Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Sambucus nigra agglutinin (SNA, recognizes α2,6-linked sialic acid) and Maackia amurensis agglutinin II (MAA II, recognizes α2,3-linked sialic acid) ligand expression in WT BV2 cells (orange), WT BV2 cells pretreated with sialidase (blue), and CMAS KO cells (red) assessed by flow cytometry. Sialidase treatment and CMAS KO reduce sialic acid ligands on the cell surface. b , Western blot showing SHP1 protein expression in WT and PTPN6 KO BV2 cells. For raw source image, see . c , Percent confluence of control (gray), CMAS KO (red), and PTPN6 KO (blue) BV2 cells during time-lapse microscopy phagocytosis assays (n=3, mean +/− s.e.m.). d , e , Phagocytosis of pH-sensitive fluorescent beads by untreated (black) and sialidase-treated (red) BV2 cells (d) or vehicle-treated and 3F ax -Neu5Ac-treated BV2 cells prior to phagocytosis (n=3, ** P <0.005, two-sided t-test; mean +/− s.e.m). f , Phagocytosis of pH-sensitive fluorescent beads by WT (black), WT + sialidase (red), CD22 KO (blue), or CD22 KO + sialidase (green) BV2 cells (n=3, * P <0.05, N.S. not significant, one-way ANOVA with Tukey’s multiple comparisons correction; mean +/− s.e.m.). g , Microglia were acutely isolated from the brains of aged (18 m.o.) WT (left) or CD22 −/− (right) mice, treated with or without sialidase, and incubated with pH-sensitive fluorescent latex beads. Microglia specific phagocytosis was measured using flow cytometry (n=6, * P <0.05, N.S. not significant, paired two-sided t-test). h , Representative images of BV2 cells coated with AlexaFluor 488 conjugated glycopolymers (green) and stained with a plasma membrane-specific dye (CellMask, red) showing overlap (yellow). Scale bar = 25 microns. i , Recombinant mouse CD22-human Fc fusion protein was pre-complexed with AF647 anti-human Fc secondary antibody, treated with various concentrations of IgG (black) or anti-CD22 (blue, red), and subsequently allowed to bind to ligands on the surface of BV2 cells or BV2 cells pretreated with sialidase (red). Binding was measured by flow cytometry. j , Internalization of IgG (black), function blocking anti-CD22 (clone Cy34.1, blue), and non-function-blocking anti-CD22 (clone OX96, green) conjugated to a pH-sensitive fluorescent dye by BV2 cells assessed by time-lapse microscopy (n=3, mean +/− s.e.m.). k , Western blot quantification of ratio of active pSHP1 to total SHP1 protein in BV2 cells pretreated with various concentrations of anti-CD22. Blue line represents the fitted variable slope inhibitor-response curve. For raw source image, see . All data were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Expressing, Flow Cytometry, Western Blot, Control, Time-lapse Microscopy, Isolation, Incubation, Staining, Clinical Proteomics, Membrane, Recombinant, Binding Assay, Blocking Assay

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Myelin debris labeled with a fluorescent dye (AF555) was stereotactically co-injected with anti-CD22 or IgG into the cortex on opposite hemispheres of the same aged (14-16 m.o.) mouse. b , Representative images of AF555-labed myelin (red, top row) overlaid with the myeloid marker Iba1 (green, bottom row) at the injection sites of IgG (left) or anti-CD22 (right) treated hemispheres of the same aged brain. Scale bar = 100 microns. c , Clearance of myelin debris in IgG (black) or anti-CD22 (green) treated hemispheres of aged mice assessed 48 hours post-injection (n=8, ** P <0.005, paired two-sided t-test). d , Flow cytometry quantification of ex vivo phagocytosis of pH-sensitive beads by aged microglia pretreated with IgG or anti-CD22 (n=6, ** P <0.005, paired two-sided t-test). e , Labeled myelin debris was stereotactically injected into the cortices of aged (12–14 m.o.) WT or CD22 −/− mice. f , Clearance of myelin debris in cortices of aged WT (black) vs CD22 −/− (green) mice assessed 48 hours post-injection (n=4, * P <0.05, two-sided t-test, mean +/− s.e.m.). g , Clearance of myelin debris in IgG (black) or anti-CD22 (green) treated hemispheres of young mice assessed 48 hours post-injection (n=4, N.S. not significant, paired two-sided t-test). h , Clearance of Aβ oligomers in IgG (black) or anti-CD22 (green) treated hemispheres of aged mice assessed 48 hours post-injection (n=8, ** P <0.005, paired two-sided t-test). i , Percent area of residual Aβ oligomers that were CypHer5E+, indicating localization to acidified lysosomes (n=8, ** P <0.005, paired two-sided t-test). All data were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Labeling, Injection, Marker, Flow Cytometry, Ex Vivo

