Graduation Year

2020

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Paul R. Sanberg, Ph.D., D.Sc

Co-Major Professor

Paula C. Bickford, Ph.D.

Committee Member

Kevin Nash, Ph.D.

Committee Member

Dominic D'Agostino, Ph.D.

Committee Member

Brent Small, Ph.D.

Keywords

aging, bioinformatics, inflammation, proteome, serum

Abstract

Aging is a complex physiological process that leads to the deterioration of all cells and tissues throughout the body. Aging is a major risk factor for the onset of many degenerative diseases in both the central nervous system (CNS) and the periphery, but even nonpathological aging (“normal” aging) is associated with chronic inflammation, oxidative stress, and decreased stem cell proliferation and regenerative capacity. This decreased regenerative capacity in stem cell niches is thought to be a key component underlying the aging process and many disease states associated with aging.

While the exact biological mechanisms underlying impaired stem cell proliferation and regeneration with age remain unclear, two areas of investigation are being explored: cell-autonomous changes and cell non-autonomous changes. Cell autonomous changes involve the senescence of genes such as p53, p16INK4A, critical proteins involved in canonical pathways such as the Wnt/β-catenin pathway, and others. Cell non-autonomous effects on stem cell niches refers to various circulating factors, such as pro-inflammatory cytokines, chemokines, and other proteins that may influence the microenvironment.

Highly relevant early examples of circulating factors impacting stem cell proliferation come from heterochronic parabiosis studies. Heterochronic parabiosis entails the surgical union of the circulatory systems of a young mouse and an old mouse. It was observed that exposure to blood from old mice had a deleterious impact on stem cell proliferation in young mice, however blood from young mice had a positive effect on old mice. Similarly, when young mice were injected with plasma from old mice, their performance on cognitive behavioral paradigms such as contextual fear conditioning and the radial arm water maze was impaired, thus underscoring the fact that circulating factors in the aging systemic milieu can impact the CNS.

While parabiosis studies have proven useful for the study of circulating factors, they are not highly translational. We and others have found that nutraceutical intervention is able to reverse some of the age-related decline in the CNS observed in rats and human subjects. In particular, we have demonstrated that a particular nutraceutical called NT-020, a proprietary blend of blueberries, green tea, vitamin D3, and carnosine, is able to rescue age-related cognitive decline, reverse impaired neurogenesis, and exert anti-inflammatory effects. In vitro, it has been previously demonstrated that rat hippocampal neural progenitor cells (NPCs) and bone marrow-derived mesenchymal stem cells (MSCs) cultured with serum from aged rats showed decreased proliferation as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-dephyltetrazolium bromide (MTT) and 5-bromo-2’-deoxyuridine (BrdU) assays. However, when NPCs and MSCs were cultured with serum from aged rats given a diet supplemented with NT-020, the proliferation rates were not different from those of cells cultured with serum from young rats.

In studies with rats maintained on a standard diet or an NT-020-supplemented diet for one month, aged rats showed delayed ability to locate the platform in the Morris Water Maze paradigm, whereas the performance of aged rats maintained on a diet with NT-020 did not differ significantly from that of young rats (Acosta et al. 2010). This suggests that NT-020 may have rescued learning and memory in the aged rats. Furthermore, when the brains were collected and subjected to immunostaining, it was observed that aged rats on an NT-020-supplemented diet had a greater number of dividing cells in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, as determined by cell cycle marker Ki67 compared to the aged control. Additionally, NT-020-treated aged rats showed decreased activated microglia as indicated by fewer cells that expressed major histocompatibility complex II (MHCII) (by OX6 staining) in the DG than aged controls. Finally, NT-020-treated aged rats also showed increased proliferation of neural progenitors in the SVZ, as indicated by increased doublecortin+ (DCX) staining compared to aged controls. In a double-blind, placebo-controlled trial with older adults 65-85 years of age, those taking the NT-020 supplement improved on two measures of processing speed within the two-month test period, but did not show significant improvement on other cognitive performance tests. These results collectively suggest that NT-020 exerts therapeutic effects by reversing inflammation and increasing stem cell proliferation in the neurogenic niches. While some of the therapeutic mechanisms of action for NT-020 have been defined, a complete profile of circulating factors being affected by NT-020 supplementation has yet to be elucidated.

The goal of the current study was to generate a complete list of factors from the systemic milieu that could be altered as a consequence of aging and rescued by NT-020 supplementation. We used bottom-up, discovery-based proteomics to create a profile for the entire proteome for serum derived from aged rats given NT-020 or a normal diet and young rats given NT-020 or a normal diet. Our data suggest that there are age-related molecular changes that can be rescued by NT-020 supplementation.

We designed a discovery-, mass spectrometry-based proteomics study to generate a profile of the entire rat proteome for serum derived from young (3-6 months) control, old (20-22 months) control, and old male Fisher 344 rats given a diet supplemented with NT-020. We identified some proteins that were differentially expressed between the old control and old diet groups, specifically proteins that are involved in aging and inflammatory pathways such as autophagy and complement pathways. While the findings suggest that NT-020 is exerting anti-aging effects, depletion of albumin, the most highly abundant protein in serum, presented a methodological challenge in the study. It is likely that, in spite of depletion steps undertaken to remove high abundance protein from the samples, much of the albumin and other high abundance proteins remained and may have prevented other proteins from detected.

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