Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) MS4A4A and MS4A6A were detected by Western from non-targeted control (NTC), MS4A4A KO and MS4A6A KO macrophages. Representative image of n=3 donors. (B) Surface and (C) soluble TREM2 were measured from NTC, MS4A4A KO and MS4A6A KO macrophages. Representative of n=4 donors. (D) Surface and (E) soluble TREM2 levels in macrophages (n=3 donors) treated with V312, TD1 or isotype control for 48 hours. (F) MS4A4A4, MS4A6A and TREM2 levels in macrophages treated with 1 ug/mL of V312, TD1 or isotype control antibodies for 4, 24 or 48 hours. Representative blot and representative quantification from n=4 donors. (G) Surface TREM2 in a WT or MS4A4A KO iMG line (Lupe). Representative of two experiments. (H) Soluble TREM2 from six iMG lines treated with 1 μg/mL TD1 or isotype control for 48 hours. (I, J) Solub;e TREM2 was measured from co-cultures of cerebral organoids and microglia from MSN38 and MSN9 lines treated with 1 µg/mL TD1 (I), V312 (J) or isotype control for 6, 24 or 48 hours. Data shows mean + SEM of at least 6 organoids per cell line. p values were calculated using T-tests.

Article Snippet: Plates were washed three times, then 50 mL of 100 ng/mL goat anti-human biotinylated TREM2 detection antibody (R&D Systems #BAF1828) was added.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) MS4A6A was immunoblotted after MS4A4A immunoprecipitation. Representative image of n=3 macrophage donors. (B,C) TREM2 and DAP12 were immunoblotted from MS4A4A (B) or MS4A6A (C) immunoprecipitates. Representative image of n=3 donors. (D) Untransfected (UT) HEK293 and cells transfected with DAP12-GFP, MS4A6A-HA, or both were immunoblotted for DAP12 and MS4A6A after MS4A6A immunopreciptation. Whole cell lysate (10% immunoprecipitation input) was loaded as a control. Representative of n=2 experiments. (E) Macrophages were treated with 1 ug/mL TD1 or isotype control for 4 hours, then TREM2 and DAP12 were immunoblotted from DAP12 immunoprecipitates. n=3 donors. (F). Proximity ligation assay detecting the association between DAP12 and TREM2 after 48-hour treatment with 1 µg/mL TD1 or isotype control.. (G) MDL1 levels were measured on FACS after treatment with 1 µg/mL TD1 or isotype control for 48 hour. n=6 donors. (H) Macrophages were primed with 1 µg/mL TD1 or isotype control for 48 hours, then cells were treated with a TREM2 activator or a control for 5 minutes. Phosphorylated Syk was measured from the lysates. n=6 donors (I) Model of MS4A4A and MS4A4A-degrading antibodies mechanism of action on DAP12-associated receptors and effects of downstream functions. (D-G) Data in graphs show mean±SEM. p values were calculated by t-test (D,E) or one-way ANOVA (F, G).

Article Snippet: Plates were washed three times, then 50 mL of 100 ng/mL goat anti-human biotinylated TREM2 detection antibody (R&D Systems #BAF1828) was added.

Techniques: Immunoprecipitation, Transfection, Control, Proximity Ligation Assay

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) Experimental design of the study (V312 n=10 or isotype control n=8. (B) UMAP plot visualizing 11,167 high quality single human microglial cells sorted from mouse brain (CD11b + hCD45 + ) after treatment with V312 or isotype control. (C) Volcano plot of differentially expressed genes average (log2Fold Change ≥ 0.2 and adjusted p-value ≤ 0.05) where each dot represents a cell. Labelled genes are differentially expressed and part of the top 30 marker genes of a cluster ranked on average log2Fold Change; (D) Comparison of V312 treatment with TREM2 KO dataset (E) Comparison of our differentially expressed genes with selected genes from aducanumab-treated animals in showing enhanced MHC (HLA) response male mice.(F,G) Representative images (scale bar 50 μm) stained for P2RY12 (HM microglia) and X-34 (Aβ plaques) and either HLA-DR/DQ (HLA microglia) (F) or LAMP2 (G) as proxy for phagocytosis. Quantification of HLA (F) and LAMP2 (G) markers at the site of Aβ plaques was performed with modified Sholl analysis. n=8 isotype control-treated mice and n=10 V312-treated mice. p values were calculated with one-tailed t-test.

Article Snippet: Plates were washed three times, then 50 mL of 100 ng/mL goat anti-human biotinylated TREM2 detection antibody (R&D Systems #BAF1828) was added.

Techniques: Control, Marker, Comparison, Staining, Modification, One-tailed Test

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) Study 1 design; (B) PK parameters (C) sTREM2 levels at the indicated time points in the serum (top) and CSF (bottom). (D) TREM2 protein levels in the frontal cortex and hippocampus 48 hours after the final dose of TD1. (E) Effect of TD1 on microglia gene expression in cortex at takedown. (F) Effect of TD1 on CSF proteins 24 and 48 hours after the first dose of antibody. (G) OPN and (I) IL1RA levels in the CSF at the indicated time points. (H) OPN and (J) sCSF1R protein levels in the frontal cortex and hippocampus 48 hours after final dose of TD1 Data shows mean±SEM for n=5 animals/group. p values were calculated with T-tests (D, H, JI) or Wilcoxon rank-sum test. (E) or two-Way ANOVA (F).

Article Snippet: Plates were washed three times, then 50 mL of 100 ng/mL goat anti-human biotinylated TREM2 detection antibody (R&D Systems #BAF1828) was added.

Techniques: Expressing

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) MS4A4A and MS4A6A were detected by Western from non-targeted control (NTC), MS4A4A KO and MS4A6A KO macrophages. Representative image of n=3 donors. (B) Surface and (C) soluble TREM2 were measured from NTC, MS4A4A KO and MS4A6A KO macrophages. Representative of n=4 donors. (D) Surface and (E) soluble TREM2 levels in macrophages (n=3 donors) treated with V312, TD1 or isotype control for 48 hours. (F) MS4A4A4, MS4A6A and TREM2 levels in macrophages treated with 1 ug/mL of V312, TD1 or isotype control antibodies for 4, 24 or 48 hours. Representative blot and representative quantification from n=4 donors. (G) Surface TREM2 in a WT or MS4A4A KO iMG line (Lupe). Representative of two experiments. (H) Soluble TREM2 from six iMG lines treated with 1 μg/mL TD1 or isotype control for 48 hours. (I, J) Solub;e TREM2 was measured from co-cultures of cerebral organoids and microglia from MSN38 and MSN9 lines treated with 1 µg/mL TD1 (I), V312 (J) or isotype control for 6, 24 or 48 hours. Data shows mean + SEM of at least 6 organoids per cell line. p values were calculated using T-tests.

Article Snippet: Whole cell lysates or immunoprecipitation samples were separated using Any kDa Mini-PROTEAN® TGX Stain-Free™ gels (Bio-Rad # 4568123 and #4568125) and transferred to PVDF membrane using the Trans-Blot Turbo kit (Bio-Rad #1704156).The membrane was blocked with 5% milk in 1X TBS with 0.1% Tween for 1 hour at room temperature and then incubated with primary antibodies against MS4A4A (1:300 dilution, Alector, Clone Gigi), MS4A6A (1:1,000 dilution, Abcam #ab189983), DAP12 (1:1,000 dilution, Santa Cruz Biotechnology #sc-133174 or Cell signaling Technology #12492) or TREM2 (1:1,000 dilution, R&D Systems #BAF1828) overnight at 4°C.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) MS4A6A was immunoblotted after MS4A4A immunoprecipitation. Representative image of n=3 macrophage donors. (B,C) TREM2 and DAP12 were immunoblotted from MS4A4A (B) or MS4A6A (C) immunoprecipitates. Representative image of n=3 donors. (D) Untransfected (UT) HEK293 and cells transfected with DAP12-GFP, MS4A6A-HA, or both were immunoblotted for DAP12 and MS4A6A after MS4A6A immunopreciptation. Whole cell lysate (10% immunoprecipitation input) was loaded as a control. Representative of n=2 experiments. (E) Macrophages were treated with 1 ug/mL TD1 or isotype control for 4 hours, then TREM2 and DAP12 were immunoblotted from DAP12 immunoprecipitates. n=3 donors. (F). Proximity ligation assay detecting the association between DAP12 and TREM2 after 48-hour treatment with 1 µg/mL TD1 or isotype control.. (G) MDL1 levels were measured on FACS after treatment with 1 µg/mL TD1 or isotype control for 48 hour. n=6 donors. (H) Macrophages were primed with 1 µg/mL TD1 or isotype control for 48 hours, then cells were treated with a TREM2 activator or a control for 5 minutes. Phosphorylated Syk was measured from the lysates. n=6 donors (I) Model of MS4A4A and MS4A4A-degrading antibodies mechanism of action on DAP12-associated receptors and effects of downstream functions. (D-G) Data in graphs show mean±SEM. p values were calculated by t-test (D,E) or one-way ANOVA (F, G).

