Location: Jean Mayer Human Nutrition Research Center On Aging
2023 Annual Report
Objectives
Objective 1: Determine the effects of genetic, molecular and environmental influences on the aging brain, and the modifying impact of specific phytonutrients on neural cell function and behavior, including cognition.
Sub-Objective 1a: Characterize genetic and molecular signatures, especially pro-inflammatory markers, of normal adult brain stem/progenitor and differentiated cells, including neurons and microglia, in vitro and following the introduction of whole berry fruits and a combination phytonutrient: polymolecular botanical compound (PBC).
Sub-Objective 1b: Characterize genetic and molecular signatures of normal neural stem/progenitor and differentiated cells from Sub-Objective 1a in vivo following their grafting to the forebrains of immunocompromised mice, and subsequent feeding of the phytonutrients assayed in the in vitro model of Sub-Objective 1a.
Sub-Objective 1c: Characterize the genetic and molecular signatures, especially those associated with chronic inflammatory pathways, and the cognitive behavioral profile in aging models in rodents following feeding of phytonutrient compounds studied in Sub-Objectives 1a and b.
Sub-Objective 1d: Analyze biomarkers, especially those related to chronic inflammation and the cognitive behavioral profile, from liquid biopsies (e.g., serum) collected in human studies following phytonutrient supplementation with the candidate fruit and plant compounds studied in Sub-Objectives 1a-c.
Objective 2: Characterize in vitro and in vivo models that manifest aspects of human age-related neurological diseases, such as Parkinson’s and Alzheimer’s disease, for screening combinations of phytonutrient that can prevent or delay chronic inflammation and other deleterious micro-environmental conditions that contribute to cell degeneration in the neurodegenerative disorders.
Sub-Objective 2a: Characterize, in vitro, the genetic and molecular signatures, especially those associated with chronic inflammatory pathways, of stem/progenitor and differentiated neural and microglial cells (and exosomes isolated from them) isolated from patients with Parkinson’s Disease, following phytonutrient treatments studied in Objective 1.
Sub-Objective 2b: Characterize, in vivo, the genetic and molecular signatures (including mutant LRRK2-associated inflammation, stem cell and cell death/protection gene pathways) of cells, and exosomes derived from them, following xenotransplantation to the forebrain of immunocompromised mice and feeding of the phytonutrients studied in Objective 1.
Sub-Objective 2c: Characterize the genetic and molecular signatures of neural cells at-risk for abnormal functioning and cell death in transgenic mouse models of Parkinson’s disease, including behavioral studies, following feeding of candidate phytonutrient compounds studied in Objective 1.
Approach
As Americans are living longer, the incidence of age-related neurological disorders is a growing burden for older adults and the healthcare system. Our lab studies how plant-derived phytonutrients benefit the aging brain, especially in maintaining mobility and cognitive function and slowing the progression of neurological disease. Specifically, we look at the ways phytonutrients can counteract the changes in the aging brain that make it more susceptible to neurological disorders. We focus on the persistent activation of inflammatory pathways that reduce brain plasticity and, over time, contribute to destructive cellular changes which affect the nervous system’s functioning and ability to adapt to new experiences. We will analyze the anti-inflammatory properties of phytonutrient combinations and berries that contain numerous beneficial bioactives that target aging processes involving cellular communication and the propagation of disease. In vitro and in vivo bioassays utilizing human stem/progenitor cells and the brain’s innate immune cells, microglia, will be used to test combinations of phytonutrient components in normal aging and neuropathological models (i.e. Parkinson’s disease in the proposed studies here). Exosome microvesicles isolated from these assays are used as sensitive biomarkers for gene and protein expression patterns in interactive anti-inflammatory, neurogenic, and cell survival networks. Phytonutrient screening along with molecular and behavioral findings from cell culture, in vivo xenotransplantation, and human studies will establish phytonutrient effects that help counter neurodegeneration.
Progress Report
a) All cell lines established under Sub-objective 1a continued to be used as in vitro assays for treatments and evaluations. Specifically, adult human neuronal progenitor cells (AHNPs) derived from hippocampus, rodent primary neurons and microglial cell lines were tested during this reporting period. We performed several studies using these in vitro assays to investigate the effects of various treatments on cell survival and differentiation, as well as the potential cellular and molecular mechanisms underlying these effects. Two of them were completed and published. Our data indicated that blueberry (BB) and polymolecular botanical compound (PBC) treatments have beneficial effects on the survival and proliferation of the human hippocampal AHNPs and rat microglial cells, especially under stressed conditions. In another published study, we showed that cellular inflammatory stress increases the expression levels of certain oxidative stress and inflammation markers in rat microglia, and that blueberry treatments, applied both before and after the stress, are able to decrease their expression levels. Evaluation of the effects of other potentially beneficial bioactive compounds, such as myo-Inositol and DHA, on normal and patient derived AHNPs, and the underlying cellular mechanisms, are currently underway.
b) Under Sub-objective 1b, control hiPSCs proposed to be used as donor cells for the xenograft study were established and characterized. The human neural stem/progenitor cell xenograft model was also established, and baseline information related to the survival and differentiation of the donor cells was obtained for future comparison with xenografted animals undergoing diet treatments. However, performing follow up treatment studies on xenografted animals has not been initiated and, based on preliminary data, this aim was no longer scientifically relevant with respect to the PBC and blueberry intervention.
