Cognitive function declines as the brain ages, especially its hippocampus. In age-related diseases, neurogenesis is inhibited in the hippocampal dentate gyrus and the microglia are activated(Morel et al.,, 2015). Furthermore, aging exerts negative effects on neuronal growth and function through dysregulating the expression of genes in the hippocampus (Yan et al.,, 2015). In the present study, we explored age-dependent gene expression in the hippocampus and its functional changes. After bioinformatics analysis, TREM2 and CD163 were identified to express in an age-dependent manner. RT-qPCR and WB results were consistent with those of bioinformatics analysis.
We found a wide distribution in gene expression levels calculated by binary logarithm of fold-change (log2(FC)). Based on microarray data, we screened out 120 DEGs between two groups, with 4 down-regulated and 116 up-regulated, as shown in the volcanic plot (Fig. 1A) and heatmap (Fig. 1B). GO enrichment analysis indicated that these DEGs were mainly enriched in plasma membrane and cell surface (CC), immune response, inflammatory response, phagocytosis (BP) (Table I), which were consistent with the results of KEGG analysis (Table II). The PPI network was constructed using STRING database and visualized by Cytoscape software (Fig. 2). Among the 18 hub genes screened by the intersection of four algorithms in Cytoscape MCODE (Table III. Figure 3), most of the up-regulated genes were related to immune and inflammatory responses, which was further validated by the high-level IL-1β and IL-6 in the aging brain (Figure.5ADE). TREM2 and CD163 up-regulation can promote phagocytosis and the release anti-inflammatory mediators from the microglia. Therefore, we hypothesized that the TREM2 and CD163 may counter microglia-mediated neuroinflammation, thus mitigating age-related cognitive decline. Our RT-qPCR and WB experiments also confirmed the up-regulation of TREM2, CD163 and high-level IL-1β and IL-6 in the hippocampus of aged mice (Fig. 4, Figure.5).
The aged brain demonstrate accumulation of endogenous factors (such as fibrillar Aβ, pro-inflammatory cytokines) and up-regulation of complement components, inflammasomes and toll-like receptors (David et al.,, 2012). Microglia protect against neurodegenerative disorders by phagocytosing debris, removing protein aggregates, and assisting neural repair (Puigdell¨ªvol et al.,, 2020). Microglial activation is a hallmark of aged brain; meanwhile, their neuroprotective effects are impaired (Soto et al.,, 2015, Michelle et al.,, 2007). Microglia show a moderately activated phenotype in the normal human brain. In addition, reactive microglia are indispensable for neuroinflammation in AD, posing a high risk of cognitive decline (Azam et al.,, 2021, Hui-Ming et al.,, 2003, Mandrekar-Colucci and Landreth, 2010).
The triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor of the immunoglobulin superfamily, is mainly expressed in the myeloid cells and uniquely expressed by microglia in the central nervous system (Frank et al.,, 2008). The cytoplasmic portion of TREM2 associates with DNAX-activating protein of 12 kDa (DAP12, also called TYROBP), which is a type I transmembrane adaptor protein, forming a molecular complex for its signaling and functions (Daws et al.,, 2001, Humphrey et al.,, 2006). Studies have found that activated TREM2 can regulate the functions of microglia, including stimulating phagocytosis and suppressing cytokine production. Recent evidence also proves the crucial role of TREM2 in maintaining the expansion and survival of microglia(Kawabori et al.,, 2015, Zhong et al.,, 2015). Previous genome-wide association studies have shown that the rare mutation (R47H) of TREM2 is a risk for AD (Colonna and Wang, 2016, Guerreiro et al.,, 2013, Tanzi, 2015); in addition, the silence of TREM2 in microglia impairs their capacity to phagocytize cell membrane debris, and increases the production of pro-inflammatory cytokines (Hsieh et al.,, 2009, Kleinberger et al.,, 2014). Microglia are also activated in the brain of cognitively normal elderly (Takahashi and K, 2005). Previous research has found the expression levels of TREM2 in the brain of AD patients and AD mice are positively correlated with age, and higher than those in the normal controls of the same age(Zhao, 2019, Piccio et al.,, 2016).
In our study, we found that the expression level of TREM2 was significantly up-regulated in the hippocampus of the Aged group, compared with the Young group (Fig. 4A, Fig. 5AB). This result can be explained by that the up-regulation of TREM2 compensatively enhances the ability of microglia to clear debris, reduce the stimulating effect of endogenous debris, and suppress the generation of pro-inflammatory factor. It also promotes the accumulation of microglia and strengthens their response to the breakdown of neurons astrocytes, oligodendrocytes and myelin, thus mitigating the inflammation in the aging brain. Based on this evidence, we hypothesize that TREM2 protects from age-related cognitive degeneration.
CD163, a member of scavenger receptor cysteine-rich (SRCR) family of class B, is expressed in macrophages, monocytes and microglia (Fabriek et al.,, 2005, Moestrup and M?ller, 2004). CD163 has a homeostatic capacity, and the soluble CD163 generated via the shedding of the outer domain can exert anti-inflammatory effects (Pey et al.,, 2014). Endogenous pro-inflammatory cytokines, such as IFN-γ and tumor necrosis factor-alpha (TNF-α) classic activation macrophages/microglia, can decrease the expression of CD163; while glucocorticoids, IL-6 and IL-10 can increase CD163 expression (Christa et al.,, 2000). In recent years, CD163 has been considered as a specific marker with strong anti-inflammatory properties in microglia (Gorp et al.,, 2010), monocytes and macrophages. CD163 is the haemoglobin-haptoglobin (HbHp) complex receptor, whereas its metabolites, such as bilirubin, free iron and CO released by HbHp degradation upon the action of HO-1 enzyme, have strong anti-oxidative and anti-inflammatory effects (Otterbein et al.,, 2003, Soares Miguel and Bach Fritz, 2009). CD163 not only acts as an anti-inflammatory marker, but also binds to its ligands to transduce signals, leading to the release of anti-inflammatory mediators, such as interleukin-10 (IL-10). The IL-10 further up-regulates the expression of CD163 and HO-1 in an autocrine or paracrine manner, forming a positive feedback loop involved in the clearance of haemoglobin (Hb) and prevention of extracellular Hb triggering inflammatory responses. In addition, the increases of CD163 immunoreactive microglia is a specific immune response to AD neuropathology. In the present study, we found a significant difference in CD163 expression between the Aged and Young groups (P < 0.05) (Fig. 4B, Figure.5AC). The upregulation of CD163 and its positive feedback loop with IL-10 are prominent in aged people, suggesting that the protection of CD163 against Hb-induced inflammation is associated not only with the scavenging of HbHp complexes from the extracellular milieu, but also with the active release of IL-10 and haem metabolites.
With age, more microglia are activated and their ability is reduced to engulf dead neurons, neuron residues or β-amyloid protein, thereby inducing oxidative stress and the overproduction of pro-inflammatory mediators. In the aged, the up-regulation of TREM2 and CD163 may pose a compensatory effect on the pro-inflammatory environment in hippocampal microglial cells, which curbs the decline of cognitive function. The molecular mechanisms of TREM2 and CD163 in regulating microglia-mediated inflammatory milieu should be further explored, in order that appropriate interventions can be designed to counter age-related neuroinflammation and reverse age-related cognitive degeneration.