Elsevier

Free Radical Biology and Medicine

Volume 65, December 2013, Pages 324-334
Free Radical Biology and Medicine

Original Contribution
Dietary vitamin D deficiency in rats from middle to old age leads to elevated tyrosine nitration and proteomics changes in levels of key proteins in brain: Implications for low vitamin D-dependent age-related cognitive decline

https://doi.org/10.1016/j.freeradbiomed.2013.07.019Get rights and content

Highlights

  • VitD deficiency and cognitive decline are highly prevalent among the elderly.

  • Low VitD during brain aging leads to elevated 3-NT, glycolytic enzymes, and peroxidases.

  • Results correlate with VitD involvement in VDR–RXR and NF-κB signaling.

  • VitD supplementation may help protect against cognitive decline in adults.

Abstract

In addition to the well-known effects of vitamin D (VitD) in maintaining bone health, there is increasing appreciation that this vitamin may serve important roles in other organs and tissues, including the brain. Given that VitD deficiency is especially widespread among the elderly, it is important to understand how the range of serum VitD levels that mimic those found in humans (from low to high) affects the brain during aging from middle age to old age. To address this issue, 27 male F344 rats were split into three groups and fed isocaloric diets containing low (100 IU/kg food), control (1000 IU/kg food), or high (10,000 IU/kg food) VitD beginning at middle age (12 months) and continued for a period of 4–5months. We compared the effects of these dietary VitD manipulations on oxidative and nitrosative stress measures in posterior brain cortices. The low-VitD group showed global elevation of 3-nitrotyrosine compared to control and high-VitD-treated groups. Further investigation showed that this elevation may involve dysregulation of the nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) pathway and NF-κB-mediated transcription of inducible nitric oxide synthase (iNOS) as indicated by translocation of NF-κB to the nucleus and elevation of iNOS levels. Proteomics techniques were used to provide insight into potential mechanisms underlying these effects. Several brain proteins were found at significantly elevated levels in the low-VitD group compared to the control and high-VitD groups. Three of these proteins, 6-phosphofructokinase, triose phosphate isomerase, and pyruvate kinase, are involved directly in glycolysis. Two others, peroxiredoxin-3 and DJ-1/PARK7, have peroxidase activity and are found in mitochondria. Peptidyl–prolyl cis–trans isomerase A (cyclophilin A) has been shown to have multiple roles, including protein folding, regulation of protein kinases and phosphatases, immunoregulation, cell signaling, and redox status. Together, these results suggest that dietary VitD deficiency contributes to significant nitrosative stress in brain and may promote cognitive decline in middle-aged and elderly adults.

Graphical abstract

The figure depicts a potential mechanism for the protein nitration regulatory effects of VitD in brain and the consequences of VitD deficiency. Results obtained in this study supporting this mechanism are marked with wide green arrows, up arrows designating a found increase and down arrows designating a found decrease.

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Introduction

The steroid hormone vitamin D (VitD) can be produced by the body or obtained through the diet. VitD is synthesized in the skin from the cholesterol precursor 7-dehydrocholesterol and is converted to cholecalciferol (VitD3) upon exposure to sunlight [1]. VitD3 can also be obtained through several dietary sources and is transported in the blood via vitamin D-binding protein. In the liver, VitD3 is converted to calcidiol, 25-hydroxyvitamin D (25-OH VitD), followed by further conversion to calcitriol, 1α,25-dihydroxyvitamin D (1α,25-(OH)2 VitD), primarily in the kidneys, where it helps to regulate calcium homeostasis [2], [3]. VitD also plays roles in autoimmunity [4], mental health [3], [5], [6], [7], [8], and inhibition of tumor growth through reductions in proliferation and angiogenesis [9], [10], [11], [12].

VitD deficiency has long been associated with osteoporosis, brittle bones, and muscle weakness, but recently low levels of VitD have been linked to increased overall mortality [13], [14]. VitD status is typically assessed using serum concentration of 25-OH VitD because it is longer lived than the biologically active 1α,25-(OH)2 VitD [13], [15], [16].

VitD deficiency is highly prevalent in Europe and North America [1], [17], with the elderly particularly at risk [11], [13], [18], [19], [20]. Current estimates suggest that as many as 40–100% of the elderly populations in these areas are VitD deficient [21]. Poor diet and lower exposure to UV-B from the sun limits VitD synthesis in the skin and an age-related decrease in the VitD synthesis machinery may contribute to the observed lower VitD levels [15].

The elderly represent those at greatest risk of age-related cognitive decline and neurodegenerative disorders [22]. Recent retrospective studies on elderly human subjects provide correlative evidence that those with VitD deficiency have a much higher incidence of cognitive impairment than those with normal VitD levels [23], [24]. Thus, it seems that VitD deficiency may accelerate cognitive decline in aging [25]. A recent meta-analysis also shows that patients with Alzheimer disease (AD) typically have lower serum concentrations of VitD [26]. AD is associated with defects in amyloid-β (Aβ) processing and an upregulation of inflammatory cytokines and nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) [8]. Interestingly, 1α,25(OH)2 VitD helped to reverse soluble Aβ and inflammatory issues [8]. In addition to these actions, VitD is neuroprotective against Ca2+-mediated excitotoxicity, reduces biomarkers of brain aging associated with Ca2+ dyshomeostasis [3], [5], and helps to regulate levels of glutathione, a primary antioxidant in the brain, by modulating γ-glutamyltranspeptidase activity [27]. VitD also prevents onset of autoimmune demyelination in animal models of multiple sclerosis [28], [29].

Here, we manipulated serum VitD status by dietary supplementation with low, moderate/control, or high levels of VitD to identify changes in the VitD-dependent proteome in the brains of rats from middle to old age. Prior studies have shown that cognitively impaired subjects have significant levels of mitochondrial dysfunction and oxidative protein damage. In particular, nitration of protein-resident tyrosine residues is a common marker observed in brain of cognitively impaired subjects [30], [31], [32], [33], [34].Therefore, we tested the hypothesis that manipulating serum VitD levels would alter protein nitration and key protein markers of mitochondrial function. Our results identify several possible targets of VitD action that may mechanistically link circulating VitD levels with risk for age-related cognitive decline.

Section snippets

Chemicals

Criterion precast polyacrylamide gels, Tris–glycine–sodium dodecyl sulfate (SDS) (TGS) and Mes electrophoresis running buffers, ReadyStrip IPG strips, mineral oil, Precision Plus Protein All Blue standards, SYPRO Ruby protein stain, nitrocellulose membranes, dithiothreitol (DTT), iodoacetamide (IA), Biolytes, and urea were purchased from Bio-Rad (Hercules, CA, USA). Chemicals, proteases, protease inhibitors, and antibodies used in this study were purchased from Sigma–Aldrich (St. Louis, MO,

Vitamin D deficiency leads to increased nitrosative protein damage in brain

Tyrosine nitration is a common indicator/biomarker of the aging brain and of age-related neurodegenerative disorders [30], [34], both of which typically are accompanied by different extents of cognitive deficit. Here, we tested for indicators of oxidative and nitrosative stress in brain tissue samples from rats in which we manipulated serum VitD levels from middle age to old age. Significantly increased global 3-NT (Fig. 2) in the brains of rats on a low-VitD diet compared to rats on control or

Discussion

We have previously shown that tyrosine nitration occurs early in neurodegenerative processes, i.e., in mild cognitive impairment, arguably the earliest form of AD [31]. Nitration of tyrosine occurs from the reaction of NOradical dot with O2radical dot through the reactive intermediate ONOO in the presence of CO2 [32], [58], leading to tyrosine nitration by the NO2radical dot radical. Nitrosative stress measures on these cortical samples showed approximately a 25% elevation in 3-NT globally in brain protein in the low-VitD

Conclusions

This study is the first to demonstrate that a chronic low-VitD diet and consequential low levels of VitD in the bloodstream result in significant increases in tyrosine nitration in brain proteins, alterations in glucose metabolism, and mitochondrial changes in brain of elderly rats, an animal model of brain in older human subjects (Fig. 8). A shift from the TCA cycle to glycolysis may be indicative of metabolic dysfunction. Further, ATP generated from glycolysis is important for maintaining a

Acknowledgment

This work was supported by the following grants from the National Institute on Aging: AG05119 (D.A.B.), AG010836 (P.W. Landfield, D.A.B., N.M.P.), AG034605 (P.W. Landfield, N.M.P.), and T32 AG0000242 (C.S.L., G. Gerhardt).

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      Citation Excerpt :

      Then, in a rapid radical-radical recombination reaction, NO2 formed as from above binds to the free electron in the 3-position of Tyr to form 3-NT (Fig. 2), which is often detected in samples immunochemically by reaction with specific antibodies to protein-resident 3-NT [14]. Specificity of the antibody used is shown by the absence of binding to samples that had been pretreated with the powerful reducing reagent, Na2S2O4 that converts 3-NT to 3-amino-Tyr that is no longer recognized by antibodies against 3-NT [15]. When present on proteins, steric hindrance of 3-NT of the OH group on the aromatic ring of Tyr interferes with cell-signaling processes involving receptor tyrosine kinases, i.e.,3-NT is highly detrimental to intracellular signaling [16].

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