Skip to main content
PMC home page
Frontiers in Aging Neuroscience logoLink to Frontiers in Aging Neuroscience
. 2020 Dec 3;12:589196. doi: 10.3389/fnagi.2020.589196

Do Alzheimer's Disease Risk Gene Products Actually Act in Microglia?

Sadayuki Hashioka 1,*, Ken Inoue 2, Haruo Takeshita 3, Masatoshi Inagaki 1
PMCID: PMC7744292  PMID: 33343331

Introduction

The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allele ε4 of the apolipoprotein E (ApoE) gene, which was discovered in 1993 (Corder et al., 1993). In around 2010, human genome-wide association studies (GWASs) of late-onset AD cases identified novel AD risk genes, the majority of which are considered to be expressed selectively or highly in microglia (Hansen et al., 2018). These include the triggering receptor expressed on myeloid cell 2 (TREM2) (Jonsson et al., 2013), complement receptor 1 (CR1) (Lambert et al., 2009), ATP-binding cassette sub-family A membrane 7 (ABCA7) (Hollingworth et al., 2011), and CD33 (Hollingworth et al., 2011). It is of note that the strength of the effect of the TREM2 variant (odds ratio of 2.36-2.71) is similar to that of ApoE ε4 (odds ratio of 3.08) (Villegas-Llerena et al., 2016). Of these genes, ApoE, TREM2, and CD33 have been identified as key genes involved in the intermediate state of disease-associated microglia (DAM, also referred to as microglial neurodegenerative phenotype) by most recent single-cell RNA sequencing studies of microglia from AD-transgenic (Tg) mice (Keren-Shaul et al., 2017; Krasemann et al., 2017). Specifically, ApoE and TREM2 are up-regulated while CD33 is down-regulated during DAM activation (Keren-Shaul et al., 2017; Krasemann et al., 2017). Furthermore, the DAM signature is dependent on the TREM2-ApoE pathway in AD-Tg mice (Krasemann et al., 2017). Immunohistochemistry on human AD brain tissue has shown that even though DAM-like microglia (labeled with only one type of DAM marker) are located in close proximity to Aβ plaques, they are not found in non-plaque areas (Keren-Shaul et al., 2017; Krasemann et al., 2017). These genetic and immunohistochemistry findings imply that the functional changes of ApoE, TREM2, and CD33 caused by the variants of GWAS-identified AD risk genes, which overlap with DAM-associated genes, alter microglial functions and consequently play important roles in AD pathogenesis. Nevertheless, it should be taken into account that the protein expression of these molecules in human brains is fundamentally different from that in rodent brains.

TREM2 Expression In Microglia

A number of animal studies have demonstrated microglial immunoreactivity for TREM2 in the rodent brain (Thrash et al., 2009; Kawabori et al., 2015). In the human brain, however, there has been controversy about the microglial expression of TREM2. Even though human microglia isolated from AD brains show up-regulated expression of TREM2 mRNA (Gosselin et al., 2017), this increase cannot be readily confirmed at the protein level. Using the same polyclonal anti-TREM2 antibody (Sigma HPA010917), two independent immunohistochemical studies on 312 postmortem cases, including 92 AD cases, have shown that human microglia do not express the TREM2 protein (Satoh et al., 2013; Fahrenhold et al., 2018). These studies have also demonstrated that the TREM2-immunoreactive cells are recruited monocytes in brain blood vessels, not microglia or perivascular macrophages (Satoh et al., 2013; Fahrenhold et al., 2018). On the other hand, a study on 33 cases, including 11 AD cases, reported TREM2-immunoreactive microglia associated with plaques; this study employed a different polyclonal anti-TREM2 antibody (R&D AF1828) (Lue et al., 2015). Although we have no convincing reason to explain this inconsistency in microglial immunoreactivity, the HPA010917 antibody appears to be more reliable than the AF1828 antibody, based on immunoreacting ability. Whereas AF1828 can recognize human recombinant TREM2 in immunoblotting, it is not able to label monocytes, macrophages, dendritic cells, or osteoclasts in human spleen and bone marrow (Satoh et al., 2013). The discrepancy in the human microglial expression of TREM2 between mRNA levels and protein levels could be attributed to the possibility that human TREM2 protein in microglia is post-translationally modified. For example, HPA010917 may not bind to a protein modified by glycosylation. Human TREM2 has been demonstrated to undergo glycosylation (Park et al., 2015), even though it is not known whether this kind of post-translational modification changes the TREM2 antigenicity. Another possibility is that the antibody recognizes human TREM2, but since the protein is soluble in humans, it is no longer detected by immunohistochemical methods. Soluble TREM2 resulting from ectodomain shedding has been detected in human cerebrospinal fluid by the enzyme-linked immunosorbent assay (Suarez-Calvet et al., 2019). The immunohistochemical finding that human TREM2-immunoreactive cells are largely restricted to blood circulation, not to the brain, implies that an altered immune response in the periphery may play a role in the development and progression of AD.

