Astrocytes in aged nonhuman primate brain gray matter synthesize excess hyaluronan

Abstract

The glycosaminoglycan hyaluronan (HA) accumulates in central nervous system lesions where it limits astrogliosis but also inhibits oligodendrocyte progenitor cell (OPC) maturation. The role of hyaluronan in normative brain aging has not been previously investigated. Here, we tested the hypothesis that HA accumulates in the aging nonhuman primate brain. We found that HA levels significantly increase with age in the gray matter of rhesus macaques. HA accumulation was linked to age-related increases in the transcription of HA synthase-1 (HAS1) expressed by reactive astrocytes but not changes in the expression of other HAS genes or hyaluronidases. HA accumulation was accompanied by increased expression of CD44, a transmembrane HA receptor. Areas of gray matter with elevated HA in older animals demonstrated increased numbers of olig2(+) OPCs, consistent with the notion that HA may influence OPC expansion or maturation. Collectively, these data indicate that HAS1 and CD44 are transcriptionally upregulated in astrocytes during normative aging and are linked to HA accumulation in gray matter.

Fig. 1

Astrogliosis as a function of age in the nonhuman primate prefrontal cortex (area 46) gray matter (A, C, E) and white matter (B, D, F). GFAP immunoreactivity (DAB-nickel), in young Japanese macaques was high in the glial limitans (A, arrow) and white matter (B). Increased GFAP immunoreactivity was observed in the mid-cortical layers by middle-age (C, arrow), and progressed with age (old, E). Levels remained elevated in the white matter of middle-aged and old animals (D and F, respectively). The area of GFAP coverage (pixels) in the prefrontal cortex increased significantly (ANOVA, p<0.05) and progressively with age (old>middle-aged>young, p<0.01 for all comparisons).

Fig. 2

CD44 is transcriptionally upregulated in gray matter with aging. (A) Real-time RT-PCR analysis of CD44 transcript levels in gray matter RNA. (B) Real-time RT-PCR analysis of CD44 transcripts in RNA from white matter. (C) Left panel: Western blots of prefrontal cortex lysates showing levels of CD44s (the standard form of CD44) from animals at different ages. Actin is used as a loading control. Note that lanes ran somewhat unevenly due to a “smile effect”. Right panel: Examples of the full blots from a young and an old animal, overexposed to reveal low, unchanging levels of apparent CD44 splice variants (CD44v) and a band corresponding to the size of underglycosylated CD44 (CD44u). (D–I) Immunohistochemical analysis of CD44 expression in a 30-year-old animal (D, G) in prefrontal cortex gray matter, showing co-localization with areas of elevated GFAP (E, H) immunoreactivity (merged images, F, I). D–F are 10x images; G–I are 20x images. Insets in G–I are 40x images of glial processes. Arrowheads show areas of CD44 and GFAP co-localization. Arrows show areas of diffuse CD44 expression not associated with cells.

Fig. 3

Colocalization of GFAP and CD44 in the vasculature of the macaque prefrontal cortex. In areas containing a low density of immunoreactivity, blood vessels containing both GFAP (A, D, G) and CD44 (B, E, H) could be observed, with some overlap (C, F, I). These isolated structures were seen at a very low frequency. Arrows indicate blood vessels near the middle of gliotic areas. Scale bars: A–F, 10 μm; G–I, 2 μm.

Fig. 4

HA accumulates with aging in prefrontal cortex gray matter. (A) ELISA assay for total HA concentrations in prefrontal cortex gray matter. (B) ELISA assay for HA concentrations in white matter. (C–E) Immunohistochemical staining (10x) for CD44 (green) of area 46 in young (C), middle-aged (D), and old (E) animals. Insets show high power (40x) images taken from the cortex. (F–H) Immunohistochemical staining of area 46 for GFAP (green) and HA (red) in young (F), middle-aged (G) and old (H). Overlapping labeling is shown in yellow. The insets are high power (40x) images of cortex. Exposure times for all images were equivalent.

Fig. 5

HAS1 is transcriptionally upregulated with age in macaque prefrontal cortex gray matter. (A) Real time RT-PCR analysis of HAS1, HAS2, and HAS3 expression in RNA from prefrontal cortex gray matter. (B) Real time RT-PCR analysis of HYAL1, HYAL2, and HYAL3 expression in RNA from prefrontal cortex gray matter. (C–H) Immunohistochemical analysis of HAS1 expression in sections of prefrontal cortex gray matter from an old (age 30) animal. HAS1 (C; red) is predominantly expressed by GFAP-immunolabeled astrocytes (D; green) as shown by double labeling (E). (F) Cells in the oligodendrocyte lineage (Olig2⁺, red) did not co-localize with HAS1 (green). (G) Neurons (red) also did not express detectable levels of HAS1 (green). (H) Microglia (green) also did not appear to express HAS1 (red). Scale bar = 100 μm.

Fig. 6

Olig2+ cells accumulate in areas with elevated HA. (A–C) Immunohistochemical labeling for HA (A; red), olig2 (B; green) and both HA and olig2 with DAPI (blue) to label cell nuclei in prefrontal cortex of a 30-year-old animal. The percentages of olig2+ nuclei in HA-high (encircled) and HA-low (areas outside of encircled areas) were calculated and compared (D). *p<0.01.