Journal: eLife

Article Title: A non-mosaic transchromosomic mouse model of Down syndrome carrying the long arm of human chromosome 21

doi: 10.7554/eLife.56223

Figure Lengend Snippet: ( A–C ) APP protein levels and amyloid plaques in brains of 15–24 month-old TcMAC21 and Eu (n = 6 per group). ( A ) western blot of total APP in hippocampus and cortex. ( B ) ELISA of total Aβ40 and Aβ42 levels in hippocampus and cortex. ( C ) amyloid plaques visualized by immunostaining with β-amyloid antibody 6E10; APPswe/PS1∆E9 mouse is a positive control for plaque formation, scale bar (1 mm). ( D ) CFU level of GM and GEMM in TcMAC21 spleen and bone marrow (n = 6 per group). GM, granulocyte/macrophage; GEMM, granulocyte/erythroid/monocyte/megakaryocyte. ( E–G ) Chromatid and chromosome aberrations in bone marrow cells after X-ray irradiation (n = 3 per group). ( E ) Chromatid aberration. ( F ) Chromosome aberration. ( G ) Chromatid and/or chromosome exchange. chtg (chromatid gap); chtb (chromatid break); chrg (chromosome gap); chrb (chromosome break); ace (acentric fragment); chte (chromatid exchange); chre (chromosome exchange); mar (marker chromosome, including dicentric chromosome, ring chromosome, robertsonian translocation, and other abnormal size of chromosome). All data are analyzed by two-tailed t-test and expressed as mean ± SEM. Figure 5—source data 1. Peripheral blood analyses in TcMAC21 and Eu.

Article Snippet: Commercial assay or kit , Human/rat β amyloid (Aβ40) , Wako , 294–64701 , .

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining, Positive Control, Irradiation, Marker, Translocation Assay, Two Tailed Test

Journal: eLife

Article Title: A non-mosaic transchromosomic mouse model of Down syndrome carrying the long arm of human chromosome 21

doi: 10.7554/eLife.56223

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Human/rat β amyloid (Aβ40) , Wako , 294–64701 , .

Techniques: Expressing, Isolation, Transfection, Software

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a ) Treatment scheme. App NL-G-F mice (APP) were fed PLX3397 from 1 month until analysis at 4 months of age (1-4M PLX) or a control diet (4M ctrl). ( b ) Representative confocal images of amyloid plaques in the brain, stained with X34 (fibrillar plaques) or 82E1 (total Aβ). ( c ) Quantification of amyloid plaques in the whole brain (%X34 area: unpaired t-test, n(ctrl)=12, n(PLX)=12, p<0.0001; total number of X34 plaques: Mann-Whitney test, n(ctrl)=12, n(PLX)=12), p<0.0001; average size X34 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=12, p=0.5098; %82E1 area: unpaired t-test, n(ctrl)=11, n(PLX)=12, p<0.0001; total number of 82E1 plaques: unpaired t-test with Welch’s correction, n(ctrl)=11, n(PLX)=12), p=0.012; average size 82E1 plaques: unpaired t-test, n(ctrl)=11, n(PLX)=12, p= 0.1225). ( d ) Higher magnification images of amyloid plaques and microglia in control and PLX3397-treated mice. ( e ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=12), p=0.0098; sol. Aβ40: Mann-Whitney test, n(ctrl)=12, n(PLX)=12), p=0.1725; sol. Aβ42: unpaired t-test, n(ctrl)=12, n(PLX)=12), p= 0.2571; insol. Aβ38: unpaired t-test with Welch’s correction, n(ctrl)=11, n(PLX)=12, p<0.0001; insol. Aβ40: unpaired t-test, n(ctrl)=11, n(PLX)=12, p<0.0001; insol. Aβ42: unpaired t-test, n(ctrl)=11, n(PLX)=12, p<0.0001). ( g ) Quantifications of dystrophic neurites around amyloid plaques (%LAMP1 area: unpaired t-test, n(ctrl)=12, n(PLX)=12), p<0.0001; total number of LAMP1 dystrophic neurites: unpaired t-test, n(ctrl)=12, n(PLX)=12), p=0.0317; average size of LAMP1 dystrophic neurites: unpaired t-test, n(ctrl)=12, n(PLX)=12), p=0.1321). ( f ) Representative images of LAMP1 + dystrophic neurites in the brain. White dots represent female mice and black dots represent male mice. Scale bars 500 μm (b, g), 30 μm (d, h). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, Staining, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a ) Representative images and microglia depletion efficiency of early PLX3397-treatment in App NL-G-F mice (APP) (unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=11, p<0.0001). ( b ) Representative images and quantification of microglia depletion efficiency of late PLX3397-treatment in App NL-G-F mice (unpaired t-test, n(ctrl)=12, n(PLX)=14, p<0.0001). ( c ) Linear regression of 82E1 + plaque staining and LAMP1 + dystrophic neurite staining across early and late microglia depletion cohorts (R 2 =0.7706, p<0.0001). ( d ) Treatment scheme for sustained microglia depletion. App NL-G-F mice (APP) were fed PLX3397 from 1 month until 7 months of age (1-7M PLX) or control diet (7M ctrl). ( e ) Representative images of amyloid plaques in the brain, stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( f ) Image quantifications of X34 + and 82E1 + plaques in the whole brain (%X34 area: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=13, p<0.0001; total number of X34 plaques: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=13, p<0.0001; average size X34 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=13, p=0.0023; %82E1 area: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=13, p=0.0005; total number of 82E1 plaques: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=13, p<0.0001; average size 82E1 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=13, p<0.0001). ( g ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=13, p=0.1392; sol. Aβ40: unpaired t-test, n(ctrl)=12, n(PLX)=13, p=0.4911; sol. Aβ42: unpaired t-test, n(ctrl)=12, n(PLX)=13, p=0.0098; insol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=13, p<0.0001; insol. Aβ40: unpaired t-test, n(ctrl)=12, n(PLX)=13, p<0.0001; insol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=13, p<0.0001). ( h ) Representative images and quantification of microglia depletion efficiency after sustained PLX3397 treatment (unpaired t-test, n(ctrl)=12, n(PLX)=13, p<0.0001). White dots represent female mice and black dots represent male mice. Scale bars 100 μm (a, b, h) and 500 μm (e). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a ) Treatment scheme. App NL-G-F mice (APP) were fed PLX3397 from 3 months until analysis at 7 months of age (3-7M PLX) or control diet (7M ctrl). ( b ) Amyloid plaques in the brain, stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( c ) Image quantifications of amyloid plaques in the whole brain (%X34 area: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.4926; total number of X34 plaques: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=14, p=0.1575; average size X34 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0034; %82E1 area: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0027; total number of 82E1 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=14, p= 0.4042; average size 82E1 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=14, p<0.0001). ( d ) Higher magnification images of amyloid plaques and microglia in control and PLX3397-treated mice. ( e ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.7964; sol. Aβ40: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.5302; sol. Aβ42: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0638; insol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=14, p<0.0001; insol. Aβ40: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0887; insol. Aβ38: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.1904). ( f ) Quantifications of dystrophic neurites around amyloid plaques (%LAMP1 area: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0172; total number of LAMP1 dystrophic neurites: unpaired t-test, n(ctrl)=12, n(PLX)=14, p=0.0003; average size of LAMP1 dystrophic neurites: unpaired t-test, n(ctrl)=12, n(PLX)=14, p<0.0001). ( g ) LAMP1 + dystrophic neurites in the brain. White dots represent female mice and black dots represent male mice. Scale bars 500 μm (b, g), 30 μm (d, h). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a-c ) Immunodeficient App NL-G-F /Rag2 -/- mice (APP/R2) were fed PLX3397 from 1 month until analysis at 4 months of age (1-4M PLX) or control diet (4M ctrl). ( a ) Quantifications of amyloid plaques in the whole brain of immunodeficient App NL-G-F /Rag2 -/- mice (%X34 area: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=9, p<0.0001; total number of X34 plaques: Mann-Whitney test, n(ctrl)=12, n(PLX)=9, p<0.0001; average size X34 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=9, p<0.0001; %82E1 area: unpaired t-test, n(ctrl)=12, n(PLX)=9, p<0.0001; total number of 82E1 plaques: unpaired t-test with Welch’s correction, n(ctrl)=12, n(PLX)=9, p<0.0001; average size 82E1 plaques: unpaired t-test, n(ctrl)=12, n(PLX)=9,- p=0.0926). ( b ) Representative images of amyloid plaques in the brain, stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( c ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=13, n(PLX)=11, p= 0.3824; sol. Aβ40: unpaired t-test, n(ctrl)=13, n(PLX)=11, p= 0.0558; sol. Aβ42: unpaired t-test, n(ctrl)=13, n(PLX)=11, p= 0.0029; insol. Aβ38: unpaired t-test, n(ctrl)=13, n(PLX)=11, p<0.0001; insol. Aβ40: Mann-Whitney test, n(ctrl)=13, n(PLX)=11, p<0.0001; insol. Aβ38: unpaired t-test, n(ctrl)=13, n(PLX)=11, p<0.0001). ( d-f ) Immunodeficient App NL-G-F /Rag2 -/- mice (APP/R2) were treated with PLX3397 from 3 months until analysis at 7 months of age (3-7M PLX) or control diet (7M ctrl). ( d ) Image quantifications of amyloid plaques in the whole brain (%X34 area: unpaired t-test, n(ctrl)=16, n(PLX)=13, p=0.0036; total number of X34 plaques: unpaired t-test with Welch’s correction, n(ctrl)=16, n(PLX)=13, p<0.0001; average size X34 plaques: unpaired t-test, n(ctrl)=16, n(PLX)=13, p<0.0001; %82E1 area: unpaired t-test, n(ctrl)=16, n(PLX)=13, p<0.0001; total number of 82E1 plaques: Mann-Whitney test, n(ctrl)=16, n(PLX)=13, p=0.0056; average size 82E1 plaques: unpaired t-test, n(ctrl)=16, n(PLX)=13, p<0.0001). ( e ) Representative images of amyloid plaques in the brain of immunodeficient App NL-G-F /Rag2 -/- mice, stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( f ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=18, n(PLX)=13, p=0.9646; sol. Aβ40: unpaired t-test with Welch’s correction, n(ctrl)=18, n(PLX)=13, p=0.4748; sol. Aβ42: unpaired t-test, n(ctrl)=18, n(PLX)=13, p0.1756; insol. Aβ38: unpaired t-test with Welch’s correction, n(ctrl)=18, n(PLX)=13, p=0.0006; insol. Aβ40: unpaired t-test, n(ctrl)=18, n(PLX)=13, p=0.2682; insol. Aβ38: unpaired t-test, n(ctrl)=17, n(PLX)=13, p=0.1715). White dots represent female mice and black dots represent male mice. Scale bars 500 μm (a, d). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, MANN-WHITNEY, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: (a) Microglia depletion efficiency of early PLX3397-treatment in App NL-G-F /Rag2 -/- mice (APP/R2) (unpaired t-test, n(ctrl)=12, n(PLX)=9, p<0.0001) . (b) Representative images and quantification of microglia depletion efficiency of late PLX3397-treatment in App NL-G-F /Rag2 -/- mice (APP/R2) (unpaired t-test, n(ctrl)=18, n(PLX)=13, p<0.0001). ( c ) Treatment scheme for sustained microglia depletion. App NL-G-F /Rag2 -/- mice (APP/R2) were fed PLX3397 from 1 month until 7 months of age (1-7M PLX) or control diet (7M ctrl). ( d ) Representative images of amyloid plaques in the brain, stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( e ) Image quantifications of X34 + and 82E1 + plaques in the whole brain (%X34 area: unpaired t-test, n(ctrl)=16, n(PLX)=13, p<0.0001; total number of X34 plaques: unpaired t-test with Welch’s correction, n(ctrl)=16, n(PLX)=13, p<0.0001; average size X34 plaques: unpaired t-test, n(ctrl)=16, n(PLX)=13, p=0.1188; %82E1 area: unpaired t-test, n(ctrl)=16, n(PLX)=13, p=0.0013; total number of 82E1 plaques: Mann-Whitney test, n(ctrl)=16, n(PLX)=13, p<0.0001; average size 82E1 plaques: unpaired t-test, n(ctrl)=16, n(PLX)=13, p<0.0001). ( f ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(ctrl)=18, n(PLX)=13, p=0.0146; sol. Aβ40: unpaired t-test, n(ctrl)=18, n(PLX)=13, p=0.8164; sol. Aβ42: Mann-Whitney test, n(ctrl)=18, n(PLX)=13, p=0.0073; insol. Aβ38: unpaired t-test with Welch’s correction, n(ctrl)=18, n(PLX)=12, p<0.0001; insol. Aβ40: unpaired t-test, n(ctrl)=18, n(PLX)=13, p<0.0001; insol. Aβ38: unpaired t-test, n(ctrl)=17, n(PLX)=12, p<0.0001). ( g ) Representative images and quantification of microglia depletion efficiency after sustained PLX3397 treatment (unpaired t-test, n(ctrl)=18, n(PLX)=13, p<0.0001). White dots represent female mice and black dots represent male mice. Scale bars 100 μm (a, b, g) and 500 μm (d). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, Staining, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a ) Overview and higher magnification images of amyloid plaques in the brain and microglia in 6-7-week-old control mice ( App NL-G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI , with mouse microglia) and FIRE mice ( App NL-G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI ; Csf1r ΔFIRE/ΔFIRE , no microglia). ( b ) Aβ ELISA of soluble and insoluble cortex extracts in 6-7-week-old control and FIRE mice (sol. Aβ38: unpaired t-test with Welch’s correction, n(ctrl)=10, n(FIRE)=11, p=0.1936; sol. Aβ40: unpaired t-test, n(ctrl)=10, n(FIRE)=12, p=0.0016; sol. Aβ42: unpaired t-test, n(ctrl)=10, n(FIRE)=12, p=0.0665; insol. Aβ38: unpaired t-test, n(ctrl)=9, n(FIRE)=12, p=0.017; insol. Aβ40: unpaired t-test, n(ctrl)=9, n(FIRE)=12, p=0.0107; insol. Aβ42: unpaired t-test with Welch’s correction, n(ctrl)=9, n(FIRE)=12, p<0.0001). ( c ) Representative images of amyloid plaques in the brains of 3-month-old control mice, FIRE mice, and FIRE mice xenografted with human microglia (FIRE + hMG), stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( d ) Quantifications of amyloid plaques in the whole brain (%X34 area: Welch’s ANOVA test, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=7, p<0.0001; total number of X34 plaques: Kruskal-Wallis test, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=7, p= 0.0001; average size X34 plaques: one-way ANOVA, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=7, p<0.0001; %82E1 area: Welch’s ANOVA test, n(ctrl)=10, n(FIRE)=12, n(FIRE +hMG)=7, p<0.0001; total number of 82E1 plaques: Kruskal-Wallis test, n(ctrl)=10, n(FIRE)=12, n(FIRE +hMG)=7, p<0.0001; average size 82E1 plaques: Welch’s ANOVA test, n(ctrl)=10, n(FIRE)=12, n(FIRE +hMG)=7, p<0.0001). ( e ) Aβ ELISA of soluble and insoluble cortex extracts (sol. Aβ38: one-way ANOVA, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=8, p=0.0237; sol. Aβ40: one-way ANOVA, n(ctrl)=10, n(FIRE)=12, n(FIRE +hMG)=8, p=0.1868; sol. Aβ42: one-way ANOVA, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=8, p=0.0125; insol. Aβ38: one-way ANOVA, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=8, p<0.0001; insol. Aβ40: one-way ANOVA, n(ctrl)=11, n(FIRE)=11, n(FIRE +hMG)=8, p<0.0001; insol. Aβ38: one-way ANOVA, n(ctrl)=11, n(FIRE)=12, n(FIRE +hMG)=8, p<0.0001). White dots represent female mice and black dots represent male mice. Scale bars 500 μm (a,c) and 30 μm (a). All data are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: ( a ) Images of X34 + and 82E1 + amyloid plaques in the whole brain of control mice ( App NL- G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI , with mouse microglia), FIRE mice ( App NL-G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI ; Csf1r ΔFIRE/ΔFIRE , no microglia), and xenografted FIRE mice (FIRE + hMG, human microglia). ( b ) Image quantifications of amyloid plaques in the whole brain (%X34 area: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; total number of X34 plaques: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE+hMG)=8, p<0.0001; average size X34 plaques: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; %82E1 area: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; total number of 82E1 plaques: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; average size 82E1 plaques: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.0594). ( c ) ELISA of soluble and insoluble Aβ levels in the cortex (sol. Aβ38: Kruskal-Wallis test, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.6434; sol. Aβ40: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.6971; sol. Aβ42:m one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.2007; insol. Aβ38: Kruskal-Wallis test, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; insol. Aβ40: Kruskal-Wallis test, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001; insol. Aβ38: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p<0.0001). ( d ) Images of LAMP1 + dystrophic neurites in the whole brain. ( e ) Image quantifications LAMP1 + dystrophic neurites in the whole brain (%LAMP1 area: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.0118; total number of LAMP1 dystrophic neurites: one-way ANOVA, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.2678; average size of LAMP1 dystrophic neurites: Kruskal-Wallis test, n(ctrl)=10, n(FIRE)=10, n(FIRE +hMG)=8, p=0.8563). White dots represent female mice and black dots represent male mice. Scale bars 500 μm (a,d). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: FIRE mice ( App NL-G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI ; Csf1r ΔFIRE/ΔFIRE ) were xenotransplanted with human WT microglia (WT) and human microglia harboring the Trem2 R47H/R47H (R47H) risk gene at P4 and analyzed at 6-7 weeks of age. ( a ) Overview images of amyloid plaques in the cortex of WT and R47H microglia grafted mice. ( b ) Higher magnification images on amyloid plaques, surrounded by human microglia. Early amyloid plaques do not yet show LAMP1 + dystrophic neurites. ( c ) ELISA of soluble and insoluble Aβ extracts of the cortex (sol. Aβ38: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.6948; sol. Aβ40: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.5737; sol. Aβ42: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.7904; insol. Aβ38: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.5686; insol. Aβ40: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.7534; insol. Aβ38: unpaired t-test, n(WT)=4, n(R47H)=13, p=0.7714). White dots represent female mice and black dots represent male mice. Scale bars 500 μm (a) and 30µm (b). All data is presented as mean ± SD.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Microglia initially seed and later reshape amyloid plaques in Alzheimer’s disease

doi: 10.1101/2024.08.06.606783

Figure Lengend Snippet: FIRE mice ( App NL-G-F ; Rag2 −/− ; IL2rg −/− ; hCSF1 KI ; Csf1r ΔFIRE/ΔFIRE ) were xenotransplanted with human WT microglia (TREM2 WT) and human microglia harboring the TREM2 R47H/R47H risk gene (TREM2 R47H) at P4 and analyzed at 3 months of age. ( a ) Image quantification of X34 + and 82E1 + amyloid plaques in the whole brain (%X34 area: Mann-Whitney test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.6282; total number of X34 plaques: Mann-Whitney test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.014; average size X34 plaques: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.0072; %82E1 area: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.1734; total number of 82E1 plaques: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.2909; average size 82E1 plaques: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.0201). ( b ) Representative images of amyloid plaques in the brain stained with X34 (fibrillar plaques) and 82E1 (total Aβ). ( c ) ELISA of amyloid levels in soluble and insoluble cortex extracts (sol. Aβ38: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.2712; sol. Aβ40: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=6, p=0.434; sol. Aβ42: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.9832; insol. Aβ38: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.1897; insol. Aβ40: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p= 0.4699; insol. Aβ38: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p= 0.0295). ( d ) Zoom in on plaques, dystrophic neurites, and human microglia staining in 3-month-old xenografted FIRE mice. ( e ) Quantification of LAMP1 + dystrophic neurites in the whole brain (%LAMP1 area: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.0002; total number of LAMP1 dystrophic neurites: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.2023; average size of LAMP1 dystrophic neurites: unpaired t-test, n(TREM2 WT)=6, n(TREM2 R47H)=7, p=0.2574). ( f ) Representative images of LAMP1 + dystrophic neurites in the whole brain. White dots represent female mice and black dots represent male mice. Scale bars 500 μm (b, f), 30 μm (d). All data is presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Aβ38, Aβ40, and Aβ42 levels in the soluble and insoluble brain extracts were quantified by Meso Scale Discovery (MSD).

Techniques: MANN-WHITNEY, Staining, Enzyme-linked Immunosorbent Assay

Journal: Fluids and Barriers of the CNS

Article Title: Amyloid-beta transporter expression at the blood-CSF barrier is age-dependent

doi: 10.1186/2045-8118-8-21

Figure Lengend Snippet: Aβ42 concentration in the CP epithelium with age . (A) Semi-quantitative IHC for Aβ42 deposition. Mean staining intensity in grayscale units (GU) was significantly decreased ( p < 0.05) for the age groups, n = 5 per age group. (B) IHC of Aβ42 at 3 mo (left), 20 mo (center), and 36 mo (right) old rats. Staining is granular and primarily cytosolic (asterisks), though also found along the apical membrane (arrows).

Article Snippet: After washing in 0.05 M Tris-buffered saline with 0.05% Tween-20 (TBST), pH 7.6, sections were incubated overnight at 4°C with their appropriate primary antibody: rabbit polyclonal to Aβ40 (Linaris, Wertheim-Bettingen, Germany; Cat. # PAK6012, diluted 1:100), rabbit polyclonal to Aβ42 (Linaris, # PAK6023, diluted 1:200), rabbit polyclonal to LRP-1 (Orbigen, San Diego, CA, USA; # PAB-10774, diluted 1:1000), rabbit polyclonal to LRP-2 (Orbigen, # PAB-10775, diluted 1:2000), mouse monoclonal to P-glycoprotein (Abcam, Cambridge, MA, USA; # ab3364, diluted 1:100), or goat polyclonal to RAGE (Abcam, # ab7764, diluted 1:400).

Techniques: Concentration Assay, Staining, Membrane

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: 1. Erythrocyte clearance of Aβ

doi: 10.1016/j.jalz.2017.03.010

Figure Lengend Snippet: A1) Aggregated Aβ42 was incubated with NHS, then assayed by ELISA for production of C3a, a cleavage product generated from C3 following C3 activation. A significant dose-dependent response was obtained. Incubation of Aβ and serum with 10 mM EDTA, which blocks complement activation, abolished the response to Aβ and gave only background readings. A2) Aβ40 gave similar results. B) These findings were also extended to the terminal step in classical and alternative pathway activation, formation of C5b-9, the membrane attack complex, and its soluble form, sC5b-9. Significant dose-dependent activation was observed in all experiments.

Article Snippet: 2.04 Aβ preparation Human synthetic Aβ40 and Aβ42 (Bachem, Torrence, CA, or Genscript, Piscataway, NJ) were solubilized in 100% DMSO at 10 mg/ml, gradually diluted in ddH 2 O to 2 mg/ml, and then brought to a 1 mg/ml concentration in 0.1M Tris buffer (pH 7.4).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Generated, Activation Assay, Membrane

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: 1. Erythrocyte clearance of Aβ

doi: 10.1016/j.jalz.2017.03.010

Figure Lengend Snippet: Consistent with previous studies [8,9], plasma levels of Aβ40 spiked almost immediately after inoculation and rapidly declined over the next 15–20 minutes (top panel). Erythrocyte levels followed a nearly identical pattern and were significantly correlated with plasma levels at both doses of Aβ (bottom panel). Clearance from the erythrocyte pathway was rapid, also consistent with previous studies [19], and was capable of reducing the high dose of Aβ from nearly 10-fold normal levels to normal levels in 15–20 minutes (bottom panel).

Article Snippet: 2.04 Aβ preparation Human synthetic Aβ40 and Aβ42 (Bachem, Torrence, CA, or Genscript, Piscataway, NJ) were solubilized in 100% DMSO at 10 mg/ml, gradually diluted in ddH 2 O to 2 mg/ml, and then brought to a 1 mg/ml concentration in 0.1M Tris buffer (pH 7.4).

Techniques: Clinical Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Aβ40(42), IAPP, and synthetic IAPP mutants (left, short names); C-terminal amides and Cys2–Cys7 disulfide bridge not shown. Similar/identical residues between Aβ and IAPP are underlined (7). Previously identified “hot segments” of IAPP self-assembly and its hetero-assembly with Aβ are in boldface type (12), and introduced Ala substituents are shown in red.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: K d (app) values of interactions of IAPP(8–18), IAPP(22–28), and their Ala mutants with IAPP or Aβ40 as determined by fluorescence titration assays N-terminal fluorescein-labeled IAPP segments (5 n m ) were titrated with IAPP or Aβ40 (pH 7.4). K d (app) values were determined from one or three binding curves, as indicated; shown are the S.D. values from three binding curves. NB, no binding up to 2 μ m IAPP or Aβ40 (mutants in boldface type).

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Fluorescence, Titration, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Determination of Kd(app) values of interactions of IAPP and selected Ala mutants with Aβ40 by fluorescence spectroscopic titrations. Fluorescence emission spectra of N-terminal fluorescein-labeled IAPP or mutants (Fluos-peptide, 5 nm) alone and after titration with Aβ40 (molar ratios of Fluos-peptide/Aβ40 as indicated) are shown for the following peptides: IAPP (A), 8A (B), 4A (C), A15,23,26 (D), A15,23 (E), A23,26 (F), A23 (G), A26 (H), and A16 (I). In the insets, the binding curves are shown; data are means ± S.D. (error bars) from three binding curves. Calculated Kd(app) values are shown in Table 2. a.u., arbitrary units.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Fluorescence, Labeling, Titration, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: K d (app) values of interactions of IAPP and its Ala mutants with IAPP, Aβ40, and Aβ42 as determined by fluorescence spectroscopic titrations N α -amino-terminal fluorescein-labeled IAPP or IAPP mutants (5 n m ) were titrated with IAPP, Aβ40, or Aβ42 (pH 7.4). K d (app) values are from three binding curves; shown are S.D. values from three binding curves except for the values of IAPP-IAPP/-Aβ40 interactions, for which S.E. values are shown (see Footnote a ). NB, no binding at IAPP or Aβ40(42) concentrations ≤ 5 μ m . Mutants/values in boldface type indicate ≥19-fold weaker binding than IAPP toward IAPP and/or Aβ40 and/or Aβ42.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Factorial decrease of apparent binding affinity ( K d (mut) / K d (wt) ) of mutants to IAPP, Aβ40, and Aβ42 as compared with wild-type IAPP Apparent K d values determined by fluorescence spectroscopic titrations ( ): N α -terminal fluorescein-labeled IAPP mutants (5 n m ) were titrated with IAPP, Aβ40, or Aβ42 (pH 7.4). Mutants/values in boldface type indicate ≥19-fold weaker binding than IAPP.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Binding Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Identification of hot spots and key residues of IAPP self-assembly and its hetero-assembly with Aβ40(42) via fluorescence spectroscopic titrations (A–C). Differences in binding free energy of Ala mutants (mut) and wild-type (wt) IAPP (ΔΔGmut − wt or ΔΔG) toward wild-type IAPP (A), Aβ40 (B), or Aβ42 (C) (means ± S.D. (error bars); n = 3 assays). Mutants in boldface type are those with strongly diminished binding affinity to IAPP, Aβ40, or Aβ42 as compared with IAPP.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Identification of hot spots and key residues of IAPP hetero-assembly with Aβ40 via pulldown assays. A, pulldown assays of biotin-4A– and biotin-IAPP–Aβ40 hetero-assemblies. Top, anti-Aβ40 Western blot analysis (WB) of mixtures or their components as indicated (Aβ40, 5 μm; biotin-peptide, 2.5 μm) following biotin pulldown and peptide dissociation from beads. Bottom, anti-biotin WB of the same incubations as above. B, biotin-A23- versus biotin-IAPP–Aβ40 pull-down assay. Top, anti-Aβ40 WB of mixtures or their components as indicated (Aβ40, 5 μm; biotin-peptide, 2.5 μm) following biotin pulldown and peptide dissociation from beads. Bottom, anti-biotin Western blotting of the above incubations. Aβ40 control lane (A and B), Aβ40 not incubated with beads. Results shown are representative of three assays.

