Fig 1.
Representation of CR1 structure, ligand binding sites and location of antibody epitopes.
CYT: cytoplasmic domain, TM: transmembrane domain. Red dot indicates location of rs4844609 (Ser1610Thr). Diagram modified from[16]; Antibody reactivity modified from[17].
Table 1.
Electrophoretic mobility (Mr) and gene frequency of the allelic size isoforms of CR1.
Table 2.
Case Characteristics for Analyzed Brain Samples.
Table 3.
Characteristics for Analyzed Plasma and Reb Blood Cell CR1 Samples.
Fig 2.
CR1 expressed by CHO cells is recognized by anti-CR1 antibodies.
CR1 immunofluorescence staining using mouse mAbs 8C9.1(A), E11 (B) or J3B11 (C) on fixed CHO cells expressing 1x106 (high) (upper panels) or 1x105 (moderate) (middle panels) receptors/cell. Bottom panels show isotype control on high CHO-CR1 (A), or antibody reactivity on non-transfected CHO cells (B, C). Scale bar: 50 μm.
Fig 3.
CR1/CD35 expression in human brain.
Representative micrographs of brain sections stained with monoclonal anti-CR1 Abs 8C9.1 (A, B, D-F), J3B11 (G-I) or mIgG control (C) in frontal cortex of control (CTR) and Alzheimer’s disease (AD) cases. Genotype for the two GWAS SNPs rs4844609 (T or A) and rs6656401 (G or A) are presented in each panel. Scale bar 100 μm.
Fig 4.
Anti-CR1 immunoreactivity with astrocytes but not neurons is preabsorbed by rhCR1.
Immunostaining with monoclonal anti-CR1 8C9.1 (A, B) or J3B11 (C) without (upper panels) or with (lower panels) preabsorption with recombinant human CR1. Neuronal staining (B) was not preabsorbed with rhCR1. Scale bar: A, C: 50 μm, B: 100 μm.
Fig 5.
CR1 staining colocalizes with a subset of astrocytes.
Immunofluorescent dual labelling of CR1 (8C9.1, red) and astrocytes (anti-GFAP, green) in an AD case (A-F). Merged images show colocalization (C) or lack of colocalization (F) of CR1 and GFAP immunofluorescence labelling. Scale bar: 50 μm.
Fig 6.
CR1 expression by human AD brain-derived astrocytes.
Representative confocal photomicrographs of astrocytes isolated from post-mortem human brain stained with 8C9.1 (red) (C, D) and J3B11 (red) (E, F) mAbs and anti-GFAP (green) (D, F). A, B negative controls in which only secondary Ab labeled with Alexa555 (A) or Alexa488 (B) but no primary antibodies were added. Scale bar 20 μm.
Fig 7.
WB and immunoprecipitation of CR1 with mAbs E11, 8C9.1 and J3B11.
(A) WB of RBC samples with and without SNP rs6656401 (loaded 0.9 ng CR1 per lane) using E11 mAb. Lanes: 1 RBC control, 2 C (nondemented) (AT/AA), 3 MCI (mild cognitive impairment) (AT/AG), 4 C (AT/AG), 5 MCI (AT/AG), 6 C (TT/GG), 7 MCI (TT/GG), 8 C (TT/AG), 9 MCI (TT/GG). (B) WB of brain extracts from donors with and without rs6656401 (loaded 100 μg brain extract protein per lane 3–9) probed with E11 mAb. Lane: 1 sCR1 (1 ng), 2 RBC (5 μg), 3 C (TT/AG), 4 AD (AT/AA), 5 AD (AT), 6 AD (TT/AA), 7 AD (TT/GG), 8 C (TT/GG), 9 C (AT/AG). Exposure time in lanes 1 and 2 was 5 sec and in lanes 3–9, 7 min. (C) Immunoprecipitates of E11 (4 μg) (lanes 3, 5, 7) or IgG1 (4 μg) (lanes 2, 4, 6) with sCR1 (2 μg) (lanes 2,3), RBC from a MCI subject (TT/GG) (50 μg protein, lane 4, 5) or brain extracts from cognitively intact subject (AT/AG) (250 μg, lane 6, 7). sCR1 is loaded at 1 ng (lane 1). (D) Representative immunoprecipitations of brain lysates C (TT/GG) (250 μg protein) (lanes 3–6) with 8C9.1 (lane 4), J3B11 (lane 6), or corresponding isotype IgG (lanes 3, 5). Control sCR1 loaded at 1 ng (lane 1) and RBC extract loaded at 1 ng CR1 (lane 2). C and D. Blots were probed with polyclonal anti-CR1 Ab.
Fig 8.
Soluble CR1 concentration (ng/ml) in plasma from donors with different CR1 SNPs and diagnosis.
Soluble CR1 was measured by ELISA and plotted vs. A. SNP [rs6656401 only (n = 21) or rs6656401 and rs4844609 (n = 16) and without the rs6656401 and rs4844609 SNPs (n = 22)] or B. categorized by diagnosis [nondemented (n = 22),), MCI (n = 14) and AD (n = 19)]. Recombinant CR1 was used to generate the standard curve. Black symbols indicate samples with no variant SNP (rs6656401 and rs4844609), red reflects donors with two SNPs, and blue is donors with rs6656401 only. Data points are from multiple experiments in which samples were run in triplicate and normalized to standard CR1 samples across experiments.
Fig 9.
C1q-CR1 and C3b-CR1 relative binding activity.
CR1 partially purified from RBC was assessed for binding to immobilized C1q (A, B) or C3b (C, D, E, F). A. UCI cohort: CR1- C1q relative binding for CR1with rs6656401 (n = 10) only, with rs6656401 and rs4844609 (n = 8), or without rs6656401 or rs4844609 (n = 11) SNPs. Black, green and purple symbols indicate patients clinically diagnosed as cognitively normal, mild cognitive impairment (MCI), and demented (AD) respectively. B. UCI cohort: CR1-C1q relative binding vs diagnosis: Normal (n = 12), MCI (n = 11) and Dementia (n = 16). In C and E, CR1-C3b relative binding is plotted by CR1 isoform (determined by WB): donors with common allele (fast form or F form) (n = 12) or with extra C3b-binding domain (n = 17) (slow form or S form) in UCI cohort (C) or F form (n = 38), S form (n = 17) for SRI cohort (E). In D and F, CR1-C3b relative binding plotted vs diagnosis: Normal (n = 12), MCI (n = 11), Dementia (n = 6) in UCI cohort (D) or normal (n = 30), MCI (n = 13), Dementia (n = 20) in SRI cohort (F). (Reference CR1 samples were used to standardize values among assays performed on different dates). In B-F: samples with no variant SNP (black); red, donors with two SNPs (rs6656401 or rs4844609); blue, donors with rs6656401 only and green open square is donor with unknown SNP genotype. Subjects in whom the CR1 SNP analysis was discordant with the F/F WB results are visible as blue circles in Fig 9C and E. *p<0.01–0.05, **p<0.01–0.001, calculated using 1 way ANOVA. Data points are from multiple experiments in which samples were run in triplicate and normalized to standard CR1 samples across experiments.