Age-related and cuprizone-induced changes in myelin and transcription factor gene expression and in oligodendrocyte cell densities in the rostral corpus callosum of mice - PubMed (original) (raw)

J Ronald Doucette et al. Cell Mol Neurobiol. 2010 May.

Abstract

During aging, there is a decrease both in the stability of central nervous system (CNS) myelin once formed and in the efficiency of its repair by oligodendrocytes (OLs). To study CNS remyelination during aging, we used the cuprizone (a copper chelator) mouse model. Inclusion of cuprizone in the diet kills mature OLs and demyelinates axons in the rostral corpus callosum (CC) of mice, which enabled us to characterize age-related changes (i.e., 2-16 months of age) in glial cell response during the recruitment (i.e., demyelination) and differentiation (i.e., remyelination) phases of myelin repair. We have found that the time between 12 and 16 months of age is a critical period during which there is an age-related decrease in the number of OL lineage cells (Olig2(Nuc)+ve/GFAP-ve cells) in the rostral CC of both control mice and mice recovering from cuprizone-induced demyelination. Our results also show there was an age-related impaired recruitment of progenitor cells to replace lost OLs in spite of there being no major age-related decrease in the size of the progenitor cell pool (PDGFalphaR+ve/GFAP-ve, and Olig2(Nuc) +ve/PDGFalphaR+ve cells). However, there were cuprizone-induced increased numbers of astrocyte progenitor cells (Olig2(Cyto)+ve/PDGFalphaR+ve) in these same mice; thus PDGFalphaR+ve progenitor cells in mice as old as 16 months of age retain the ability to differentiate into astrocytes, with this fate choice occurring following cytoplasmic translocation of Olig2. These data reveal for the first time age-related differences in the differentiation of PDGFalphaR+ve progenitor cells into OLs and astrocytes and lead us to suggest that during aging there must be a transcriptional switch mechanism in the progenitor cell fate choice in favor of astrocytes. This may at least partially explain the age-related decrease in efficiency of OL myelination and remyelination.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

A diagram illustrating experimental design of the study. There were three treatment groups: a control mice, which were fed normal milled chow for 6 weeks; b cuprizone mice, which were fed a 0.2% cuprizone-containing diet for 6 weeks; and c cuprizone recovery mice, which were fed 0.2% cuprizone diet for the first 6 weeks followed by normal milled chow for the next 3 weeks. Six mice were used for each of the three treatments at each of the four ages. Half of the mice for each treatment and age were used for immunofluorescent microscopy and the remaining half for collecting tissue for quantitative RT–PCR (qRT–PCR)

Fig. 2

Fig. 2

A diagram summarizing how the brain was dissected for cutting sagittal and coronal cryostat sections. aThe brain is removed from the skull and separated down the midline,b thus dividing the brain into a right and a left half. c A midsagittal section of the right cerebral hemisphere, including the genu (arrow) and body of the CC. Myelin protein immunostaining was done on cryostat sagittal tissue sections to see age-related differences in cuprizone-induced demyelination in the CC and the extent of myelin repair in the cuprizone recovery mice. The long, thin rectangle passes through the rostral part of the body of the CC and indicates the plane of section for the drawing in d, which shows the coronal plane through the brain. This coronal plane was used for all of the immunostaining for quantifying cell densities, with the quantification done on digital images captured from the medial part of the CC, which is indicated by the boxed area in d. The CC tissue used for qRT-PCR was obtained from a comparable area within the medial aspect of this myelinated fiber tract

Fig. 3

Fig. 3

Histograms depicting age-related and treatment-related changes in body weight. Three mice were used for each treatment and for each age.a The average day zero body weight (in grams) in 2–16 month-old controls, cuprizone and cuprizone recovery mice.b and c Week 3 and Week 6, respectively, average body weights of 2–16-month-old mice expressed as a percentage of day zero weight.d Week 6 and Week 9 average body weights of 2–16 month-old cuprizone recovery mice expressed as a percentage of day zero body weight. The brackets in color denote significant age-related differences within a group (red = control, green = cuprizone, blue = cuprizone recovery), whereas black brackets denote significant treatment-related differences and brown brackets denote significant difference between cuprizone recovery groups (Bonferroni’s post-test; significance level of P ≤ 0.05). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

Fig. 4

Fig. 4

Immunohistochemical analysis of myelin basic protein (MBP) in coronal sections through the rostral CC of 2, 6, 12, and 16 month-old mice, untreated control (a, d, g, andj), 6 week cuprizone treated (b, e, h, and k), and 6 week cuprizone treatment followed by 3 week recovery (c, f, i, and l). Cuprizone-fed mice show decreased expression of MBP in rostral CC (b, e,h, kcompared to a, d, g, j) that recovers in 2 and 6 month-old mice (c, f compared toa, d) after a period of 3 weeks but only partially recovers in the 12 and 16 month-old mice (i, l compared to g,j and c, f). Thus, when the cuprizone model is used on mice of 2, 6, 12, and 16 months of age, the impaired efficiency of OL remyelination is first evident at 12 months of age. n = 3, bar = 50 μm

Fig. 5

Fig. 5

Immunohistochemical analysis of myelin oligodendrocyte glycoprotein (MOG) in coronal (a–l) and sagittal (m–x) sections through the CC of 2, 6, 12, and 16 month-old mice: control (a, d,g, j,m, p,s, and v), cuprizone (b, e, h, k, n, q, t, andw), cuprizone recovery (c, f, i, l, o, r, u, and x) mice. The boxes in m to r indicate the areas in the rostral CC shown in higher power in s to x, respectively. n = 3, bar in a = 100 μm (applies to a to l), bar inm = 100 μm (applies to m to r), bar ins = 100 μm (applies to s to x)

Fig. 6

Fig. 6

Histograms depicting the gene expression profiles (quantitative [q] RT-PCR) for MOG a, and CGT b in the rostral CC of 2–16 month-old control, cuprizone and cuprizone recovery mice. Three mice were used for each treatment and for each age. The qRT-PCR data are expressed as normalized RQ values, with all RQ values for a gene being normalized to the mean RQ value of the respective 2-month-old control mice. The brackets in color denote significant age-related differences within a group (red = control, blue = cuprizone recovery), whereas black brackets denote significant treatment-related differences (Bonferroni’s post-test; significance level of P ≤ 0.05). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

Fig. 7

Fig. 7

a Histogram depicting the mean number (±SEM) of Hoechst+ve nuclei in the rostral CC of 2-16 month-old control, cuprizone and cuprizone recovery mice. Three mice were used for each treatment and for each age. The brackets in color denote significant age-related differences within a group (green = cuprizone, blue = cuprizone recovery), whereas black brackets denote significant treatment-related differences (Bonferroni’s post-test; significance level of P < 0.05). b–e Hoechst+ve nuclei in coronal sections of CC from a 2 month-old (2C) control mouse b, a 16 month-old (16C) control mouse c, a 2 month-old (2TrR) cuprizone recovery moused and a 16 month-old (16TrR) cuprizone recovery mouse e. Bar in b = 50 μm (applies to b–e). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

Fig. 8

Fig. 8

Histograms depicting the gene expression profiles (quantitative [q] RT-PCR) for PDGFαR a, Olig2 b, and Nkx2.2 c in the rostral CC of 2–16 month-old control, cuprizone and cuprizone recovery mice. Three mice were used for each treatment and for each age. The qRT-PCR data are expressed as normalized RQ values, with all RQ values for a gene being normalized to the mean RQ value of the respective 2-month-old control mice. The brackets in color denote significant age-related differences within a group (red = control, green = cuprizone, blue = cuprizone recovery), whereas black brackets denote significant treatment-related differences (Bonferroni’s post-test; significance level of P ≤ 0.05). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

Fig. 9

Fig. 9

Immunohistochemical analysis of Olig2Nuc+ve/GFAP−ve cells (a, d), Olig2Nuc+ve/PDGFαR+ve cells (b, e), PDGFαR+ve/GFAP−ve cells (c, f) in coronal sections through the rostral CC. The images were obtained from CC of 6 month-old cuprizone (a, d), cuprizone recovery (b, e), and control (c,f) mice. The white boxed area ina–cdenote the portion of each image that is shown at higher magnification ind–f, respectively. d Two Olig2Nuc+ve/GFAP−ve cells in the top center of the image (arrow). e A Olig2Nuc+ve/PDGFαR+ve cell in the center of the image (arrow). f A PDGFαR+ve/GFAP−ve cell on the left side of the image (arrow). The arrowhead in a points out an Olig2Cyto+ve/GFAP−ve cell, in b points out a Olig2Cyto+ve/PDGFαR+ve cell, and in c points out a PDGFαR−ve/GFAP+ve astrocyte. bar (a, d) = 50 μm, n = 3

Fig. 10

Fig. 10

Histograms depicting the mean number (±SEM) of progenitor (PDGFαR+ve/GFAP−ve), OL progenitor (Olig2Nuc+ve/PDGFαR+ve), and OL lineage (Olig2Nuc+ve/GFAP−ve) cells in the rostral CC of 2 (a), 6 (b), 12 (c), and 16 (d) month-old control, cuprizone and cuprizone recovery mice. Three mice were used for each treatment and for each age. The brackets in color denote significant differences within a phenotype (brown = progenitor cells,pink = OL lineage cells), whereas black brackets denote significant differences between phenotypes (Bonferroni’s post-test; significance level of P ≤ 0.05). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

Fig. 11

Fig. 11

Histograms depicting the mean number (±SEM) of astrocyte progenitor cells (Olig2Cyto+ve/PDGFαR+ve), newly formed astrocytes (Olig2Cyto+ve/GFAP+ve), and mature astrocytes (Olig2Cyto−ve/GFAP+ve) in the rostral CC of 2 (a), 6 (b), 12 (c), and 16 (d) month-old control, cuprizone and cuprizone recovery mice. Three mice were used for each treatment and for each age. The brackets in color denote significant differences within a phenotype (brown = astrocyte progenitor cells, blue = Olig2Cyto+ve astrocytes,purple = astrocytes), whereas_black_ brackets denote significant differences between phenotypes (Bonferroni’s post-test; significance level of P ≤ 0.05). For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article

References

    1. Ando S, Tanaka Y, Toyoda Y, Kon K (2003) Turnover of myelin lipids in aging brain. Neurochem Res 28:5–13 -DOI -PubMed
    1. Armstrong RC, Le TQ, Frost EE, Borke RC, Vana AC (2002) Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white matter. J Neurosci 22:8574–8585 -PMC -PubMed
    1. Arnett HA, Fancy SP, Alberta JA, Zhao C, Plant SR, Kaing S, Raine CS, Rowitch DH, Franklin RJ, Stiles CD (2004) bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS. Science 306:2111–2115 -DOI -PubMed
    1. Bakker DA, Ludwin SK (1987) Blood-brain barrier permeability during Cuprizone-induced demyelination. Implications for the pathogenesis of immune-mediated demyelinating diseases. J Neurol Sci 78:125–137 -DOI -PubMed
    1. Bartzokis G (2004a) Age-related myelin breakdown: a developmental model of cognitive decline and Alzheimer’s disease. Neurobiol Aging 25:5–18 author reply 49–62 -DOI -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources