochemical analyses of OX-6 expression to assess inflammatory response revealed that KA produced an inflammatory response
by increasing the OX-6 positive areas in the hippocampus of KA- injected rats. qRT-PCR analysis showed that KA up-regulated the expression of inflammatory cytokines IL-1beta and TNF-alpha, the neurotrophic factor IGF-1 and the transcription factor NF-kappa B. BB supplementation was found to reduce the expression of all but IGF-1. These results indicate that BB polyphenols exert anti-inflammatory actions via alteration of gene expression. 1950-51000-063-08R This report serves to document research conducted under a Reimbursable Cooperative Agreement between ARS and the University of Maryland - Baltimore County (UMBC). Exposure to heavy particle irradiation produces neurochemical and cognitive deficits in young animals that are characteristic of much older animals. As a result, it has been suggested that exposure to heavy particles can produce accelerated aging. In collaboration with scientists from the University of Maryland, Baltimore County (UMBC), we found that the doses of 56Fe
(irradiated) particles needed to affect neurobehavioral endpoints decrease according to the age of the rat. For both measurements of anxiety, using the elevated plus maze, and responsiveness to environmental contingencies, using operant responding on an ascending fixed-ratio schedule, older animals are more susceptible to the effects of irradiation than younger animals. The results also suggest that the interaction between age and exposure to 56Fe particles is not linear. The greatest effect of irradiation was seen with the middle-aged animals; i.e., those that were irradiated at 7-8 months of age. As shown most clearly with elevated plus maze performance, the effect of radiation of the middle-aged animals was to change their level of performance to that of the non-irradiated older animals. Exposing older animals (17-months) did not appear to produce a further deterioration in performance. These findings suggest that age may be a risk factor for the behavioral effects of exposure to
heavy particles. The effects are not necessarily linear (i.e., middle-aged rats seem more susceptible to the deleterious effects of 56Fe particle exposure than young or very old animals). Maintaining rats on diets containing antioxidant phytochemicals (blueberry and strawberry extract) can provide a significant degree of radiation protection, depending upon the specific diets and neurobehavioral endpoints. Maintaining rats on diets containing blueberry or strawberry extract at the time of radiation can prevent the occurrence of both the acute and late degenerative changes that result from exposure to 56Fe particles. Both strawberries and blueberries rank high in their ability to scavenge free radicals. The observation that they are capable of ameliorating the effects of exposure to 56Fe particles is consistent with the observation that exposure to HZE heavy ion particles, produces oxidative stress and that oxidative stress in the central nervous system may play a role in mediating
the neurobehavioral consequences of irradiation. The data, however, indicate that the effects of antioxidant diets on the neurobehavioral effects of exposure to 56Fe particles varies as a function of diet and endpoint. While both blueberry and strawberry diets were equally effective in preventing the 56Fe particle-induced disruption of dopamine release, the amelioration of the behavioral effects varied by diet. The strawberry diet was effective in preventing the late degenerative changes producing decreased operant responding on an ascending fixed-ratio schedule. In contrast, the effects of the diets on the deleterious effects of exposure to 56Fe particles on spatial learning and memory varied as a function of the specific task and the specific brain region mediating the task. Latency performance in the Morris water maze depends upon the integrity of the striatum, whereas spatial learning performance with the platform removed on the probe trials is a hippocampally-mediated task. The
diet containing the strawberry extract was most effective in countering the effects of irradiation of the hippocampal task; the diet containing the blueberry extract was most effective in countering the effects of irradiation of the striatal task. These results suggest that the effectiveness of antioxidant diets in countering the neurobehavioral effects of exposure to 56Fe particles may be an interactive effect of the specific diets and endpoints. The results also suggest that a dietary cocktail may be necessary to provide the maximum protection against radiation. These findings have important implications for aging, since a similar berry cocktail may be important in forestalling or reversing the deleterious effects of aging. 1950-51000-063-09T This report serves to document research conducted under a Cooperative Agreement between ARS and Pom Wonderful/the Stewart and Linda Resnick Revocable Trust of Los Angeles, CA. Animals and humans show increased motor and cognitive declines with
aging, thought to be due to increased susceptibility to the long-term effects of oxidative stress and inflammation. Previous findings have suggested that improvements in these age-related declines might be accomplished by increasing the dietary intake of polyphenolics found in fruits and vegetables, especially those identified as being high in antioxidant and anti-inflammatory activity. Therefore, we investigated whether pomegranate (POM) juice would be effective in altering motor and cognitive behavioral deficits in aged rodents that were fed water or diets supplemented with low (.33 %) or high (3.3%) amounts of pomegranate juice for 8 weeks (beginning when the rats were 19 months of age and tested at 21 months of age). To determine whether rats consuming the low and high amounts of POM juice significantly improved their cognitive performance as measured in the Morris water maze, differences were calculated between Trial 1, which measured reference (long-term) memory and Trial 2,
which measured working (short-term) memory. Trial 2 was performed 10 minutes following Trial 1. No improved performance was seen in the high POM group. The low POM juice showed improved working memory (i.e., there was a significant difference between Trial 1 and Trial 2 performance), however, Trial 1 performance in the low POM juice group was worse than the water group, i.e., the low POM juice group took longer to find the hidden platform on the reference memory trial. Working memory performance was not different between the groups. Therefore, one cannot conclude that the low POM juice had improved performance compared to the water group. Interestingly, dopamine release was significantly greater in both the low and high POM groups, compared to water, therefore, showing greater muscarinic receptor sensitivity in the supplemented groups. The doses of pomegranate (POM) juice used, .33 % (low) and 3.3% (high) may not have been high enough as they were not effective in reversing the age
effects on the behavioral parameters examined. 1950-51000-063-10T This report serves to document research conducted under a Trust Fund Cooperative Agreement between ARS and the California Strawberry Commission. The grant from the California Strawberry Commission to fund this work has just been awarded and there are no findings to report. We have recently received the standardized extract that we will use for these studies. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Over the life of the project the Nutrition and Neurocognition Laboratory has observed in human population studies that common vitamin B12 and folate deficiencies and high blood homocysteine levels are often associated with cognitive decline and dementia. It is not clear if and how these conditions contribute to the observed cognitive impairments. In animal studies scientists fed rodents experimental diets that increased blood homocysteine levels from
either folate or vitamin B12 deficiency or by high intake of methionine without vitamin deficiency. These models pave the way for elucidating the relation of dietary imbalances in these factors and cognitive decline in human populations. These findings are related to Objective 2, Milestone 6, and to National Program 107 - Human Nutrition program components: 4. Nutrient Requirements; and 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle; and to Performance Measure 4.1.2. Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. These findings, once verified in controlled trials, may lead to nutritional interventions to prevent some dementias. Please refer to the responses to question 4B for a summary of the accomplishments of the Neuroscience Laboratory. These results are related to National Program 107 - Human Nutrition, program components 5 - Health Promoting Properties of Plant
and Animal Foods; and 6. Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle; and to Performance Measure 4.1.2: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and Nutritional Value of Foods. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The Nutrition and Neurocognition Laboratory's research on B vitamin nutrition and age-related cognitive function has been reported in peer- reviewed journals and has been presented to industry groups and scientists at professional meetings. There are several organizations to whom the findings of the Neuroscience Laboratory regarding the beneficial effects of fruits and vegetables on brain aging have been transferred through CRADAs - see