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Representative images of myelin labeled with a pH-sensitive fluorescent dye (CypHer5E, white), a constitutively fluorescent dye (AF555, red) and stained for Iba1 (green). The majority of AF555 overlapping with Iba1 is also positive for CypHer5E, indicating localization to an acidified compartment. Scale bar = 100 microns. b , 3D reconstruction of microglial cell (Iba1, green) with ingested myelin (CypHer5E and AF555, white and red, yellow arrow) near un-ingested myelin (AF555, red, white arrow). Scale bar = 5 microns. c , Microgliosis, as assessed by % Iba1+ area at the injection site, was not altered by CD22 blockade (n=8, N.S. not significant, paired two-sided t-test). d , Representative images of myelin (red) overlaid with the myeloid marker Iba1 (green) at the injection site of IgG (left) or PBS (middle) treated hemispheres of the same aged brain, or an image of a stab wound control (not injected with myelin). Scale bar = 100 microns. e , Microgliosis, as assessed by % Iba1+ area at the injection site, was not altered by IgG compared to the stab wound control (n=2, N.S. not significant, paired two-sided t-test). f , Clearance of myelin debris in the IgG (black) or PBS (blue) treated hemispheres assessed 48 hours post-injection (n=4, N.S. not significant, paired two-sided t-test). g , Representative images of Iba1 (gray), a macrophage marker, and Tmem119 (magenta), a microglia-specific marker, at the injection site in IgG (left) or anti-CD22 (right) treated hemispheres of the same aged brain. Scale bar = 100 microns. h , Percent of Iba1+ phagocytes expressing Tmem119 at the injection site (n=4, N.S. not significant, paired two-sided t-test). i , Clearance of myelin debris in young (2.5 m.o.) WT (black) or CD22 −/− (green) mice was assessed 48 hours after injection (n=4, N.S. not significant, two-sided t-test; mean +/− s.e.m.). j , Representative images of total Aβ (white,), Thioflavin S+ fibrillar Aβ (green), and Iba1 (red) in transgenic mice expressing human APP with Swedish and London familial AD mutations (left) or WT mice injected with Aβ oligomers 48 hours prior to analysis (right). k , Representative images of Aβ (red, left column) and Aβ overlaid with the myeloid marker Iba1 (green, right column) at the injection site (+/− 2mm lateral, 0mm A-P, -1.5mm D-V relative to bregma) of IgG (top row) or anti-CD22 (bottom row) treated hemispheres of the same aged brain. Scale bar = 100 microns. l , Microgliosis, as assessed by % Iba1+ area at the Aβ oligomer injection site, was not altered by CD22 blockade (n=8, N.S. not significant, paired two-sided t-test). m , Representative images of α-synuclein and Iba1 at the injection site in IgG and anti-CD22 treated mice. Scale bar = 100 microns. n , Clearance of α-synuclein fibrils in the IgG (black) or anti-CD22 (green) treated hemispheres assessed 48 hours post-injection (n=7, * P <0.05, paired two-sided t-test). o , Microgliosis, as assessed by % Iba1+ area at the α-synuclein fibril injection site, was not altered by CD22 blockade (n=7, N.S. not significant, paired two-sided t-test). All data were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Labeling, Staining, Injection, Marker, Control, Expressing, Transgenic Assay

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Schematic of long-term CNS-targeted CD22 blockade. b , c , Correlation of gene fold changes between microglia from anti-CD22 (n=7) and IgG (n=7) treated mice and between microglia from aged (22 m.o., n=3) and young (3 m.o., n=3) mice ( b ) ( R = −0.47, P =1.7e-12) or between microglia from 5xFAD (8.5 m.o., n=5) and age-matched WT (8.5 m.o., n=5) mice ( c ) ( R = −0.27, P =8.5e-6, blue line, linear regression, Spearman correlation). Genes differentially expressed ( P <0.05, two-sided t-test) in either dataset (union) are shown. d , Hierarchical clustering of gene fold changes between microglia from anti-CD22 (n=7) and IgG (n=7) treated mice, between microglia from aged (22 m.o., n=3) and young (3 m.o., n=3) mice , and between microglia from 5xFAD (8.5 m.o., n=5) and age-matched WT (8.5 m.o., n=5) mice . Genes differentially expressed ( P -value<0.05, two-tailed t-test) in all three datasets (intersection) are shown. e , f , g , h , Percentage of time spent in the novel arm of the forced alternation Y-maze ( e, g ) or displaying freezing behavior in a contextual fear conditioning test ( f, h ) for aged (18 m.o.) WT and CD22 −/− mice ( e , f , n=6) or aged (18 m.o.) WT mice treated with IgG or anti-CD22 ( g , h , independent cohorts of mice in g (n=7 IgG, 9 anti-CD22) and h (n=7 for both groups)) (** P <0.005, * P <0.05, two-sided t-test; mean +/− s.e.m.). i , Representative images of dentate gyri stained for Prox1 (white) and c-Fos (red) from IgG and anti-CD22 treated mice. White arrows indicate cFos+Prox1+ active granule neurons. Scale bar = 100 microns. j , Total number of Prox1+c-Fos+ neurons in the dentate gyrus quantified over 5 tissue sections (n=7, * P <0.05, two-sided t-test; mean +/− s.e.m.). Data in a , g , h , i , j were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Two Tailed Test, Staining

