Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a C4a and C4b restore the astrocytic engulfment of microglial debris in vitro in serum-free culture medium. b Quantifications of the phagocytic influence by complement supplementation and preopsonization in serum-free culture medium. N = 11 independent biological replicates of each group. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). c Scheme of in vivo microglial depletion and time points for analysis. d Reanalysis of RNA-seq data from whole-brain homogenate (GSE108269 ) showing that Gfap and C4b are upregulated and C1qa is downregulated during microglial ablation, whereas C2 , C3 and C4a remain at low levels and are unaffected. N = 5 mice at D0 and N = 4 mice at D2 to D21. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). e qPCR further confirmed the upregulation of C4b in sorted astrocytes upon microglial depletion. N = 5 in each group. Two-tailed independent t test. f Scheme of the in vivo examination of astrocytic engulfment using AAV PHP.eB-based astrocyte labeling and microglial depletion. g Confocal orthogonal colocalization and 3D reconstruction show that C4b −/− impairs the astrocytic engulfment of microglial debris under physiological condition (D21) and upon CSF1R inhibition (D23). h Quantification of microglial debris engulfment by astrocytes. N = 7 (D21) and 8 (D23) WT mice, and N = 3 (D21) and 5 (D23) C4b −/− mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, IV intravenous, MFI mean fluorescence intensity, Ctx cortex, Hipp hippocampus, OB olfactory bulb. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: Recombinant complement C4b (Cat#: RPB305Mu01) was purchased from Cloud-Clone.

Techniques: In Vitro, In Vivo, RNA Sequencing Assay, Two Tailed Test, Labeling, Inhibition, Fluorescence

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: CR1 and A po E SNPs evaluated in the present study for their relationships to erythrocyte clearance mechanisms and AD risk

Article Snippet: C__31805085_10 , CR1L;CR1 , 1 , rs12080578.

Techniques:

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: CR1 and A po E SNPs evaluated in the present study for their relationships to erythrocyte clearance mechanisms and AD risk

Article Snippet: C__26971190_10 , CR1L;CR1 , 1 , rs6540437.

Techniques:

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: A) Direct binding and degradation of pathogens such as Aβ can occur without CR1 mediation, but is typically less effective than CR1-dependent mechanisms [52]. B) Classical immune adherence occurs when pathogen/antibody immune complexes activate complement, resulting in fixation of the immune complex with complement opsonins, particularly C3b. C3b is a primary ligand for CR1 and therefore binds the immune complex to CR1 on monocyte/macrophage and erythrocyte surfaces. Such binding is considered to be more effective/facile than direct binding [52], as is the case for Aβ [26]. After capture of the pathogen/immune complex, monocyte/macrophages then internalize and degrade the pathogen. Erythrocytes, however, ferry the bound immune complex to the liver, where the pathogen is stripped off by Kupffer cells and degraded [22,23]. C) Aβ in its aggregated state is one of a handful of peptides that can activate complement without antibody mediation [24–30]. As a result, it is tagged with complement opsonins such as C3b [29,30] and becomes subject to capture by CR1. D) The most efficacious binding and degradation of pathogens occurs when both antibody-dependent (Panel B) and antibody-independent (Panel C) mechanisms of complement opsonization occur (Panel D) [52].

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: Binding Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: CR1 is synthesized in four different structural isoforms. The F (fast migrating on electrophoresis) isoform has a molecular weight of ~190 kDa; the S (slow migrating on electrophoresis) isoform has a molecular weight of ~220 kDa. The C (~160 kDa) and D (~250 kDa) isoforms are quite rare (<1% of the population) [43]. Western blots with CR1 clone E11 antibody reveal these structural isoforms. For example, the patient in lane 3 (F/C) has one allele that expresses the F isoform and one allele that expresses the C isoform, whereas the patients in lanes 4–6 (F/F) are homozygous for the F isoform, and the patient in lane 9 (S/S) is homozygous for the S isoform. In addition to defining the structural isoform genotype for each patient, Western blots of CR1 have the further advantage of providing separate expression measures for each isoform (i.e., densitometry of each isoform b and) as well as measures of total erythrocyte CR1 (i.e., sum of the levels for each band). (MW) Molecular weight standards. (rCR1) Recombinant CR1.