c) Under Sub-objective 1c, we continued to analyze the results from our experiment in collaboration with NC State to assess the association of blueberry metabolites with motor and cognitive alterations following continuous or intermittent wild blueberry supplementation in old rats. This study was designed to assess whether the optimal intake of blueberries is continuous or intermittent consumption. For this reporting period, we used targeted metabolomics to analyze the bioavailability of 181 polyphenolics and their metabolites in four different brain regions of aged rats fed either a continuous control diet, a continuous 2% blueberry diet, or intermittent 2% blueberry diet for 7-8 weeks. For 50% of the metabolites measured in the striatum, the levels were higher in the intermittent-fed group compared to the control group. These differences were not seen in the hippocampus, cortex, or cerebellum. We are in the process of writing this manuscript for publication.
d) To expand our knowledge on how dietary compounds affect cognition and underlying mechanisms, we analyzed bioavailability of vitamin K from an animal study during this reporting period and also investigated the effects of vitamin K intake on cognition and related hippocampal neurogenesis. Our preliminary data indicate that menaquinone-4 (MK4), the VK metabolite, was significantly lower in the brains of animals with low dietary vitamin K (LVK) intakes compared to those of animals with the control diet. The LVK intake significantly impaired survival and weight gain of C57BL6 mice, especially among males. It also negatively impacted learning- and memory-related cognitive function. Further mechanistical investigation performed during this reporting period indicated that LVK intake reduced hippocampal neurogenesis and induced a loss of hippocampal synapses, both which may contribute to the LVK-induced cognitive decline. These results were presented at the American Society of Nutrition meeting.
e) In support of Sub-objective 1d, we completed study participation in a multi-center, double-blind, placebo-controlled, crossover study in older (55–70-year-old), overweight/obese (BMI 27-35) adults to study the effects of acute raspberry intake on the relationship between enhanced metabolic control and cognitive and psychomotor function. This project is in collaboration with Illinois Institute of Technology (ITT) in Chicago. The aim of this project is to determine whether restoring meal-induced metabolic/inflammatory balance via supplementation with red raspberries results in improved cognitive performance in humans. Further, we are interested in determining if the expected enhancements are mediated through improvements in vascular function. For this reporting period, we used the serum from raspberry- or control-fed people to pre-treat microglial cells subjected to LPS-induced insults to determine possible mechanisms of action through which the raspberry compounds produce their beneficial effects. Results showed that microglia treated with serum from participants who consumed raspberry demonstrated reduced LPS-induced neuroinflammation on expression of nitric oxide, COX-2, and iNOS, compared to cells treated with placebo, at both 2 and 6 hours (p<0.05). Only iNOS was lower at 2 hours compared to 6 hours, suggesting that ongoing supplementation may provide the greatest health benefits. These changes in protection also align with the biphasic increase in plasma concentrations of phenolic acid metabolites, with some peaking at 1–2 hours and others at 6 hours. These results were presented at the Society for Neuroscience meeting.
f) Under Sub-objective 2a, adult human neuronal progenitor cells (AHNPs) derived from midbrain or substantia nigra (SN) of Parkinson’s disease (PD) patients were established and characterized. For this reporting period, we evaluated the effects of bioactive compounds such as myo-inositol and DNA on these PD-derived AHNPs. Our preliminary data indicate that cellular stressors, such as H2O2 and dopamine, decrease the survival and proliferation of the PD-AHNPs, and that myo-inositol were able to alter these decreases. Our data suggest that the PD-AHNPs may respond differently to nutrient treatments compared to the control AHNPs. Studies focusing on identifying the possible mechanisms that are causing this difference, such as alterations on inflammation and oxidative stress, apoptosis, and calcium homeostasis are underway. Data of this study is being prepared to present at the American Society of Nutrition meeting.
Accomplishments
1. Sugar alcohol compound may help improve neuronal connections. The effects of dietary nutrients on the central nervous system, and more specifically, neuronal connectivity, are still in the early stages of understanding. ARS-funded researchers in Boston, Massachusetts, found a sugar alcohol compound produced by the body and naturally found in many fruits, vegetables, and wheat, such as cantaloupe, green peas, and bran products, may improve communication of neurons in the brain. The researchers tested the presence of this compound, known as myo-inositol, in three different model types and demonstrated it increased neuronal connections at different life stages. This discovery may have important implications for improving neuronal connections during development and throughout maturation in the brain.
Review Publications
Fisher, D.R., Zheng, T., Bielinski, D.F., Kelly, M.E., Cahoon, D.S., Shukitt Hale, B. 2022. Phytochemical combination is more effective than individual components in reducing stress signaling in rat hippocampal neurons and microglia. International Journal of Molecular Sciences. 23(20):12651. https://doi.org/10.3390/ijms232012651.
Rabin, B.M., Miller, M.G., Shukitt Hale, B. 2023. Effects of preexposure to a subthreshold dose of helium particles on the changes in performance produced by exposure to helium particles. Life Sciences in Space Research. 37:88-96. https://doi.org/10.1016/j.lssr.2023.03.003.
Cahoon, D.S., Fisher, D.R., Lamon-Fava, S., Wu, D., Zheng, T., Shukitt Hale, B. 2022. Blueberry treatment administered before and/or after lipopolysaccharide stimulation and oxidative stress in rat microglial cells. Nutritional Neuroscience. 26:127-137. https://doi.org/10.1080/1028415X.2021.2020404.
Zheng, T., Bielinski, D.F., Fisher, D.R., Zhang, J., Shukitt Hale, B. 2022. Protective effects of a polyphenol-rich blueberry extract on adult human neural progenitor cells. Molecules. 27(19):6152. https://doi.org/10.3390/molecules27196152.
