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Complement receptor 1 is expressed on brain cells and in the human brain

Daskoulidou, Nikoleta, Shaw, Bethany, Torvell, Megan, Watkins, Lewis, Cope, Emma L., Carpanini, Sarah M., Allen, Nicholas D., Morgan, B. Paul·2023·Glia
DOI 10.1002/glia.24355PMC PMC10953339
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Abstract

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi‐pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)‐derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC‐derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1‐specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co‐localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions.

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Figures

FIGURE 1 Representation of CR1 structure and ligand binding sites. From the amino terminus (NH2) CR1*1 comprises four long homologous repeats (LHRs) each composed of seven short consensus repeats (SCRs) of 60–70 amino acids, two additional SCRs, a transmembrane segment (TM) and an intracytoplasmic carboxy‐terminal domain (IC‐COOH). Each circular block represents an SCR (numbered 1–30). There are three C4b binding sites (SCR 1–3, 8–10, and 15–17) and two C3b binding sites (SCR 8–10 and 15–17). SCRs 22–28 bind C1q, MBL, and ficolins. CR1*2 has an additional LHR domain (LHR‐S) and consequently an extra C3b/C4b binding site. Adapted from Torvell et al. (2021).
FIGURE 1 Representation of CR1 structure and ligand binding sites. From the amino terminus (NH2) CR1*1 comprises four long homologous repeats (LHRs) each composed of seven short consensus repeats (SCRs) of 60–70 amino acids, two additional SCRs, a transmembrane segment (TM) and an intracytoplasmic carboxy‐terminal domain (IC‐COOH). Each circular block represents an SCR (numbered 1–30). There are three C4b binding sites (SCR 1–3, 8–10, and 15–17) and two C3b binding sites (SCR 8–10 and 15–17). SCRs 22–28 bind C1q, MBL, and ficolins. CR1*2 has an additional LHR domain (LHR‐S) and consequently an extra C3b/C4b binding site. Adapted from Torvell et al. (2021).CC BY · PubMed Central
FIGURE 2 CR1 expression in HMC3 and IMhu microglial cell lines. (a) Dual immunofluorescence staining of HMC3 (i–iii) and IMhu (iv–vi) cells with anti‐CR1 antibodies: affinity‐purified polyclonal anti‐CR1 (i; iv), mAb MBI38 (ii) and mAb MBI35 (v) with microglial markers IBA1 (ii, iii, v, vi) or CD45 (i, iv). Impact of pre‐adsorption with excess sCR1 on staining with mAb MBI38 (iii) and mAb MBI35 (vi) are shown. Nuclei are stained with Hoechst 33342 in the merged images for each set of plates. All images are captured at 40× except set (iii) which are at 80×; scale bar shown in right image for each set is 50 μm in all. (b) CR1 protein was detected in HMC3 and IMhu cells by Western blotting using all anti‐CR1 antibodies (polyclonal shown as example). Protein lysate from red blood cells (RBC) from a CR1*1/CR1*2 donor was used as a positive control. (c) CR1 mRNA was detected by qRT‐PCR using intron‐spanning primers (Table S2) in HMC3, IMhu, THP‐1 (human monocytic cell line) but not SH‐SY5Y (neuronal) cells. The reaction without reverse transcriptase (no RT) was used as a negative control.
FIGURE 2 CR1 expression in HMC3 and IMhu microglial cell lines. (a) Dual immunofluorescence staining of HMC3 (i–iii) and IMhu (iv–vi) cells with anti‐CR1 antibodies: affinity‐purified polyclonal anti‐CR1 (i; iv), mAb MBI38 (ii) and mAb MBI35 (v) with microglial markers IBA1 (ii, iii, v, vi) or CD45 (i, iv). Impact of pre‐adsorption with excess sCR1 on staining with mAb MBI38 (iii) and mAb MBI35 (vi) are shown. Nuclei are stained with Hoechst 33342 in the merged images for each set of plates. All images are captured at 40× except set (iii) which are at 80×; scale bar shown in right image for each set is 50 μm in all. (b) CR1 protein was detected in HMC3 and IMhu cells by Western blotting using all anti‐CR1 antibodies (polyclonal shown as example). Protein lysate from red blood cells (RBC) from a CR1*1/CR1*2 donor was used as a positive control. (c) CR1 mRNA was detected by qRT‐PCR using intron‐spanning primers (Table S2) in HMC3, IMhu, THP‐1 (human monocytic cell line) but not SH‐SY5Y (neuronal) cells. The reaction without reverse transcriptase (no RT) was used as a negative control.CC BY · PubMed Central
FIGURE 3 CR1 expression in KOLF2‐derived microglia. (a) CR1 expression in iPSC‐microglia in WT KOLF2‐derived microglia shown by immunofluorescence. CR1 was expressed in a granular, membrane‐associated pattern in KOLF2‐derived microglia but was not detected in CR1 KO KOLF2. Cells are co‐stained with microglial marker CD11b. Maximum projections of Z‐stacks are presented. Scale bar: 30 μm. (b) CR1 protein was detected in WT iPSC‐microglia by western blotting with the different anti‐CR1 antibodies (polyclonal shown here). Protein lysate from RBC from a CR1*1/CR1*2 donor was used as positive control. CR1 KO cells showed trace staining. (c) CR1 transcript was quantified by qRT‐PCR using intron‐spanning primers (Table S2) in WT and CR1 KO KOLF2 iPSC and iPSC‐microglia. Histogram shows the relative mRNA expression normalized to non‐differentiated cells (iPSC); **p < .01.
FIGURE 3 CR1 expression in KOLF2‐derived microglia. (a) CR1 expression in iPSC‐microglia in WT KOLF2‐derived microglia shown by immunofluorescence. CR1 was expressed in a granular, membrane‐associated pattern in KOLF2‐derived microglia but was not detected in CR1 KO KOLF2. Cells are co‐stained with microglial marker CD11b. Maximum projections of Z‐stacks are presented. Scale bar: 30 μm. (b) CR1 protein was detected in WT iPSC‐microglia by western blotting with the different anti‐CR1 antibodies (polyclonal shown here). Protein lysate from RBC from a CR1*1/CR1*2 donor was used as positive control. CR1 KO cells showed trace staining. (c) CR1 transcript was quantified by qRT‐PCR using intron‐spanning primers (Table S2) in WT and CR1 KO KOLF2 iPSC and iPSC‐microglia. Histogram shows the relative mRNA expression normalized to non‐differentiated cells (iPSC); **p < .01.CC BY · PubMed Central
FIGURE 4 CR1 expression in donor‐derived iPSC‐microglia. (a) CR1 expression in MPC and iPSC‐microglia is shown by immunofluorescence. Cells showed membrane‐associated staining with the pAb against CR1 and the anti‐CR1 mAbs, MBI38, and 3E10; sCR1 pre‐adsorption ablated staining for each antibody (MBI38 shown as example). Maximum projections of Z‐stacks are presented. Representative examples of lines homozygous for CR1*1 and CR1*2 variants are shown. Scale bar: 50 μm. (b) CR1 protein was detected in MPC and iPSC‐microglia expressing the CR1*1 and CR1*2 variants by western blotting with the different anti‐CR1 antibodies (polyclonal anti‐CR1 shown as example). Protein lysate from RBC from a CR1*1/CR1*2 donor was used as a positive control and anti‐tubulin as the loading control. (c) CR1 transcripts were detected by qRT‐PCR using intron‐crossing primers introns (Table S2) in iPSC (1), MPC (2), and iPSC‐microglia (3) expressing the CR1*1 and CR1*2 variants. Histograms show the relative mRNA expression normalized to non‐differentiated cells (iPSC). *p < .05; **p < .01; ***p < .001.
FIGURE 4 CR1 expression in donor‐derived iPSC‐microglia. (a) CR1 expression in MPC and iPSC‐microglia is shown by immunofluorescence. Cells showed membrane‐associated staining with the pAb against CR1 and the anti‐CR1 mAbs, MBI38, and 3E10; sCR1 pre‐adsorption ablated staining for each antibody (MBI38 shown as example). Maximum projections of Z‐stacks are presented. Representative examples of lines homozygous for CR1*1 and CR1*2 variants are shown. Scale bar: 50 μm. (b) CR1 protein was detected in MPC and iPSC‐microglia expressing the CR1*1 and CR1*2 variants by western blotting with the different anti‐CR1 antibodies (polyclonal anti‐CR1 shown as example). Protein lysate from RBC from a CR1*1/CR1*2 donor was used as a positive control and anti‐tubulin as the loading control. (c) CR1 transcripts were detected by qRT‐PCR using intron‐crossing primers introns (Table S2) in iPSC (1), MPC (2), and iPSC‐microglia (3) expressing the CR1*1 and CR1*2 variants. Histograms show the relative mRNA expression normalized to non‐differentiated cells (iPSC). *p < .05; **p < .01; ***p < .001.CC BY · PubMed Central
FIGURE 5 CR1 protein expression in the human brain. Immunofluorescent staining of frozen sections of control (a) and AD (b) human brain tissue (region BA41/42) with anti‐CR1 antibodies (anti‐CR1 mAb MBI35 shown as example) demonstrated expression of CR1 in IBA1‐positive microglia and GFAP‐positive astrocytes but not in HuC/D‐positive neurons. Scale bar: 30 μm in main and 20 μm in insert figures. (c) Pre‐adsorption of MBI35 anti‐CR1 with sCR1 abolished microglial and astroglial staining (scale bars in right panels). Top panel scale bar: 100 μm; bottom scale bar: 30 μm. (d) Immunofluorescent staining of AD human brain tissue with anti‐Aβ (6E10) and anti‐CR1 (MBI35) demonstrated expression of CR1 in Aβ plaques and surrounding glia. Scale bar: 30 μm in main and 20 μm in insert figure. (e) Representative low power views (scale bar: 150 μm) of whole sections to show CR1 staining in AD and control brain gray matter (GM). Staining was clearly increased in AD cases compared to controls. (f) Quantitation of MBI35‐positive cell density per field of view (fov) in GM showed increased staining in AD brain in all ROIs, significantly greater compared to controls in deep and mid GM ROIs. *p < .05; data from five AD and five control brains.
FIGURE 5 CR1 protein expression in the human brain. Immunofluorescent staining of frozen sections of control (a) and AD (b) human brain tissue (region BA41/42) with anti‐CR1 antibodies (anti‐CR1 mAb MBI35 shown as example) demonstrated expression of CR1 in IBA1‐positive microglia and GFAP‐positive astrocytes but not in HuC/D‐positive neurons. Scale bar: 30 μm in main and 20 μm in insert figures. (c) Pre‐adsorption of MBI35 anti‐CR1 with sCR1 abolished microglial and astroglial staining (scale bars in right panels). Top panel scale bar: 100 μm; bottom scale bar: 30 μm. (d) Immunofluorescent staining of AD human brain tissue with anti‐Aβ (6E10) and anti‐CR1 (MBI35) demonstrated expression of CR1 in Aβ plaques and surrounding glia. Scale bar: 30 μm in main and 20 μm in insert figure. (e) Representative low power views (scale bar: 150 μm) of whole sections to show CR1 staining in AD and control brain gray matter (GM). Staining was clearly increased in AD cases compared to controls. (f) Quantitation of MBI35‐positive cell density per field of view (fov) in GM showed increased staining in AD brain in all ROIs, significantly greater compared to controls in deep and mid GM ROIs. *p < .05; data from five AD and five control brains.CC BY · PubMed Central
FIGURE 6 CR1 mRNA expression in the human brain. (a) Agarose gel demonstrating detection of CR1 transcripts in control and AD human brain tissue using specific primers. Transcripts for the cell type‐specific markers IBA1, GFAP, and RBFOX3 and the housekeeping gene SDHA were also present in the brain extracts. (b) Relative CR1, IBA1, GFAP and RBFOX3 mRNA expression (‐fold) in control and AD human brain tissue; CR1 mRNA showing ~5‐fold greater expression in AD brain. **p < .01; ***p < .001, data from five AD and five control brains.
FIGURE 6 CR1 mRNA expression in the human brain. (a) Agarose gel demonstrating detection of CR1 transcripts in control and AD human brain tissue using specific primers. Transcripts for the cell type‐specific markers IBA1, GFAP, and RBFOX3 and the housekeeping gene SDHA were also present in the brain extracts. (b) Relative CR1, IBA1, GFAP and RBFOX3 mRNA expression (‐fold) in control and AD human brain tissue; CR1 mRNA showing ~5‐fold greater expression in AD brain. **p < .01; ***p < .001, data from five AD and five control brains.CC BY · PubMed Central

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Metadata

Authors
Daskoulidou, N., Shaw, B., Torvell, M., Watkins, L., Cope, E. L., Carpanini, S. M., Allen, N. D., & Morgan, B. P.
Affiliations
  • UK Dementia Research Institute, Cardiff University Cardiff UK
  • School of Biosciences, Cardiff University Cardiff UK
ORCID
Venue
Glia
Publisher
Wiley
Published
2023-02-24
Volume
71
Issue
6
Pages
1522-1535
ISSN
  • 0894-1491
  • 1098-1136
DOI
10.1002/glia.24355
PMC
PMC10953339
Cited by
40
References
8
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Alzheimer's Society (AS‐PG‐17‐005)
  • UK Dementia Research Institute
  • Medical Research Council

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