2009 Annual Report 1a.Objectives (from AD-416) LAB:Nutrition and Cognition Determine whether and how nutritional factors, especially B vitamins, can be employed in the understanding and prevention of age-related cognitive impairment in humans and in human populations. Charecterize the mechanisms by which nutritionally induced hyperhomocysteinemia affects neuronal function and cognitive performance using transgenic mouse models of human cognitive decline. LAB:Neuroscience Identify the structural and compositional difference among muscarinic receptor subtypes and the lipid microenvironment (lipid rafts) or their combination that contribute to increased vulnerability to oxidative stress and inflammation in aging in the COS-7 cell model. Assess the protective capability of berryfruit polyphenolic extracts and determine the most effective component polyphenol(s) against oxidative stress and inflammatory agents in a muscarinic receptor transfected COS-7 cell model. Assess the vulnerability to oxidative and inflammatory stressors in microglia cells (which may affect loss of neuronal function in aging), or hippocampal cells (which may be involved in memory function) and determine the effects of polyphonolic and berryfruit extracts. Establish the effects of dietary berryfruit extracts and the most effective component polyphenolics on neuronal function in aging by determining the effects on motor cognitive behaviors as a function of age. (a) Identify brain regional localization of berryfruit compounds and correlating the amounts seen with the behavioral performance. (b) Determine their effects on signaling and the generation of new neurons in aging. 1b.Approach (from AD-416) LAB:Nutrition and Cognition Collaboration will continue with the Framingham Heart Study cohorts to examine prospective relationships among nutritional status, cognitive performance, homocysteine, and brain morphology by imaging. Using the homebound elderly population, we will study these relationships first cross-sectionally, and then prospectively. We will add other factors, including antioxidant vitamin status and its relation to inflammatory markers and adhesion molecules in the vasculature. We have designed a trial as an adjunct to the large homocysteine lowering intervention on subjects who have undergone renal transplantation to measure the impact of homocysteine lowering on cognitive decline and performance. The animal models we intend to use are the apoE knockout, the APP/PSI and an APP/London, in which we can modify nutritional status with respect to B vitamins and homocysteine levels and examine the relationships between increased sensitivity to behavioral decline. We will compare age-related status of membrane sphingolipids and vitamins K & A in brain regions controlling behavior in aged Fisher rats. We will also compare the status of membrane sphingolipids, one carbon metabolism, and neuronal viability in a human neuronal cell culture model. LAB:Neuroscience COS-7 cells transfected with various muscarinic receptor subtypes and their chimerics will be used to identify the structural and membrane lipid raft differences contributing to increased oxidative stress vulnerability by exposing them to dopamine and assessing alterations in calcium flux and viability. Motor and cognitive assessments will be undertaken in senescent rats given diets supplemented with whole berryfruits or extracts derived from them. Correlative determinations of neurogenesis, neuronal signaling and gene activation or inhibition will also be undertaken in various brain regions of these control and supplemented animals. 3.Progress Report 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). 4.Accomplishments 1. Blueberry/Strawberry Extract Prevent Oxidative Stress and Inflammation Signals in Cells. (LAB: Neuroscience). Supplementation with berry fruits such as blueberries (BB) or strawberries (SB) can enhance cognitive and motor function in aged animals and can modulate function in cells, possibly by decreasing inflammatory and oxidative stress. Recent experiments have shown that deficits in calcium function induced by the oxidant stressors dopamine or amyloid beta (AB) in cells are blocked by BB extract and that BB pretreatment can reduce both stress- and inflammatory- induced neuronal dysfunction. A related study in cells indicated that at least part of BB-induced reductions in stress mediated signaling might involve decreases in nitric oxide which is a powerful stressor. Experiments were carried out with BB and SB to determine if similar findings could be seen in co-cultures of different but related cells sensitive to oxidative stress and inflammation. Results indicated that in the cells the effectiveness of the various treatments are dependent upon the stressor that we used such as dopamine, suggesting that it is better to eat a variety of berries. We showed for the first time that mechanism other than the inactivation of free radicals (molecules producing oxidative stress) are involved in the beneficial effects of the berry fruits. Discovery of these additional mechanisms might lead to important dietary information for an aging population. 