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: Synthesized, Electrophoresis, Molecular Weight, Western Blot, Expressing, Recombinant

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: CR1 and A po E SNPs evaluated in the present study for their relationships to erythrocyte clearance mechanisms and AD risk

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques:

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: A) Representative immunocytochemistry of erythrocyte ghosts from a normal elderly subject using a conventional, commercial CR1 antibody (#376223, clone E11, US Biologicals, Salem, MA) directed at an epitope in the C3b/C4b binding region of CR1. CR1 immunoreactivity was readily detected at the cell surface and in clusters, characteristics of this cell surface receptor [53]. B) Representative immunohistochemistry of a <4 hours postmortem cortical brain section from an AD case using the same antibody and conditions as in Panel A. No material staining of any cell type was observed in any of the 4 AD or 4 ND patients studied, except for staining of cells that clearly lay in the brain vasculature (inset). Similar results were obtained with the three other CR1 antibodies employed in the experiments. C) In addition to erythrocytes, other blood cells known to express CR1 were detected in the brain vasculature by these methods. For example, CR1 immunoreactive eosinophils could be identified based on their multi-nucleated morphology (inset). D) In Western blots with CR1 clone E11 antibody and conventional 20 μg/ml/lane protein loads, CR1 immunoreactive bands were readily detected in erythrocyte samples (E) (lane 10) at the same molecular weight as recombinant CR1 (rCR1) (lane 9). Under the same conditions, only faint traces of immunoreactive CR1 were observed for the neocortical samples even at extremely high, 100 μg/ml/lane loads. E) Nearly identical results were obtained for the same samples in parallel blots of GPVI, a platelet marker that is expressly not found in brain [54], strongly suggesting that it (and putative brain CR1) are simply vascular contaminants that are inherently trapped in brain homogenates. Autofluorescent and immunoreactive hemoglobin could also be observed at these high protein concentrations (not shown). (P) Plasma sample showing bands for GPVI, which appear as multimers of the 62 kDa monomer form [55].

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: Immunocytochemistry, Binding Assay, Cell Surface Receptor Assay, Immunohistochemistry, Staining, Western Blot, Molecular Weight, Recombinant, Marker, Clinical Proteomics

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: A) Log plot of qPCR for CR1 mRNA and mRNAs for other blood and brain proteins in brain samples. At 30 amplification cycles, mRNAs for brain-specific markers such as GFAP and ALDH1L1 were readily detected in brain homogenates from four AD and four ND subjects, whereas, in the same samples, there was no detectable mRNA for CR1 with either of two different primers. Likewise, mRNAs for the hemoglobin marker HBA2 [42] exhibited only trace values. Nonetheless, in these same samples, small but consistent amounts of mRNAs for CR1 and HBA2 began to be observed after 40 amplification cycles, although their copy numbers were still some 2 orders of magnitude less than those for the brain-specific markers. B) As expected, using the same primers, mRNAs for CR1 and HBA2 were readily detected in blood samples at both 30 and 40 amplification cycles. By contrast, neither of the brain-specific markers had detectable mRNAs at 30 amplification cycles, and only trace amounts of astrocyte-specific GFAP mRNA were observed at 40 cycles. C) In single cell laser captured microglia (N = 21,600) from 6 AD, 6 ND, and 6 PD cases, CR1 mRNA expression was essentially background (mean FPKM = 0.04 ± 0.03), with 0 values for 94% of samples, whereas mRNAs for other markers known to be expressed by microglia were readily observed in all samples.

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: Amplification, Marker, Expressing

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: A) Erythrocyte CR1 expression as measured by densitometry of Western blots using AD and ND erythrocyte membrane preparations. B) Erythrocyte Aβ levels as measured by flow cytometry. These data confirm our earlier reports of deficient erythrocyte Aβ capture using ELISA methods [24–26]. C) Relationship of erythrocyte CR1 expression to the structural isoforms of CR1. Possession of the F isoform in one or both alleles significantly increased total erythrocyte CR1 levels, in keeping with previous findings [16]. Homozygosity for the F allele (F/F) was particularly potent in this regard. D) Homozygosity for the CR1-enhancing F/F genotype was significantly under-represented in AD compared to ND subjects. E) Likewise, expression levels of the F isoform overall were significantly deficient in AD compared to ND subjects.

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: Expressing, Western Blot, Membrane, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: polymorphisms, structure, and function of complement receptor 1

doi: 10.1016/j.jalz.2018.04.003

Figure Lengend Snippet: Effect of CR1 SNPs on Erythrocyte CR1 Levels, Erythrocyte CR1 Structural Isoforms, and AD Risk

Article Snippet: C__12080026_10 , CR1 , 1 , rs646817.

Techniques: