1a. Objectives (from AD-416):
Objective 1: Characterize the effects of genetic, metabolic, and environmental influences on the aging brain and vasculature and the modifying impact of nutrition on neuronal function, cognition, and behavioral outcomes. Sub-objective 1A: Use human observational data to evaluate the relationships between nutrients/ bioactives, age-related cognitive decline, and neurodegenerative diseases, and characterize interactions between diet and genes, and environmental influences on these outcomes. Sub-objective 1B: Use human intervention studies as translational studies to determine the effects of intervention with specific nutrients or bioactives on cognitive and behavioral function. Sub-objective 1C: Use animal and cell models to develop mechanistic interpretations of the benefits of nutrients and bioactives to the brain in aging models in rodents. Determine the mechanisms related to the behavioral effects of nutrients and bioactives on biomarkers of inflammation and oxidative response. Objective 2: Characterize molecular, cellular, and physiological mechanisms by which food and nutritional factors affect the Central Nervous System regulation of aging processes and energy homeostasis. Sub-objective 2A: Assess whether age-related inflammation affects the cell genesis and cellular structure of the hypothalamus. Sub-objective 2B: Evaluate whether dietary intervention and natural products can be used to counteract age-related biochemical and structural changes in the hypothalamus. Sub-objective 2C: Assess whether dietary/natural product intervention attenuates systemic aging.
1b. Approach (from AD-416):
This project will utilize in vitro and in vivo studies on the mechanisms of action of nutrients and bioactives in improving brain function during aging. In particular, with a focus on age-related cognitive decline, including that associated with Alzheimer’s Disease, two objectives will focus on 1. The effects of genetic, metabolic, and environmental influences on the aging brain and vasculature and the modifying impact of nutrition on neuronal function, cognition, and behavioral outcomes. In addition to human observational data, cell and animal models will help resolve mechanisms underlying the benefits of nutrients and bioactives in the aging brain using rodent models. These studies will rely on inflammatory and oxidative stress molecular biomarkers as read outs of the preventative and potential disease attenuating attributes of diet and nutrition; and, 2. The characterization of molecular, cellular and physiological mechanisms by which food and particular combinations of phytocompounds act as nutritional and disease preventing/ treating factors for the aging central nervous system. In particular, the testing of a new theory of interactive brain and body aging will define age-related inflammation effects on cell genesis and cellular structure of the hypothalamus. In all, the studies proposed here, utilizing state of the art genetic, molecular, cellular and behavioral methodologies, will elucidate whether and how dietary/natural product intervention attenuates negative aspects of systemic aging.
3. Progress Report:
The Neuroscience and Aging Laboratory seeks to identify applications of nutrition-based preventatives and therapeutics for reversing or slowing age-associated cognitive decline, neurodegenerative disorders and age-related brain neoplastic disorders using an integrative approach of cellular, animal and human models. This translational research takes findings from basic science to design human intervention studies as the next step towards the goal of real-world applications benefiting human health and quality of life. Studies related to Objective 1, are being completed for clinical studies investigating the effects of intervention with whole foods – such as blueberries, strawberries, and avocados – on cognitive outcomes and have found that supplementation with these foods increased cognitive function in healthy older adults. To examine how polyphenols in blueberries and strawberries were absorbed and whether this absorption was related to cognition, we measured circulating levels of polyphenolics in plasma, including anthocyanins, ellagitannins, and phenolic acids, after an overnight fast and 2 hours postprandial. We found that these compounds were significantly altered as a result of berry consumption, as the polyphenols were absorbed and extensively metabolized, resulting in the production of various phenolic acid derivatives and their conjugates. Additionally, changes in circulating levels of specific phenolic compounds were correlated with the observed changes in cognition. Therefore, the addition of easily achievable quantities of berry fruit to the diets of older adults can improve some aspects of cognition, which is likely due to increased levels of circulating berry phenolics and their metabolites. Studies are underway for Objective 2 to examine the mechanisms of combined bioactive nutrient compounds to provide insights into diet and nutrient molecular pathways that help to prevent and treat age-related neurological disease. The first proof of principle is the compound EpidiferphaneTM (EDP) and its ability to attenuate biomarkers of inflammation using cell and animal models. At least part of the loss of cognitive function in aging may be dependent upon a dysregulation in calcium homeostasis, and this loss affects numerous signaling pathways. We conducted a study to examine whether the polyphenolics from blueberries (BB) or EpidiferphaneTM (EDP,) a combination of phytochemicals incorporating green tea catechin (epigallocatechin gallate, EGCG,) curcumin from turmeric, and broccoli sprouts which contain the isothiocyanate sulforaphane, could enhance calcium buffering in neurons and/or reduce stress signaling in microglial cells. Both BB and EDP were able to protect against deficits in calcium buffering induced by a dopamine stressor, showing that pre-treatment with these compounds can reduce both stress- and inflammatory-induced neuronal dysfunction. Additionally, human neural stem/progenitor cell models, including cells derived from Parkinson’s and Alzheimer’s patients and normal controls, are being established in the lab. We utilized these in vitro models for testing the effects of EDP and second, third, etc. generation combinations of whole food-derived nutrients on progenitor cells’ viability, proliferation and differentiation. While EDP as well as its individual components did not show significant effects on viability, proliferation and differentiation of adult human neural progenitor cells, “AHNPs,” that we discovered in the human brain gray matter throughout the lifespan, they did demonstrate neuroprotection for the AHNPs following cellular stress induced by a dopamine stressor. In both studies, the individual components of EDP were not as effective as the whole compound, showing that the individual polyphenols in the different components may be acting synergistically or exerting their effects through different and/or independent mechanisms. Dietary blueberry also attenuated radiation-induced declines in protein carbonyl content, a marker for oxidative protein degradation, as well as reduced the oxidative and inflammatory load, and enhanced endogenous protective signaling. Therefore, dietary intervention with compounds such as those found in blueberries, green tea, turmeric, or broccoli sprouts can play a role in reducing the age-related central nervous system inflammation, microglial activation, and stimulation of immune pathways that reduce neurogenesis and impair cognitive function. Additionally, we began an animal study to examine the mechanisms related to the behavioral effects of nutrients and bioactives, specifically EDP as our first generation of polymolecular botanical compound combinations, on biomarkers of inflammation. Long-term consumption of a high fat diet (HFD) has been shown to increase inflammation and oxidative stress (OS) in the brains of rodents, and consumption of a HFD produces behavioral deficits. We initiated a study to see if Epidiferphane™ (EDP,) a combination of antioxidant and anti-inflammatory phytochemicals, could combat the negative effects of a HFD by decreasing inflammation and OS. Therefore, the goal of this study is to show whether consumption of an EDP-supplemented HFD will mitigate the brain alterations and behavioral dysfunction associated with HFD. Additionally, we completed two studies using tart cherries, which are high in polyphenols. Tart cherries improved cognitive behavior in aged rats and were efficacious in reducing inflammatory and OS signaling in a microglia cell model. This protection might be one mechanism by which dietary supplementation of tart cherries can improve age-related deficits in behavioral and neuronal functioning. We have also established protocols to analyze the bioavailability of EDP as well as their individual components in both in-vivo and in-vitro cell culture studies. For in vivo studies, animals were fed with EDP daily for about 12 weeks. At the end of the feeding period, the animals were sacrificed, and their tissues such as liver, kidney, spleen, brain, serum, heart and lungs were collected and stored. Individual components of EDP were extracted for HPLC (high-performance liquid chromatography) analysis to identify the bioavailability of EDP’s principle bioactive components. For in vitro studies, AHNPs were harvested following a 4-day treatment of EDP, and concentration of components of EDP were examined in the cell lysates. Optimized detection methods were successfully developed and significantly reduced the time to run each sample as well the solvents required. With these methods, the concentrations of curcumin and ECGC were detected both in liver as well as kidney tissues, indicating the relatively high bioavailability of these compounds. These findings support the role of diet in combatting the negative effects of exposure to inflammation and oxidative stress through the consumption of polyphenolic-rich and omics-informed anti-inflammatory network foods. A notion of neuroregenerative nutrition is a focus of new bioassays for our lab. With both in vitro and in vivo models of age-related neurological disease, including cellular components that are both at-risk in neurodegenerative disease and brain cancer, along with patient-matched immune cells, stem cell biology is being used as a major focus for combining foods and particular nutrients that can counteract the loss of cellular potency and plasticity associated with a chronic inflammatory tissue microenvironment that accompanies pathological aging, diseases and injuries, as well as standard of care therapies. It is anticipated that our new bioassays and molecular biomarkers for monitoring age-related neurological disease will provide essential data on the roles for diet and nutrition in supporting healthy brain aging and cognitive function in the later third of life. We now have new cellular reagents under development that best reflect the at-risk cellular components of Alzheimer’s Disease: the brain’s innate immune cells, microglia. Using state-of-the-art induced pluripotent stem cells derived from Alzheimer’s versus control skin fibroblasts, microglia are being generated in collaboration with the Massachusetts Institute of Technology-Whitehead Institute that will provide a sensitive and accurate disease target for reducing chronic inflammation as a result of assaying next generation polymolecular botanical food compounds. These compounds are in combinations that target inflammatory pathways that interfere with stem cell repair and regeneration in the brain and other organs and tissues. We are studying the relationship between brain and body aging in ways where nutrient combinations hold promise for supporting the best cell and tissue health. Next generations of EDP are currently under consideration for analysis for a variety of age-related neurological diseases, including Parkinson’s, where stem cell pathologies are potent targets of nutrient preventative and treatment therapies. Mechanisms of action of individual and combination phytonutrients will be elucidated using our next generation cell/organoid/cultures and personalized, precision food and medicine animal models. Using exosomes/microvesicles as the most sensitive biomarkers of disease state and response to standard of care and integrated medicine therapies and preventatives, current studies focus on the roles of diet and nutrient supplementation to reverse chronic inflammatory gene and protein networks involved in susceptibility to age-related neurological disease and progression once disease has passed the prodromal stage.
Poulose, S.M., Rabin, B.M., Bielinski, D.F., Kelly, M.E., Miller, M.G., Thangthaeng, N., Shukitt Hale, B. 2017. Neurochemical differences in learning and memory paradigms among rats supplemented with anthocyanin-rich blueberry diets and exposed to acute doses of 56Fe particles. Life Sciences in Space Research. 12:16-23.
Carey, A., Miller, M.G., Fisher, D.R., Bielinski, D., Gilman, C.K., Poulose, S.M., Shukitt Hale, B. 2017. Dietary supplementation with the polyphenol-rich açaí pulps (Euterpe oleracea Mart. and Euterpe precatoria Mart.) improves cognition in aged rats and attenuates inflammatory signaling in BV-2 microglial cells. Nutritional Neuroscience. 20:238-245.
Miller, M.G., Hamilton, D.A., Joseph, J.A., Shukitt Hale, B. 2017. Dietary blueberry improves cognition among older adults in a randomized, double-blind, placebo-controlled trial. European Journal of Nutrition. 57:1169-1180. https://doi.org/10.1007/s00394-017-1400-8.
Miller, M.G., Thangthaeng, N., Shukitt Hale, B. 2017. A clinically relevant frailty index for aging rats. Journal of Gerontology, Series A, Biological Sciences and Medical Sciences. doi:10.1093/gerona/glw338.
Thangthaeng, N., Miller, M., Gomes, S., Shukitt Hale, B. 2015. Daily supplementation with mushroom (Agaricus bisporus) improves balance and working memory in aged rats. Nutrition Research. 35:1079-1084.
Poulose, S.M., Bielinski, D.F., Carey, A., Schauss, A.G., Shukitt Hale, B. 2016. Modulation of oxidative stress, inflammation, autophagy and expression of Nrf2 in hippocampus and frontal cortex of rats fed with acai-enriched diets. Nutritional Neuroscience. doi: 10.1080/1028415X.2015.1125654.
Thangthaeng, N., Poulose, S., Gomes, S.M., Miller, M.G., Bielinski, D., Shukitt Hale, B. 2016. Tart cherry supplementation improves working memory, hippocampal inflammation and autophagy in aged rats. Age. 38:393-404. doi: 10.1007/s11357-016-9945-7.
Wong, J.C., Scott, T., Wilde, P., Li, Y., Tucker, K., Gao, Z. 2016. Food insecurity is associated with subsequent cognitive decline in the Boston Puerto Rican Health Study. Journal of Nutrition. doi: 10.3945/jn.115.228700.
Scott, T., Rogers, G., Weiner, D.E., Livingston, K., Selhub, J., Jacques, P.F., Rosenberg, I.H., Troen, A. 2017. B-vitamin therapy for kidney transplant recipients lowers homocysteine and improves selective cognitive outcomes in the randomized FAVORIT ancillary cognitive trial. The Journal of Prevention of Alzheimer's Disease. 3:1-9.
Shukitt Hale, B., Kelly, M.E., Bielinski, D.F., Fisher, D.R. 2016. Tart cherry extracts reduce inflammatory and oxidative stress signaling in microglial cells. Antioxidants. doi: 10.3390/antiox5040033.