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Representative images of CD19+ B-cells (red, top row), DAPI (blue, middle row), and merged (bottom row) in the spleen (left, positive control) and hippocampus (right) of a mouse treated with anti-CD22 via intracerebroventricular osmotic pump. b , Concentration of trans-cyclooctene-labeled anti-CD22 in the plasma 7 days after administration of 200 μg anti-CD22 via IP injection (n=1) or intracerebroventricular osmotic pump infusion (n=4), assessed by in-gel fluorescence and quantification based on a standard curve (mean +/− s.e.m). For raw source image, see . c , Representative images of coronal brain sections of untreated (left column) and IgG treated (right column) mice. IgG was labeled with an AlexaFluor647 NHS-ester (top row, white) to assess antibody distribution throughout the brain (bottom row, DAPI, blue). In addition to the para-ventricular areas, antibodies penetrated the thalamus and hippocampus. d , Flow cytometry analysis of AF647-labeled antibody on microglia isolated from untreated (black), IgG (red), or anti-CD22 (blue) infused mice. Microglia from anti-CD22 treated mice display elevated AF647 signal, indicative of antibody target engagement. Data in a, c, d were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Positive Control, Concentration Assay, Labeling, Clinical Proteomics, Injection, Fluorescence, Flow Cytometry, Isolation, Drug discovery

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Venn diagram showing the lack of any intersection among 315 genes differentially expressed between IgG (n=7) and anti-CD22 (n=7) treated microglia and 40 genes differentially expressed between untreated (n=2) and IgG (n=7) treated microglia at an FDR cutoff of 10% (Benjamini-Hochberg method). b , Hierarchical clustering of normalized read counts from IgG and anti-CD22 treated microglia, normalized by row mean. The top-100 differentially expressed genes are shown (n=7). c , Enrichr gene-ontology analysis of genes upregulated (red) and downregulated (blue) by anti-CD22 treatment (Fisher’s exact test, Benjamini-Hochberg FDR). d , Gene set enrichment analysis (GSEA) showing normalized enrichment score for microglia genes modulated by anti-CD22 treatment within the gene signature for: aging microglia (this study), disease-associate microglia (DAM, Keren-Shaul, et al. 2017), microglial neurodegenerative phenotype (MGnD, Krasemann, et al. 2017), and microglia from lipopolysaccharide treated mice (LPS, Bennett, et al. 2016) (*FDR<0.05). e , f , g , h , GSEA showing enrichment distribution for microglia genes modulated by anti-CD22 treatment within the gene signature for aging microglia (e), DAM (f), MGnD (g), and LPS-activated microglia (h).

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques:

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Western blot for Sall1 and α-Tubulin (loading control) in whole hippocampus lysates from IgG and anti-CD22 treated mice. For raw source image, see . b , Quantification of (a) showing upregulation of Sall1 protein in anti-CD22 hippocampi (n=3, * P <0.05, two-sided t-test, mean +/− s.e.m.). c , Protein concentration of CCL3 in the supernatant of acutely isolated aged microglia treated for 8 hours with IgG or anti-CD22 in the absence or presence of Aβ oligomers (n=4, N.S. not significant, *P<0.05, ANOVA with Sidak’s multiple hypothesis correction, mean +/− s.e.m.). d , Representative images of p-CREB expression (red) in the dentate gyrus of IgG (left) and anti-CD22 (right) treated mice. e , Quantification of p-CREB mean intensity in the dentate gyrus of IgG (black) and anti-CD22 (green) treated mice (n=7, two-sided t-test, mean +/− s.e.m.). f , Quantification of total doublecortin-positive cells in three equally-spaced dentate gyrus sections of IgG (black) and anti-CD22 (green) treated mice (n=3, N.S. not significant, two-sided t-test, mean + s.e.m.). g , h , i , Quantification of C1q (g), synaptophysin (h, pre-synaptic marker), and PSD-95 (i, post-synaptic marker) mean intensity in the hippocampus of IgG (black) and anti-CD22 (green) treated mice (n=3, N.S. not significant, two-sided t-test, mean + s.e.m.). All data were replicated in at least 2 independent experiments.

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Western Blot, Control, Protein Concentration, Isolation, Expressing, Marker

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Working memory and exploratory behavior in aged (18 m.o.) mice treated with IgG (black) or anti-CD22 (green) via IP injection weekly for one month as assessed by % of time spent in the novel arm in a forced alternation Y-maze test (n=6, N.S. not significant, two-sided t-test, mean +/− s.e.m.). b , Contextual memory aged (18 m.o.) mice treated with IgG (black) or anti-CD22 (green) via IP injection weekly for one month as assessed by % of time displaying freezing behavior in a contextual fear conditioning test (n=6, N.S. not significant, two-sided t-test, mean +/− s.e.m.).

Article Snippet: Cells were treated with rat anti-mouse CD16/CD32 Fc Blocker (2.4G2, BD) then incubated with the following antibodies at 1ug/mL: mouse IgG1 isotype control antibody (MOPC21, BioXCell), mouse anti-CD22 (Cy34, BioXCell) and rat anti-CD22 (OX-96, Bio-Rad).

Techniques: Injection