ApoE Expression In Microglia

ApoE is believed to be derived mainly from astrocytes in the human brain (Murakami et al., 1988). In normal human brains, ApoE is predominantly expressed by astrocytes at both mRNA (Hansen et al., 2018) and protein (Murakami et al., 1988) levels. Microglial ApoE expression is induced in AD model mice (Hansen et al., 2018). However, in human AD brains, only a portion of microglia show the immunoreactivity for ApoE (Uchihara et al., 1995), while microglial expression of the ApoE gene is up-regulated (Mathys et al., 2019). Such inconsistency in ApoE expression by AD microglia may stem from post-translational modification of the apolipoprotein. ApoE has recently been shown to bind to human TREM2 with a specific high affinity (Atagi et al., 2015). However, the exact relationship between ApoE and microglia in the human brain remains unknown, since microglia appear not to express the TREM2 protein in the human brain, as mentioned above. In the periphery, a significant amount of circulating ApoE is produced by macrophages as well as by the liver. It has been demonstrated that human monocyte-derived macrophages from ε4/ε4 subjects secret a large amount of ApoE but lack effective cholesterol efflux (Cullen et al., 1998). The ε4/ε4 macrophages appear to facilitate the development of hypercholesterolemia and to contribute considerably to AD pathogenesis since hypercholesterolemia is a major risk factor for late-onset AD (Meng et al., 2014).

CD33 Expression In Microglia

CD33 (also referred to as sialic acid binding immunoglobulin-like lectin-3) used to be considered a myeloid-specific immunomodulatory receptor. Today, it is known that CD33 is also expressed by microglia at both mRNA (Galatro et al., 2017; Gosselin et al., 2017) and protein (Malik et al., 2013) levels in normal human brains. In human AD brains, the number of CD33-immunoreactive microglia is increased with a positive correlation with the insoluble Aβ42 burden (Griciuc et al., 2013). CD33 overexpression associated with the rs3865444C risk allele is linked to increased AD risk, while other single nucleotide polymorphisms (i.e., rs3865444A and rs12459419T) yield non-functional CD33 protein and reduce AD risk (Bradshaw et al., 2013; Malik et al., 2013). These genetic findings suggest that CD33 mutations cause dichotomous results. Although the protection against AD onset could be explained by adaptive loss-of-function (Siddiqui et al., 2017), it is still unknown how CD33 mutations contribute to AD pathogenesis. As a result of the fact that CD33 mutations also change the immunological functions of peripheral monocytes (Bradshaw et al., 2013), the CD33 mutation-altered peripheral immune system may contribute to AD pathogenesis to some extent, as do altered microglia functions.

Discussion

As mentioned above, ApoE, TREM2, and CD33 are human GWAS-identified AD risk factors and also DAM-associated genes identified by single-cell RNA analyses on microglia from AD model mice. Based on the cellular expression pattern of the proteins, ApoE, TREM2, and CD33 in humans, the possibility cannot be excluded that functional changes of these molecules due to gene mutations have little to do with microglia in AD pathogenesis. Instead, the altered functions of ApoE, TREM2, and CD33 may have a close link to peripheral monocytes expressing these proteins and cause a dysregulated immune response, such as chronic systemic inflammation. Furthermore, such functional changes in ApoE, TREM2, and CD33 may indirectly induce the neurotoxic activation of microglia via chronic systemic inflammation, which has been shown to induce neuroinflammation associated with microglial activation (reviewed in Hashioka et al., 2019).

Author Contributions

SH wrote the manuscript. All authors discussed and edited the manuscript, and read and approved the final manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

Sincere appreciation is extended to Drs. Patrick and Edith McGeer for their invaluable support.

Footnotes

Funding. This study was supported by JSPS KAKENHI Grant Number 19K08018 (SH).

References

  1. Atagi Y., Liu C. C., Painter M. M., Chen X. F., Verbeeck C., Zheng H., et al. (2015). Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2). J. Biol. Chem. 290, 26043–26050. 10.1074/jbc.M115.679043 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradshaw E. M., Chibnik L. B., Keenan B. T., Ottoboni L., Raj T., Tang A., et al. (2013). CD33 Alzheimer's disease locus: altered monocyte function and amyloid biology. Nat. Neurosci. 16, 848–850. 10.1038/nn.3435 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Corder E. H., Saunders A. M., Strittmatter W. J., Schmechel D. E., Gaskell P. C., Small G. W., et al. (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261, 921–923. 10.1126/science.8346443 [DOI] [PubMed] [Google Scholar]
  4. Cullen P., Cignarella A., Brennhausen B., Mohr S., Assmann G., Von Eckardstein A. (1998). Phenotype-dependent differences in apolipoprotein E metabolism and in cholesterol homeostasis in human monocyte-derived macrophages. J. Clin. Invest. 101, 1670–1677. 10.1172/JCI119887 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fahrenhold M., Rakic S., Classey J., Brayne C., Ince P. G., Nicoll J., et al. (2018). TREM2 expression in the human brain: a marker of monocyte recruitment? Brain Pathol. 28, 595–602. 10.1111/bpa.12564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galatro T. F., Holtman I. R., Lerario A. M., Vainchtein I. D., Brouwer N., Sola P. R., et al. (2017). Transcriptomic analysis of purified human cortical microglia reveals age-associated changes. Nat. Neurosci. 20, 1162–1171. 10.1038/nn.4597 [DOI] [PubMed] [Google Scholar]
  7. Gosselin D., Skola D., Coufal N. G., Holtman I. R., Schlachetzki J. C. M., Sajti E., et al. (2017). An environment-dependent transcriptional network specifies human microglia identity. Science 356:eaal3222. 10.1126/science.aal3222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Griciuc A., Serrano-Pozo A., Parrado A. R., Lesinski A. N., Asselin C. N., Mullin K., et al. (2013). Alzheimer's disease risk gene CD33 inhibits microglial uptake of amyloid beta. Neuron 78, 631–643. 10.1016/j.neuron.2013.04.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hansen D. V., Hanson J. E., Sheng M. (2018). Microglia in Alzheimer's disease. J C.ell Biol. 217, 459–472. 10.1083/jcb.201709069 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hashioka S., Inoue K., Miyaoka T., Hayashida M., Wake R., Oh-Nishi A., et al. (2019). The Possible causal link of periodontitis to neuropsychiatric disorders: more than psychosocial mechanisms. Int. J. Mol. Sci. 20:3723. 10.3390/ijms20153723 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hollingworth P., Harold D., Sims R., Gerrish A., Lambert J. C., Carrasquillo M. M., et al. (2011). Common variants at ABCA7, MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer's disease. Nat. Genet. 43, 429–435. 10.1038/ng.803 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jonsson T., Stefansson H., Steinberg S., Jonsdottir I., Jonsson P. V., Snaedal J., et al. (2013). Variant of TREM2 associated with the risk of Alzheimer's disease. N. Engl. J. Med. 368, 107–116. 10.1056/NEJMoa1211103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kawabori M., Kacimi R., Kauppinen T., Calosing C., Kim J. Y., Hsieh C. L., et al. (2015). Triggering receptor expressed on myeloid cells 2 (TREM2) deficiency attenuates phagocytic activities of microglia and exacerbates ischemic damage in experimental stroke. J. Neurosci. 35, 3384–3396. 10.1523/JNEUROSCI.2620-14.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keren-Shaul H., Spinrad A., Weiner A., Matcovitch-Natan O., Dvir-Szternfeld R., Ulland T. K., et al. (2017). A unique microglia type associated with restricting development of Alzheimer's disease. Cell 169, 1276–1290.e1217. 10.1016/j.cell.2017.05.018 [DOI] [PubMed] [Google Scholar]
