Location: Jean Mayer Human Nutrition Research Center On Aging
2022 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
Progress was made on Objective 1 and its subobjectives, as well as subobjective 2a, which fall under NP107, Human Nutrition.
Under Sub-Objective 1a, all cell lines needed for in vitro and in vivo studies proposed in the plan are established. Specifically, adult human neuronal progenitor cells (AHNPs) derived from various areas of human brains were used as in vitro assays for treatments and evaluations. Rodent primary neurons and microglial cell lines were also established for similar studies. Human Induced Pluripotent Stem Cells (hiPSCs) are established and maintained in the lab for proposed in vivo xenograft studies (Sub-Objective 1b). For this reporting period, we studied effects of various treatments on cell survival and differentiation, as well as the potential cellular and molecular mechanisms underlying these effects. 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 study, our data show 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 other related markers and pathways on normal and patient derived AHNPs are currently underway. We submitted three manuscripts on this data this fiscal year; two of them are in revision and one is published online.
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 as based on preliminary data, this aim was no longer scientifically relevant with respect to the PBC and blueberry intervention.
Under Sub-Objective 1c, we continued to analyze the results from our experiment to assess the association of neuro-available 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 analyzed polyphenolics and their metabolites in the brains of aged rats fed either a continuous control diet, a continuous 2% blueberry diet, or intermittent 2% blueberry diet for 7-8 weeks prior to testing on a battery of age-sensitive tests and subsequent sample collection. In collaboration with NC State, we performed targeted metabolomics using UPLC to determine which compounds in blueberries crossed the blood brain barrier. 181 metabolites were measured, including phenolic acids, anthocyanidins, and other phenolic compounds in four different brain regions: hippocampus, striatum, cerebellum, and cortex. For 50% of the metabolites measured in the striatum, the levels were higher in the intermittent-fed group compared to the control group. Specifically, as one example, there were more anthocyanin derivatives in the striatum of intermittent-fed BB animals compared to both continuous-fed BB animals or control-fed rats. These differences were not seen in the hippocampus, or the other brain regions.
To expand our knowledge on how dietary compound affects neurogenesis and cognition, we initiated an animal study during this reporting period to investigate the effects of vitamin K intake on hippocampal neurogenesis, cognition, as well as the potential mechanisms underlying those effects, based on observational studies. Our data indicated that low vitamin K intake, compared to control diet, significantly impaired survival, and weight gain of C57BL6 mice, especially among males. It also negatively impacted learning- and memory-related cognitive function. Ongoing analyses suggested that low vitamin K intake may also reduce hippocampal neurogenesis and increase cellular senescence.
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. Preliminary data of ongoing analyses suggested that microglia treated with serum from participants who consumed raspberry demonstrated reduced levels of neuroinflammation markers compared to cells treated with placebo in a time-dependent manner.
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. Human Induced Pluripotent Stem Cells (hiPSCs) obtained from PD patients with familial mutations, including patients with the G2019S LRRK2 mutation are currently maintained in the lab for proposed in vivo xenograft studies (sub-objective 2b). For this reporting period, we studied the effects of blueberry and PBC on survival and proliferation of the PD-derived AHNPs. Our preliminary data indicated that even though the cellular stressor also decrease the survival and proliferation of the PD-AHNPs, and BB and PBC appear to be able to increase the survival rate, no significance increase was found compared to the cells without treatments. However, BB was able to alter the decreases in cell proliferation and calcium buffering induced by dopamine stress. Our data suggest that the PD-AHNPs may respond differently to the BB and PBC compared to the control AHNPs. We are in the process of identifying the possible mechanisms that are causing this difference.
Accomplishments
Review Publications
Shuryak, I., Brenner, D.J., Blattnig, S.R., Shukitt Hale, B., Rabin, B.M. 2021. Modeling space radiation-induced cognitive dysfunction using targeted and non-targeted effects. Scientific Reports. 11:8845. https://doi.org/10.1038/s41598-021-88486-z.
Miller, M.G., Thangthaeng, N., Scott, T.M., Rutledge, G.A., Shukitt Hale, B. 2021. Dietary strawberry improves cognition in a randomized, double-blind, placebo-controlled trial using older adults. British Journal of Nutrition. 126(20):253-263. https://doi.org/10.1017/S0007114521000222.
Hininger-Favier, I., Thangthaeng, N., Bielinski, D.F., Fisher, D.R., Shukitt Hale, B., Poulose, S.M. 2021. Blueberries and insulin protect microglial cells against high glucose-induced inflammation and restore GLUT-1. Journal of Berry Research. 11(2):201-216. https://doi.org/10.3233/JBR-200628.
Bell, L., Shukitt Hale, B., Williams, C.M. 2022. Berry bioactives and cognition. In: Klimis-Zacas, Dorothy and Rodriguez-Mateos, Ana., editors. Berries and Berry Bioactive Compounds in Promoting Health. Cambridge, U.K. Royal Society of Chemistry. p. 185-207.