progress reports for subordinate projects 51000-063-03S and 51000-063-04T The Effects of California Dried Plums on Cognitive and Motor Deficits in Aging; 51000- 063-05T The Effects of Purple Grape Juice on Cognitive and Motor Deficits in Aging; 51000-063-06T and 51000-063-07T Localization of Fruit Polyphenolics in vitro: Beneficial Biological Actions; 51000-063-09T The Effects of Pomegranate Juice on Cognitive and Motor Deficits in Aging; and 51000-063-10T The Effects of Strawberries on Cognition and Neuronal Communication in Aging: Mechanistic Considerations). The findings from our previous CRIS cycle regarding the effects of blueberries in aged animals have been disseminated throughout the blueberry industry. Subsequently the per capita consumption of blueberries has increased significantly since the publication of our first paper in this area in 1999. Since then several other commodity groups (e.g., strawberry, walnut, avocado, etc.) have expressed interest in supporting
investigations of the possible beneficial effects of their fruits, etc. on brain/behavioral function in aging. Members of the Neuroscience Laboratory have also given talks on healthy eating to various nutrition groups who are concerned about the quality of nutrition in the US and other countries. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Nutrition and Neurocognition Laboratory Presentations: Natalia Denisova Nutrients/nutraceuticals established to improve brain function. DSM Nutritional Products AG, June 27-28, 2005, Delf, Netherlands. Kristen E. D'Anci, Arjun Vibhakar; Jordan Kanter; Caroline R. Mahoney; Holly A. Taylor Hydration Status for Optimal Cognitive Performance American Psychological Annual Meeting, May 2005, Los Angeles, CA Troen AM. Homocysteinemia and Cognitive Dysfunction: Insight from Animal Models. Plenary Lecture:
Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, Scott T, D'Anci KE, Jacques PF, Selhub J, Rosenberg IH and the FAVORIT trial consortium. (2005). Cognitive Function and Homocysteine in Renal Transplant Recipients: A FAVORIT ancillary study - Design and Cohort Description. Hematologica Reports 1(3) p.21. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, D'Anci KE, Albuquerque BM, Smith DE, Selhub J, Rosenberg IH, Kruger WD. (2005) Behavioral phenotype of adult-onset homocysteinemia in aged mice with conditional CBS deficiency. Hematologica Reports 1(3) p.28. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, D'Anci KE, Albuquerque BM, Smith DE, Rosenberg IH, Selhub J (2005). "Folate-deficiency-induced cognitive deficits in rat are ameliorated by methionine and are unrelated to homocysteinemia". Hematologica Reports 1(3) p.51. Homocysteine
Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Troen AM, D'Anci KE, Albuquerque BM, Smith DE, Rosenberg IH, Selhub J. (2005) Chronic B12 deficiency induces homocysteinemia and anemia without neurocognitive deficits in rat. Hematologica Reports 1(3) p.52. Homocysteine Metabolism - 5th International Conference Milano, Italy, June 26-30, 2005. Neuroscience Laboratory - Selected publications in the popular press 1) 33 Greatest Foods for Healthy Living, Mother Earth News, Feb/Mar 05 2) Nutrition Today, Blueberries in the American Diet, Mar/05 3) Florida Sports, Fast Food, Mar/05 4) News Journal, Making Healthful Dietary Choices, Mar/05 5) O, The Oprah Magazine, The Incredibles, Apr/05 6) Sunday Denver Post & Rocky Mtn. News, Food Hotline, Apr/05 7) News Tribune, Scale back on the weigh-ins, Apr/05 8) Courier-Journal, Color Palate, Apr/05 9) Tufts University Health & Nutrition Letter, Getting Smart about Alzheimer's, May/05 10) Arthritis Today, You
Are What you Eat, May/05 11) More Magazine, Eat, Drink and Be Healthy, May/05 12) Penthouse, Snore No More, May/05 13) Now, Upping the Anti(oxidants), May/05 14) Daily Telegraph, Does it work?, May/05 15) Scientific American, Antioxidant-Heavy Diet Provides Protection, Jun/05 16) Daily Telegraph, Berried Pleasure, Jun/05 17) Better Homes & Gardens, In Good Taste, Jul/05
Impacts
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Publications
- Smith, M.A., Zhu, X., Casadesus, G., Aliev, G., Ogawa, O., Nunomura, A., Takeda, A., Joseph, J.A., Peterson, R.B., Perry, G., 2003. Alzheimer disease: causes, consequences and surprises. Biogerontology 2003, 4 (Suppl 1): 88-89.
- Shukitt Hale, B., Meterko, V., Carey, A.N., Bielinski, D., Mcguie, T., Galli, R., Joseph, J.A. 2005. Dietary supplementation with fruit polyphenolics ameliorates age-related deficits in behavior and neuronal markers of inflammation and oxidative stress. Age. 2005, 27, 49-57.
- Rottkamp, C.A., Atwood, C.S., Joseph, J.A., Akihiko, N., Perry, G., Smith, M.A. 2002. The state versus amyloid-beta: the trail of the most wanted criminal in alzheimer's disease. Peptides 23 (2002) 1333-1341.
- Youdim, K.A., Joseph, J.A. 2003. Phytochemicals and brain aging: a multiplicity of effects. Book Chapter.
- Youdim, K.A., Shukitt Hale, B., Joseph, J.A. 2004. Flavonoids and the brain: interactions at the blood brain barrier and their physiological effect on the cns. Free Radical Biology and Medicine, VOL 37, NO11 PP. 1683-1693.
- Joseph, J.A., Fisher, D.R., Carey, A.N., Szprengiel, A. 2004. The m3 muscarinic receptor i3 domain confers oxidative stress protection on calcium regulation in transfected cos-7 cells. Aging Cell (2004)3, pp263- 271.
- Martin, A., Smith, M.A., Perry, G., Joseph, J.A. 2004. Nutritional antioxidants, vitamins, cognition and neurodegenerative disease. Principles of Gender Specific Medicine, Volume 2. Elsevier: San Diego, 2004, 813-823.
- Casadesus, G., Shukitt Hale, B., Stellwagen, H.M., Zhu, X., Lee, H., Smith, M.A., Joseph, J.A. 2004. Modulation of hippocampal plasticity and cognitive behavior by short-term blueberry supplementation in aged rats. Nutritional Neuroscience, Volume 7 Number 5/6 (October/December 2004), pp. 309-316
- Rabin, B.M., Joseph, J.A., Shukitt Hale, B., 2005. Effects of age and diet on the heavy particle-induced disruptions of operant responding produced by a ground-based model for exposure to cosmic rays. Brain Research 1036(2005) 122-129.
- Joseph, J.A., Shukitt Hale, B., Smith, M. 2004. Nutrition and communication in the aged brain: can you hear us now? Acarology International Congress Proceedings.
- Lee, H., Casadesus, G., Zhu, X., Tabaton, M., Joseph, J.A., Perry, G., Smith, M. 2004. Different perspectives on the amyloid-b cascade hypothesis. Journal of Alzheimer's Disease.
- Joseph, J.A., Fisher, D.R., Carey, A.N. 2004. Fruit extracts antagonize ab- or da-induced deficits in ca2+ flux in m1-transfected cos-7 cells. Journal of Alzheimer's Disease 6(2004) 403-411.
- Youdim, K.A., Mcdonald, J., Kalt, W., Joseph, J.A. 2002. Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults. Journal of Nutritional Biochemistry. 13:282-288.
- Obrenovich, M.E., Joseph, J.A., Atwood, C.S., Perry, G., Smith, M.A. 2002. Amyloid-2: a (life) preserver for the brain. Neurobiology of Aging.
- Rabin, B.M., Carrihill-Knoll, K.L., Carey, A.N., Shukitt Hale, B., Joseph, J.A. 2005. Effect of diet on the disruption of operant responding at different ages following exposure to fe56 particles. Age. 205, 27, 69-73.
- Shukitt Hale, B., Carey, A.N., Joseph, J.A. Phytochemicals in foods and everages: Effects on the Central Nervous System. In lieberman, H.R., Kanarek, R.B., and Prasad, C.eds. Nutritional Neuroscience. CRC Press, Taylor & Francis Group, LLC Boca Raton, FL, 2005, 393-404.
- Joseph, J.A., Bielinski, D., Fisher, D.R. 2004. Blueberry extract inhibits DA-induced increases in MAPK signaling in Muscarinic M1 and M3-transfected COS-7 cells. Soc.Neurosci. Abs. 2004, 30, 1017.17.
- Shukitt Hale, B., Carey, A.N., Belinski, D., Lau, F.C., Galli, R.L., Spangler, E.L., Ingram, D.K., Joseph, J.A. 2004. Fruit polyphenols prevent inflammatory mediated decrements in cognition. Soc. Neurosci. Abs. 2004, 30,565.5.
- Smith, M.A., Atwood, C.S., Joseph, J.A., Perry, G. 2002. Predicting the failure of amyloid-beta vaccine. Lancet. 359:1864-1865.
- Casadesus, G., Stellwagen, H.M., Shukitt Hale, B., Rabin, B.M., Joseph, J. A. 2005. Hippocampal neurogenesis and psa-ncam expression following exposure to 56fe particles mimics that seen during aging. Gerontology Journal. 40 249-254
- Joseph, J.A., Shukitt Hale, B., Casadesus, G. 2005. Reversing the deleterious effects of aging on neuronal communication and behavior: the beneficial properties of fruit polyphenolics. American Journal of Clinical Nutrition 2005, 81 (Suppl.) 313S-316S
- Andres-Lacueva, C., Shukitt Hale, B., Galli, R.L., Jaurengui, O., Lamuela- Raventos, R.M., Joseph, J.A. 2005. Anthocyanins in aged blueberry-fed rats are found centrally and may enhance memory. Nutritional Neuroscience, April 2005:8(2): 111-120.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This CRIS consists of the Nutrition and Neurocognition Laboratory and the Neuroscience Laboratory. This program aims to gain insight into the interaction between nutritional factors and age-related cognitive, motor, brain, and central nervous system declines in humans. Whereas the single most significant risk factor for cognitive decline and dementia is age, the fact that many individuals maintain their full mental faculties into old age suggests that cognitive decline is not a necessary feature of normal aging. Declining brain function may have multiple causes including processes such as vascular disease, oxidative stress and inflammation, all of which are highly influenced by nutrition. Aging may affect the brain's requirement for nutrients that are involved in normal brain metabolism; for example,
in the synthesis of neurotransmitters and maintenance of brain-cell signaling, connectivity and repair capacity. The program takes a broad approach to the problem by identifying nutritional factors that are associated with cognitive decline and neurodegenerative conditions in population studies; by investigating the mechanisms underlying those associations through nutritional intervention in genetic animal models of human dementia syndromes and in cell systems; and through efforts to translate observations from the population, animal and cell studies, into human studies of dietary intervention and brain aging. Ultimately, the program is directed towards determining the nutritional requirements for maintaining a healthy brain and for promoting neurological and cognitive function in the elderly. By the year 2050, 30 percent of the population will be over 65 years of age. There is a high probability that this group will exhibit the most common correlative motor and cognitive behavioral
changes that occur in aging. These changes may occur in concert with or in the absence of neurodegenerative diseases. In cases of severe deficits in memory or motor function, hospitalization and/or custodial care may be a likely outcome. Thus, in both financial and humane terms it is extremely important to explore methods to retard or reverse the age-related neurological deficits as well as their subsequent, behavioral manifestations. By identifying nutritional factors that are associated with risk age-related and pathological decline in brain function, and by investigating the mechanisms by which they modulate such decline, (such as determining their relationship to oxidative stress). This CRIS aims to develop nutritional methods that might maintain healthy brain function into old age. This research is related to the National Program 107 Human Nutrition program components: 1. Nutrient requirements, 2: Diet, genetics, lifestyle, and the prevention of obesity and disease, and 6:
Health promoting intervention strategies for targeted populations. This research is part of a program designed to determine the role of nutritional factors in brain aging and possible nutritional interventions that may be utilized to alter the deleterious effects of aging. 2. List the milestones (indicators of progress) from your Project Plan. Milestones - Nutrition and Neurocognition Laboratory 1a. Study the relation between brain volumes, cognitive performance, and homocysteine in the Framingham Offspring cohort over 10 years. 1b. Recruit homebound elderly for baseline studies. 1c. Recruit participants in FAVORIT cognitive outcome ancillary study. 2a. Complete and analyze cross-sectional studies of homebound elderly - MRI's cognition, and nutritional biochemistries. 2b. Complete data collection of cognitive effect of homocysteine lowering. 3. Results of studies on the effect of aging in rats on one carbon metabolism with emphasis on difference among various tissues. Publish results
of the experiments of the apoE-null mice, showing separate effects of high methionine intake vs hyperhomocysteinemia on vascular vs cognitive impairments. 4a. Further characterization of the effect of diet on vascular impairment in ApoE-null mice. 4b. Further characterization of cognitive dysfunction in ApoE-null mice for discerning between effects of homocysteine vs vitamin deficiency. Milestones - Neuroscience Laboratory Objective 1: Analyze factors in oxidative stress (OS) and inflammatory (INF) sensitivity in COS-7 cells. 1a) Develop and test procedures to construct chimerics and point mutations in M1 and M3AChR and assess sensitivity to OS and INF. 1b) Assess the effects of lipid raft modification on OS and INF sensitivity. 1c) Identify blueberry (BB) and strawberry (SB) polyphenolics that offer protection against OS and INF in M1AChR-transfected COS 7 cells. 1d) Continue studies to assess OS and INF sensitivity in M1 and M3AChR cells with point mutations in the I3 loop. 1e)
Assess the effect of these mutations on the lipid raft microenvironment. Objective 2: Behavioral evaluations following blueberry (BB), strawberry (SB), or BB or SB polyphenolic supplementation. 2a) Assess motor and cognitive behaviors in young and old rats following BB or SB supplementation with a view toward correlations with signaling, etc. parameters. 2b) Continue motor and cognitive behaviors following supplementation with BB or SB polyphenolics identified in the cell work as showing INF or OS protection. Correlate behavior with signaling parameters, etc., which show change. Objective 3: Signaling, neurogenesis, gene array, OS and INF markers. 3a) Assess brain regions for signaling, neurogenesis and OS and INF markers, after behavior is assessed. Begin gene array analysis. 3b) Continue these evaluations with additional supplementations of the polyphenolics. Utilize immunocytochemistry to validate the gene array analyses. Objective 4: Enhancements of OS and INF. 4a) Determine
protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with BB and SB. 4b) Determine protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with SB or BB polyphenolics identified in the cell work. 3. Milestones: Question 3A: The following milestones were scheduled to be addressed by Neuroscience Laboratory in FY2004: 1. Develop and test procedures to construct chimerics and point mutations in M1 and M3AChR and assess sensitivity to OS and INF. Experiments thus far have revealed that deletions of the entire i3 loop increased dopamine (DA) sensitivity (a lower % of cells showing recovery following depolarization) in both the M1 and M3 subtypes. Chimerics of M1 where the i3 loop of the M3AChR was switched with the i3 loop of the M1AChR (M1M3i3) showed that the DA sensitivity was reduced (% cells showing increases in calcium clearance)
following depolarization. In the M3 chimerics containing M1i3 (M3M1i3), the i3 loop offered no protection against DA-induced decrements in calcium buffering. We have also been assessing the effects of BB treatment on oxidative stress vulnerability (OSS) in the chimerics and wild type M1 and M3 receptors. Results, thus far, have indicated that BB reduced OSS (i.e., Ca2+ buffering following depolarization) to DA in M1-transfected cells. However, BBs were also effective in preventing these Ca2+ buffering deficits in cells transfected with M1 receptors in which the i3 loop had been removed, but only partially enhanced the protective effects of the M3 i3 loop in the M1/M3i3 chimerics. It appears that antioxidants might be targeting additional sites on these chimerics to decrease OSS. 2. Assess motor and cognitive behaviors in young and old rats following BB or SB supplementation with a view toward correlations with signaling, etc. parameters. We have begun these assessments in groups of
young and old control or BB-fed rats. 3. Determine protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with BB and SB. Four month old male F344 rats were fed a control, 2% BB, or 0.015% piroxicam (PX, an NSAID) diet for 8 weeks before Ringers (R) or kainic acid (KA, 300ng in 0.5ul R) was injected bilaterally into the hippocampus. Ten days later, rats were tested for 4 days in the Morris water maze (MWM) before measuring brain inflammatory markers. KA produced an inflammatory response as shown by increased OX-6 activation in the hippocampus, and had deleterious effects on cognitive behavior. Specifically, rats on the control diet given KA had increased latencies to find the hidden platform in the MWM on days 1-3, and on day 4 during reversal learning, compared to the R group. These deficits were improved by the BB diet; latencies to find the platform on days 2-4 in the BB-KA group were not different than the
control diet-R group. Also, the KA group utilized non-spatial strategies during the probe trials on days 2- 4: i.e., longer latencies to, fewer crossings of and less time spent in the previous platform location The BB diet, and to a lesser degree the PX diet, was able to improve performance on probe trial measures. Also important for this objective is that we have found that the striatal tissue taken from BB-fed senescent rats exposed to 50 M hydrogen peroxide showed greater protection against the loss of muscarinic receptor sensitivity than that seen in the striatal tissue from non-BB supplemented rats. Muscarinic receptor sensitivity was assessed by looking at oxotremorine-induced increases in DA release from striatal tissue obtained from these animals. Experiments to initiate the delineation of the other components of the objectives cited in 2 have not begun as yet, since the protocol was only recently approved . Question 3B: The Nutrition and Neurocognition Laboratory expects to
accomplish the following: Year 1 (FY2005): Objective 1: a. Study the relation between brain volumes, cognitive performance and homocysteine in the Framingham Offspring cohort. b. Recruit Homebound Elderly for baseline studies. c. Recruit participants in FAVORIT cognitive outcome ancillary study. Objective 2: a. Study the effect of aging in rats on one carbon metabolism with emphasis on difference among various tissues. b. Publish results of the experiments on the ApoE-null mice showing separate effects of high methionine intake vs hyperhomocysteieinmai on vascular vs cognitive impairments Year 2 (FY2006): Objective 1: a. Study the relation between brain volumes, cognitive performance and homocysteine in the Framingham Offspring cohort. b. Recruit Homebound Elderly for baseline studies. c. Recruit participants in FAVORIT cognitive outcome ancillary study. Objective 2: a. Study the effect of aging in rats on one carbon metabolism with emphasis on difference among various tissues. b.
