Lifelong calorie restriction alleviates age-related oxidative damage in peripheral nerves - PubMed (original) (raw)

Katherine Opalach et al. Rejuvenation Res. 2010 Feb.

Abstract

Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum-fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.

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Figures

FIG. 1.

FIG. 1.

A calorie-restricted (CR) diet minimizes accrual of damaged proteins within peripheral nerves during aging. (A) Western blot analysis of whole sciatic nerve lysates (20 μg/lane) from rats of the indicated months (mo) fed ad libitum (AL) or CR diets probed with an anti-ubiquitin antibody. Slow-migrating polyubiquitinated (pUbi) protein substrates are marked by a square bracket. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a protein loading control. Molecular mass in kDa is indicated on the left. (B) Quantification of pUbi band intensity normalized to GAPDH is shown (#p < 0.05, ###p < 0.001, Fisher protected least significant difference [PLSD] analysis; *p < 0.05, unpaired _t-_test, mean ± standard error of the mean [SEM], n = 3). In this and subsequent figures, the p value for Fisher protected least significant difference (PLSD) analysis is determined by comparing the 8-month-old sample with the older ages (18, 29, or 38 months) for each diet group. (C) Longitudinal sciatic nerve sections from 38-month-old animals fed ad libitum or CR diets were analyzed for ubiquitin- (red) and lysosomal associated membrane protein 1– (LAMP1) like (green) immunoreactivity. Arrows point to ubiquitin-positive protein aggregates adjacent to LAMP1-positive lysosomes (magnified 3 × in insets). Asterisks indicate intracytoplasmic vacuoles. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm.

FIG. 2.

FIG. 2.

Accumulation of lipofuscin is curtailed by a calorie-restricted (CR) diet with aging. (A) Sciatic nerve sections from 18- and 38-month-old animals fed ad libitum (AL) or CR diets were stained with 4-[2-(6-dibutylamino)-2-naphthalenyl)ethenyl]-1-(3-sulfopropyl) hydroxide (di-8 ANEPPS) dye, which labels lipofuscin content (red). Large (∼10 μm) and smaller (<10 μm) di-8 ANEPPS-positive adducts are marked with arrows and arrowheads, respectively. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm. (B) Quantification of mean di-8 ANEPPS dye-positive pixels per fixed area is shown (*p < 0.05, ***p < 0.001, unpaired _t_-test, mean ± standard error of the mean [SEM], n = 3). (Color images available online at

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FIG. 3.

FIG. 3.

Lipid peroxidation-associated modifications of proteins with age are relieved in sciatic nerve with a calorie-restricted (CR) diet. (A) Whole rat sciatic nerve lysates (20 μg/lane) were probed with a rabbit anti-malondialdehyde (MDA) antibody to detect MDA adducts to proteins (square bracket). Tubulin is shown as a protein loading control. (B) Quantification of MDA adduct band intensities normalized to tubulin is shown (###p < 0.001, Fisher protected least significant difference (PLSD) analysis; *p < 0.05, unpaired _t_-test, mean ± SEM, n = 3). (C) Biochemical analysis of tyrosine residues (Nitros) on proteins (square bracket) using an anti-nitrosylation antibody in the same lysates as in A. Tubulin is shown as a protein loading control. (D) Quantification of nitrotyrosine band intensities from Western blots normalized to tubulin is shown (##p < 0.01, Fisher's PLSD analysis; *p < 0.05, unpaired _t_-test, mean ± standard error of the mean [SEM], n = 3). (E) Western blot analysis of 4-hydroxynonenal (HNE) in the same lysates used in A and C. Tubulin is shown as a protein loading control. In A, C, and E, molecular mass is shown on the left in kDa. (F) Quantification of HNE adduct band intensities from western blots normalized to tubulin is shown (###p < 0.001, Fisher PLSD analysis; p = 0.0697, unpaired _t_-test, mean ± SEM, n = 3). (G) Immunohistochemical staining of sciatic nerve from 18- and 38-month-old animals fed ad libitum (AL) and CR diets with an antibody against HNE. Nuclei are labeled with Hoechst dye (blue). Scale bar, 20 μm. (Color images available online at

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)

FIG. 4.

FIG. 4.

Age-related increase in proinflammatory mediators is attenuated by a lifelong calorie-restricted (CR) diet. (A) Total sciatic nerve lysates (20 μg/lane) from the indicated ages and diet were analyzed with antibodies against tumor necrosis factor-α (TNF-α, p50 and p65 subunits of NF-κB, and phospho-IκB. Tubulin is shown as a loading control. Molecular mass is shown on the left in kDa. (B) Quantification of TNF-α band intensities after normalization to tubulin is represented (##p < 0.01, Fisher protected least significant difference (PLSD) analysis; *p < 0.05, unpaired _t_-test, mean ± standard error of the mean [SEM], n = 3). (C) Quantification of the p50 subunit of NF-κB band intensities normalized to tubulin is shown (##p < 0.01, ###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired _t_-test, mean ± SEM, n = 3). (D) Quantification of the p65 subunit of NF-κB band intensities normalized to tubulin is shown (p = 0.0546, ##p < 0.01, ###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired _t_-test, mean ± SEM, n = 3). (E) Phospho-IκB levels were also analyzed within the same lysates and quantification of band intensities normalized to tubulin was performed (p = 0.0830, ##p < 0.01, ###p < 0.001, Fisher PLSD analysis; **p < 0.01, unpaired _t_-test, mean ± SEM, n = 3). AL, Ad libitum.

FIG. 5.

FIG. 5.

A calorie-restricted (CR) diet diminishes macrophage infiltration of peripheral nerves with age. (A) Cryosections of sciatic nerves from 18- and 38-month-old rats either maintained on ad libitum (AL) or CR diet were immunostained with an antibody against CD11b (green). Nuclei are stained with Hoechst dye (blue). Scale bar, 20 μm. (B) Quantification of CD11b+ cells per 0.1 mm2 of nerve tissue area is shown for 18- and 38-month-old animals fed ad libitum or CR diets (***p < 0.001, unpaired _t_-test, mean ± standard error of the mean (SEM), n = 3). (C) Western blot analysis of sciatic nerve lysates (20 μg/lane) for steady-state levels of CD11b and endogenous immunoglobulin G (IgG) (heavy chain [HC] and light chain [LC]) from ad libitum and CR rats is shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a protein loading control. Molecular mass in kDa is indicated on the left. (D) Quantification of the CD11b band intensities normalized to GAPDH from western blot analyses from whole sciatic nerve lysates is shown (#p < 0.05, ###p < 0.001, Fisher PLSD analysis; **p < 0.01, unpaired _t_-test, mean ± SEM, n = 3). (E) Quantification of the IgG-HC band intensities normalized to GAPDH from whole sciatic nerve lysates of ad libitum and CR rats of the indicated ages is shown (##p < 0.01, Fisher PLSD analysis; *p < 0.05, unpaired _t-_test, mean ± SEM, n = 3). (F) Quantification of band intensities of IgG-LC normalized to GAPDH was performed (##p < 0.01, ###p < 0.001, Fisher PLSD analysis; *p < 0.05, unpaired _t_-test, mean ± SEM, n = 3). (Color images available online at

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)

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