  15. Krasemann S., Madore C., Cialic R., Baufeld C., Calcagno N., El Fatimy R., et al. (2017). The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e569. 10.1016/j.immuni.2017.08.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lambert J. C., Heath S., Even G., Campion D., Sleegers K., Hiltunen M., et al. (2009). Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease. Nat. Genet. 41, 1094–1099. 10.1038/ng.439 [DOI] [PubMed] [Google Scholar]
  17. Lue L. F., Schmitz C. T., Serrano G., Sue L. I., Beach T. G., Walker D. G. (2015). TREM2 protein expression changes correlate with alzheimer's disease neurodegenerative pathologies in post-mortem temporal cortices. Brain Pathol. 25, 469–480. 10.1111/bpa.12190 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Malik M., Simpson J. F., Parikh I., Wilfred B. R., Fardo D. W., Nelson P. T., et al. (2013). CD33 Alzheimer's risk-altering polymorphism, CD33 expression, and exon 2 splicing. J. Neurosci. 33, 13320–13325. 10.1523/JNEUROSCI.1224-13.2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mathys H., Davila-Velderrain J., Peng Z., Gao F., Mohammadi S., Young J. Z., et al. (2019). Single-cell transcriptomic analysis of Alzheimer's disease. Nature 570, 332–337. 10.1038/s41586-019-1195-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meng X. F., Yu J. T., Wang H. F., Tan M. S., Wang C., Tan C. C., et al. (2014). Midlife vascular risk factors and the risk of Alzheimer's disease: a systematic review and meta-analysis. J. Alzheimers Dis. 42, 1295–1310. 10.3233/JAD-140954 [DOI] [PubMed] [Google Scholar]
  21. Murakami M., Ushio Y., Morino Y., Ohta T., Matsukado Y. (1988). Immunohistochemical localization of apolipoprotein E in human glial neoplasms. J. Clin. Invest. 82, 177–188. 10.1172/JCI113568 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Park J. S., Ji I. J., An H. J., Kang M. J., Kang S. W., Kim D. H., et al. (2015). Disease-associated mutations of TREM2 alter the processing of N-linked oligosaccharides in the golgi apparatus. Traffic 16, 510–518. 10.1111/tra.12264 [DOI] [PubMed] [Google Scholar]
  23. Satoh J., Kawana N., Yamamoto Y., Ishida T., Saito Y., Arima K. (2013). A survey of TREM2 antibodies reveals neuronal but not microglial staining in formalin-fixed paraffin-embedded postmortem Alzheimer's brain tissues. Alzheimers Res. Ther. 5:30. 10.1186/alzrt184 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Siddiqui S. S., Springer S. A., Verhagen A., Sundaramurthy V., Alisson-Silva F., Jiang W., et al. (2017). The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool. J. Biol. Chem. 292, 15312–15320. 10.1074/jbc.M117.799346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suarez-Calvet M., Morenas-Rodriguez E., Kleinberger G., Schlepckow K., Araque Caballero M. A., Franzmeier N., et al. (2019). Early increase of CSF sTREM2 in Alzheimer's disease is associated with tau related-neurodegeneration but not with amyloid-beta pathology. Mol. Neurodegener. 14:1. 10.1186/s13024-018-0301-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thrash J. C., Torbett B. E., Carson M. J. (2009). Developmental regulation of TREM2 and DAP12 expression in the murine CNS: implications for Nasu-Hakola disease. Neurochem. Res. 34, 38–45. 10.1007/s11064-008-9657-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Uchihara T., Duyckaerts C., He Y., Kobayashi K., Seilhean D., Amouyel P., et al. (1995). ApoE immunoreactivity and microglial cells in Alzheimer's disease brain. Neurosci. Lett. 195, 5–8. 10.1016/0304-3940(95)11763-M [DOI] [PubMed] [Google Scholar]
  28. Villegas-Llerena C., Phillips A., Garcia-Reitboeck P., Hardy J., Pocock J. M. (2016). Microglial genes regulating neuroinflammation in the progression of Alzheimer's disease. Curr. Opin. Neurobiol. 36, 74–81. 10.1016/j.conb.2015.10.004 [DOI] [PubMed] [Google Scholar]

Articles from Frontiers in Aging Neuroscience are provided here courtesy of Frontiers Media SA

RESOURCES