Article Snippet: Whole cell lysates or immunoprecipitation samples were separated using Any kDa Mini-PROTEAN® TGX Stain-Free™ gels (Bio-Rad # 4568123 and #4568125) and transferred to PVDF membrane using the Trans-Blot Turbo kit (Bio-Rad #1704156).The membrane was blocked with 5% milk in 1X TBS with 0.1% Tween for 1 hour at room temperature and then incubated with primary antibodies against MS4A4A (1:300 dilution, Alector, Clone Gigi), MS4A6A (1:1,000 dilution, Abcam #ab189983), DAP12 (1:1,000 dilution, Santa Cruz Biotechnology #sc-133174 or Cell signaling Technology #12492) or TREM2 (1:1,000 dilution, R&D Systems #BAF1828) overnight at 4°C.

Techniques: Immunoprecipitation, Transfection, Control, Proximity Ligation Assay

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) Experimental design of the study (V312 n=10 or isotype control n=8. (B) UMAP plot visualizing 11,167 high quality single human microglial cells sorted from mouse brain (CD11b + hCD45 + ) after treatment with V312 or isotype control. (C) Volcano plot of differentially expressed genes average (log2Fold Change ≥ 0.2 and adjusted p-value ≤ 0.05) where each dot represents a cell. Labelled genes are differentially expressed and part of the top 30 marker genes of a cluster ranked on average log2Fold Change; (D) Comparison of V312 treatment with TREM2 KO dataset (E) Comparison of our differentially expressed genes with selected genes from aducanumab-treated animals in showing enhanced MHC (HLA) response male mice.(F,G) Representative images (scale bar 50 μm) stained for P2RY12 (HM microglia) and X-34 (Aβ plaques) and either HLA-DR/DQ (HLA microglia) (F) or LAMP2 (G) as proxy for phagocytosis. Quantification of HLA (F) and LAMP2 (G) markers at the site of Aβ plaques was performed with modified Sholl analysis. n=8 isotype control-treated mice and n=10 V312-treated mice. p values were calculated with one-tailed t-test.

Article Snippet: Whole cell lysates or immunoprecipitation samples were separated using Any kDa Mini-PROTEAN® TGX Stain-Free™ gels (Bio-Rad # 4568123 and #4568125) and transferred to PVDF membrane using the Trans-Blot Turbo kit (Bio-Rad #1704156).The membrane was blocked with 5% milk in 1X TBS with 0.1% Tween for 1 hour at room temperature and then incubated with primary antibodies against MS4A4A (1:300 dilution, Alector, Clone Gigi), MS4A6A (1:1,000 dilution, Abcam #ab189983), DAP12 (1:1,000 dilution, Santa Cruz Biotechnology #sc-133174 or Cell signaling Technology #12492) or TREM2 (1:1,000 dilution, R&D Systems #BAF1828) overnight at 4°C.

Techniques: Control, Marker, Comparison, Staining, Modification, One-tailed Test

Journal: bioRxiv

Article Title: The Alzheimer’s Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

doi: 10.1101/2024.11.23.625001

Figure Lengend Snippet: (A) Study 1 design; (B) PK parameters (C) sTREM2 levels at the indicated time points in the serum (top) and CSF (bottom). (D) TREM2 protein levels in the frontal cortex and hippocampus 48 hours after the final dose of TD1. (E) Effect of TD1 on microglia gene expression in cortex at takedown. (F) Effect of TD1 on CSF proteins 24 and 48 hours after the first dose of antibody. (G) OPN and (I) IL1RA levels in the CSF at the indicated time points. (H) OPN and (J) sCSF1R protein levels in the frontal cortex and hippocampus 48 hours after final dose of TD1 Data shows mean±SEM for n=5 animals/group. p values were calculated with T-tests (D, H, JI) or Wilcoxon rank-sum test. (E) or two-Way ANOVA (F).

Article Snippet: Whole cell lysates or immunoprecipitation samples were separated using Any kDa Mini-PROTEAN® TGX Stain-Free™ gels (Bio-Rad # 4568123 and #4568125) and transferred to PVDF membrane using the Trans-Blot Turbo kit (Bio-Rad #1704156).The membrane was blocked with 5% milk in 1X TBS with 0.1% Tween for 1 hour at room temperature and then incubated with primary antibodies against MS4A4A (1:300 dilution, Alector, Clone Gigi), MS4A6A (1:1,000 dilution, Abcam #ab189983), DAP12 (1:1,000 dilution, Santa Cruz Biotechnology #sc-133174 or Cell signaling Technology #12492) or TREM2 (1:1,000 dilution, R&D Systems #BAF1828) overnight at 4°C.

Techniques: Expressing