2006 Annual 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 – MRI’s 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 Alzheimer’s 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 Alzheimer’s type pathology directly, but rather that it may contribute to cognitive dysfunction in Alzheimer’s 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 Alzheimer’s 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 isolation—that 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 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., 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. 4c.List significant activities that support special target populations. None 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 Welch’s 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 isolation—that 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 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. 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 Alzheimer’s 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, they’re also adding muscle. Looking who’s 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