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. 2013 Oct;34(10):2310-21.
doi: 10.1016/j.neurobiolaging.2013.03.022. Epub 2013 Apr 19.

Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation

Wenxin Ma  1 , Radu Cojocaru, Norimoto Gotoh, Linn Gieser, Rafael Villasmil, Tiziana Cogliati, Anand Swaroop, Wai T Wong
Affiliations

Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation

Wenxin Ma et al. Neurobiol Aging. 2013 Oct.

Abstract

Microglia, the resident immune cells of the central nervous system (CNS), are thought to contribute to the pathogenesis of age-related neurodegenerative disorders. It has been hypothesized that microglia undergo age-related changes in gene expression patterns that give rise to pathogenic phenotypes. We compared the gene expression profiles in microglia isolated ex vivo from the retinas of mice ranging from early adulthood to late senescence. We discovered that microglial gene expression demonstrated progressive change with increasing age, and involved genes that regulate microglial supportive functions and immune activation. Molecular pathways involving immune function and regulation, angiogenesis, and neurotrophin signaling demonstrated age-related change. In particular, expression levels of complement genes, C3 and CFB, previously associated with age-related macular degeneration (AMD), increased with aging, suggesting that senescent microglia may contribute to complement dysregulation during disease pathogenesis. Taken together, senescent microglia demonstrate age-related gene expression changes capable of altering their constitutive support functions and regulation of their activation status in ways relating to neuroinflammation and neurodegeneration in the CNS.

Keywords: Activation; Aging; Angiogenesis; Complement; Gene expression; Microarray; Microglia; Neurotrophic factors; Retina; Senescence.

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Conflict of interest statement

Disclosure statement

All authors indicate that they do not have conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Hierarchical clustering heat map for 719 genes demonstrating age-related differential expression (fold-change ≥1.5x and uncorrected p-values <0.05)
Each column represents a biological replicate of a particular age (4 replicates per age group); each column row represents a single gene. Gene expression changes with respect to median changes are represented in direction and magnitude by the legend (bottom): red, up-regulated; blue, down-regulated; gray, unchanged.
Figure 2
Figure 2. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation of selected genes
Relative gene expression fold change from qRT-PCR from retinal microglia from 24-month old mice (3 -6 biological repeats were used for each experiment, with one sample replicate per biological replicate), normalized relative to those from 3-month old mice (A) is compared to the relative fold changes for the same genes predicted from microarray analysis (B). Error bars indicate ± SEM.
Figure 3
Figure 3. Age-associated gene expression changes in microglial signaling pathways involved with microglial immune function and activation as generated by Ingenuity Pathway analysis
Signaling pathways involved in the regulation of microglial immune activities were highlighted in the analyses of genes whose expression varied significantly as a function of age. Aging-related genes are indicated either in green (down-regulated) or in red (up-regulated) (for 3-month to 24-month comparisons). The most prominently represented pathways included (A) IL17 signaling, (B) ceramide signaling, (C) IL3 signaling, (D) IL1 signaling, (E) estrogen-dependent signaling, (F) nitric oxide signaling, and (G) lipopolysaccharide (LPS)-stimulated MAPK signaling.
Figure 4
Figure 4. Age-associated gene expression changes in microglial signaling pathways involved with angiogensis and neurotrophic support as generated by Ingenuity Pathway analysis
(A–B) Two particular signaling pathways involved in angiogenesis were those involving (A) thrombospondin-1 (TSP1), and (B) vascular endothelial growth factor (VEGF). (C) Neurotrophic-TRK receptor signaling pathways were also highlighted by microglial genes demonstrating age-related change. Aging-related genes are indicated either in green (down-regulated) or in red (up-regulated) (for 3-month to 24-month comparisons).
Figure 5
Figure 5. Age-related changes in the complement gene expression and complement activation in retinal microglia
Immunohistochemical staining of complement proteins, C3 (A) and CFB (B) in subretinal microglia, and complement activation product iC3b deposition in the subretinal space (C) of young (3–4 month old) and aged (20–24 month old) CX3CR1+/gfp transgenic mice. (A) Immunostaining for C3 (red) in subretinal microglia (green) demonstrated the absence of detectable immunopositivity in young mice. In aged mice, a substantial proportion of subretinal microglia showed immunopositivity, particularly in microglial processes (arrow). (B) Immunostaining for CFB (red) in subretinal microglia (green) demonstrated very weak immunopositivity in young mice but prominent staining of microglial somata in aged mice. (C) Immunopositivity for iC3b (red) was absent from the subretinal space of young mice but found distributed in a punctate manner in that of aged mice (green). (D) Quantification of the extent of immunopositivity of C3, CFB, and iC3b, demonstrating increased levels in the subretinal space in 20–24 month-old mice, relative to 3 month-old mice. (n = 6 representative 60x fields from 4–6 animals in each age group), (E) Morphological analysis of microglial soma sizes, demonstrating enlarged somata in terms of area and perimeter in 20–24 month-old mice, relative to 3 month-old mice. (F) Morphological analysis of microglial dendritic territory sizes, demonstrating decreased ramification in terms of decreased areas, perimeter, and total dendritic lengths of individual microglia in 20–24 month-old mice, relative to 3 month-old mice (n = 9 representative cells from 4–6 animals in each age group). * indicates comparisons for which p <0.05 (unpaired t-test with Welch’s correction).
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