2. Walnut Associated Fatty Acids Inhibit Inflammation. (LAB: Neuroscience). Walnuts contain high levels 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 keep cell membranes flexible, which is critical for neuronal communication. To determine the effects of PUFAs on activation of cells involved in inflammation, cells were treated either before or after exposure to the inflammatory agent lipopolysaccharide (LPS). LPS exposure increased production of pro-inflammatory mediators such as nitric oxide (NO), and other inflammatory agents. However, treatment of the cells with either PUFA prior to or following LPS activation prevented these stressor effects, such as NO production that enhances the inflammation in cells. Additionally, these cells were also treated with a combination of LA and ALA at the concentration found in our walnut extract and this combination exerted the strongest effect against the stressors, particularly when given prior to LPS treatment. Ongoing studies are investigating the extent of anti-inflammatory activity of the walnut extract and corresponding fatty acids, focusing on protein expression of enzymes known to contribute to cell-mediated neurotoxicity and modulation of cytokine expression. We showed for the first time that walnuts can inhibit inflammation, possibly because of the PUFAs contained in them, and this may be the mechanism involved in the beneficial effects of the walnuts. These findings are important in both industry and health areas, since they point once again to the importance of including nuts in the diet for promoting healthy aging. 3. Raspberries Can Improve Motor Function in Aged Rats. (LAB: Neuroscience). Aged rats show decrements in performance on motor and cognitive tasks that require the use of spatial learning and memory. In previous studies, we have shown the beneficial effects of various berry fruits (blueberries, strawberries, and blackberries) for their effectiveness in reversing age-related deficits in behavioral and neuronal function when fed to old rats. These effects may be the result of increasing antioxidant and/or anti-inflammatory levels, or by direct effects on cell signaling (necessary for learning and memory), in the brain. Red raspberries contain compounds (called polyphenols) similar to those found in other berry fruits. Thus, the present studies were carried out to determine if raspberries, fed in the rat diet at 2% (about a cup in human terms) for 8 weeks, would be efficacious in reversing the deleterious effects of aging on motor and cognitive behavior. Results for the motor testing showed that the raspberry diet improved performance on measures of motor function (e.g., balance). There was no improvement seen in cognition in the tests used. These findings are important in both industry and health areas, since they point once again to the importance of including whole berries in the diet for promoting healthy aging especially with changes in membrane lipids. 4. Anti-Inflammatory Properties of Walnuts. (LAB: Neuroscience). We have found that walnut oils (PUFA’s) can alter inflammatory signals and calcium regulation in cells. Because we have also demonstrated that dietary supplementation of aged rats with walnuts improves behavioral performance, we assessed the activity of cells involved in inflammation called microglial cells. We measured microglial activity in these animals and determined that supplementation with 6% walnuts (1oz for humans) produced fewer activated microglia in one part of the brain necessary for memory (the hippocampus). To examine the anti-inflammatory properties of walnuts in vitro, mouse microglial cells (BV-2) were pretreated with walnut extract overnight which rendered microglial cells significantly less responsive to the activating effects of an inflammatory agent, lipopolysaccharide (LPS). Mechanistically, walnut extract may induce internalization of a receptor for LPS called a toll-like receptor 4 (TLR4), that initiates the inflammatory process. Walnuts are also able to decrease an enzyme called acetylcholinesterase (AChE) which inactivates a neurotransmitter called acetylcholine (ACh). In aging and AD ACh declines, with corresponding declines in cognition, so that decreasing AChE will increase ACh levels and improve cognition. The impact of walnuts on microglia and the cholinergic system may represent an easily accessible therapeutic option to combat brain inflammation and cognitive decline in aging. 5. Dietary effects on cerebral blood circulation. (LAB: Nutrition and Cognition) We have previously shown that B-vitamin deficiency caused a structural decrease in the density of small blood vessels in the brain which are necessary for the delivery of oxygen and nutrients for normal brain function. To determine whether these structural changes have a measurable functional effect on brain blood circulation, we developed a non-invasive near infra red device (NIRS) for measuring brain blood content and oxygen concentration in rodents. This device is now allowing us to probe the functional effects of B-vitamin deficiency and other dietary imbalances on brain circulation. 6. Dietary folate and brain neurodegeneration. (LAB: Nutrition and Cognition). We have previously shown a diminishing effect of the dietary-induced folate deficiency on cognitive functions and brain membranes in young rats. To determine whether these changes have an impact on Alzheimer’s-related pathology, we will use a mouse model with genetic mutations in APP and PS1 genes. This animal model will allow us to investigate the interrelation between dietary-induced folate deficiency, cognitive functions, and brain pathology as a function of age. 5.Significant Activities that Support Special Target Populations LAB: Neuroscience None. LAB: Nutrition and Cognition None. 6.Technology Transfer Number of New CRADAS 1 Number of Active CRADAs 1 Number of New Patent Applications Filed 1 Review Publications 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.