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Western Blot, Pull Down Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Summary of identified IAPP hot spots/key residues within amyloid core segment IAPP(8–28) mediating interactions with IAPP, Aβ40, or Aβ42. Hot spots (ΔΔGmut − wt (ΔΔG) > 2 kcal/mol) are shown as red (ΔΔG > 3 kcal/mol) or pink (ΔΔG, 2–3 kcal/mol) squares. Key residues (i.e. residues whose substitution by Ala results in ΔΔG > 2 kcal/mol only in the presence of all other residues substituted by Ala marked in the same way) are shown by using, in addition to the square, a red (ΔΔG > 3 kcal/mol*) or pink (ΔΔG = 2–3 kcal/mol*) rectangle. Additional yellow rectangles indicate ΔΔG of 1–2 kcal/mol* when all residues marked in this way are substituted by Ala (mutant A(9–12)). Left, IAPP(8–28); arrows, β-strands of reported amyloidogenic conformers (12, 13, 15, 32, 33, 37).

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

doi: 10.1074/jbc.M117.774893

Figure Lengend Snippet: Schematic presentation of proposed IAPP residues and folds mediating cross-interactions between early prefibrillar IAPP and Aβ40(42) species (top) versus IAPP amyloid self-assembly (bottom) based on our current results and previous findings (8, 9, 12,–15, 29, 30, 32, 33, 38,–40, 42, 63). Here identified IAPP hot spots/key residues are indicated by red/orange symbols in early disordered conformers only. The IAPP self-assembly-mediating sequence FGAIL is shown in yellow. The disulfide bridge between Cys2 and Cys7 in IAPP is indicated by a continuous line. Only a few heterodimers under the various possible hetero-assemblies are shown; previously identified IAPP binding sequences within Aβ40(42) are shown in purple (12, 42).

Article Snippet: Peptides and peptide synthesis Aβ40 (TFA salt) was synthesized by Fmoc solid-phase synthesis on Tentagel R PHB resin (Rapp Polymere), purified by RP-HPLC, and treated as described ( 12 , 51 , 52 ).

Techniques: Sequencing, Binding Assay

Journal: Molecular Neurodegeneration

Article Title: Downregulation of CREB expression in Alzheimer's brain and in Aβ-treated rat hippocampal neurons

doi: 10.1186/1750-1326-6-60

Figure Lengend Snippet: Decrease in CREB protein in AD-postmortem brain . (A) Post-mortem hippocampal samples (12 each) of AD cases and age-matched controls with equal protein content were electrophoresed, transferred, and immunoprobed with antibodies to CREB, phospho CREB (PCREB) and β actin. Representative blots are shown. (B) Band intensities of CREB and PCREB were quantitated and corrected for β actin for 24 samples. Total and phospho CREB levels were significantly low in AD brain. *P < 0.01 compared to control. (C, D) Aβ 1-42 levels were determined in soluble and SDS-extractable fractions of post-mortem samples by sandwich ELISA. Elevated levels of soluble and SDS-extractible Aβ were observed in AD brain. *p < 0.01; **p < 0.001 vs control. (E) When the levels of SDS-soluble Aβ were plotted against CREB band intensities, an inverse correlation between the two parameters was observed. Control values are shown as open squares and AD values as filled squares.

Article Snippet: To determine Aβ-induced toxicity in cultured neurons, Aβ aggregates were prepared by the following procedure: Aβ (1-42) peptide purchased from Peptide 2.0 (Chantilly, VA) was first monomerized by resuspending in hexafluoroisopropanol followed by incubation at RT for 1 h. After evaporation of solvent under vacuum, Aβ is dissolved in DMSO and stored at -20°C.

Techniques: Sandwich ELISA