Publish results of the experiments on the ApoE-null mice showing separate effects of high methionine intake vs hyperhomocysteieinmai on vascular vs cognitive impairments. Year 3 (FY2007): Objective 1: a. Study the relation between brain volumes, cognitive performance and homocysteine in the Framingham Offspring cohort. b. Recruit Homebound Elderly for baseline studies. c. Recruit participants in FAVORIT cognitive outcome ancillary study. Objective 2: a. Further characterization of the effect of diet on vascular impairment in ApoE-null mice. b. Further characterization of cognitive dysfunction in ApoE-null mice fro discerning between effect of homocysteine vs. vitamin deficiency The Neuroscience Laboratory plans to accomplish the following: Year 1 (FY2005): Objective: 1 Analyze Factors in oxidative stress (OS) and inflammatory (INF) sensitivity in COS-7 cells. 1a. Assess the effects of lipid raft modification on OS and INF sensitivity. 1b. Identify BB and SB polyphenolics that offer
protection against OS and INF in M1AChR-transfected COS 7 cells. Objective 2: Behavioral evaluations following blueberry (BB), strawberry (SB), or BB or SB polyphenolic supplementation. Milestones: 2a. Assess motor and cognitive behaviors in young and old rats following BB or SB supplementation with a view toward correlations with signaling, etc., parameters. Objective 3: Signaling, neurogenesis, gene array, OS and INF markers. 3a. Assess brain regions for signaling, neurogenesis and OS and INF markers, after behavior is assessed. Begin gene array analysis. Objective 4: Enhancements of OS and INF. 4a. Determine protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with BB and SB. Year 2 (FY2006): 1a. Assess the effects of lipid raft modification on OS and INF sensitivity. 1b. Identify BB and SB polyphenolics that offer protection against OS and INF in M1AChR-transfected COS 7 cells. 2a. Assess motor and
cognitive behaviors in young and old rats following BB or SB supplementation with a view toward correlations with signaling, etc., parameters. 3a. Assess brain regions for signaling, neurogenesis and OS and INF markers, after behavior is assessed. Begin gene array analysis. 4a. Determine protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with BB and SB. Year 3 (FY2007): 1a. Assess the effects of lipid raft modification on OS and INF sensitivity. 1b. Identify BB and SB polyphenolics that offer protection against OS and INF in M1AChR-transfected COS 7 cells. 2a. Assess motor and cognitive behaviors in young and old rats following BB or SB supplementation with a view toward correlations with signaling, etc., parameters. 3a. Assess brain regions for signaling, neurogenesis and OS and INF markers, after behavior is assessed. Begin gene array analysis. 4a. Determine protective capacity against INF and OS insults
in tissue obtained from young and senescent control animals or those supplemented with BB and SB. 4b. Determine protective capacity against INF and OS insults in tissue obtained from young and senescent control animals or those supplemented with SB or BB polyphenolics identified in the cell work. 4. What were the most significant accomplishments this past year? This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-055-00D Dietary Effects on Neuronal Signaling. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-055-00D Dietary Effects on Neuronal Signaling. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become
available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-055-00D Dietary Effects on Neuronal Signaling. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-055-00D Dietary Effects on Neuronal Signaling.
Impacts
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Publications
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