Location: Human Nutrition Research Center on Aging
2005 Annual Report
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.
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
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
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.
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.
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.
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/05Lee, 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.
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.
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.