c3b/ic3b/c3c, mouse, mab 2/11  (Hycult Biotech)

Journal: Brain

Article Title: Brain-penetrant complement inhibition mitigates neurodegeneration in an Alzheimer’s disease mouse model

doi: 10.1093/brain/awae278

Figure Lengend Snippet: Impact of 1-week treatment with Nb62-recombinant monoclonal antibody (r-mAb) on brain parameters in APP NL-G-F mice. APP NL-G-F mice were treated with anti-C7 mAb plus either Nb62-r-mAb or control-r-mAb over 7 days; systemic inhibition of complement, inhibition of complement activation in brain, brain inflammation, and neurodegeneration were assessed. ( A and B ) Classical pathway haemolysis (CH50) assays confirm that systemic complement was inhibited in APP NL-G-F mice aged 5–6 months (control-r-mAb: n = 7, Nb62-r-mAb: n = 8) or 11–13 months (control-r-mAb: n = 5, Nb62-r-mAb: n = 6) over 1 week of systemic administration of 73D1 mAb and either r-mAb. ( C and D ) Sandwich ELISAs detecting mouse C3 fragments (C3b/iC3b/C3c; C ) and terminal complement complex (TCC; D ) in total brain homogenate (TBH). Both C3 fragments and TCC levels were significantly lower in Nb62-r-mAb-treated mice at either age. Error bars are standard errors for each dataset. Groups were compared using an unpaired two-tailed t -test. ( E ) Sandwich ELISA to measure levels of amyloid-β (Aβ) in tissue bound protein (TBP) demonstrating significantly decreased Aβ in TBP of Nb62-r-mAb-treated APP NL-G-F mice compared to controls in both age sets. ( F and G ) Immunostaining of Aβ plaques in hippocampus and cortex using anti-Aβ antibodies: 6E10 ( F ) and 4G8 ( G ) showed no significant difference in plaque coverage between Nb62-r-mAb- and control-r-mAb-treated APP NL-G-F mice at either age. ( H ) Representative confocal images of DiOIistics-labelled CA1 hippocampal dendritic segments in 5–6-month-old and 11–13-month-old APP NL-G-F mice treated with Nb62-r-mAb or control-r-mAb. Scale bar = 5 µm. ( I and J ) DiOIistics-labelled dendritic spines were analysed in prefixed coronal brain slices. Overall spine density, analysed from dendritic segments of at least 30 µm, was higher in Nb62-r-mAb-treated mice compared to controls at both ages, significant only in the 11–13-month set. Analysis of spine subtypes showed that the number of thin spines was significantly increased in Nb62-r-mAb-treated groups in both age sets. Unpaired two-tailed t -test was used to compare spine densities between groups. Error bars correspond to the standard error of the mean.

Article Snippet: To measure mouse C3b/iC3b/C3c, plates were coated with 2/11 mAb anti-mouse C3b/iC3b/C3c (5 µg/ml, HM1065, Hycult Biotech), blocked in 3% BSA-PBS-T and washed in PBS-T. TBH or TBP were diluted 1:800, sera 1:20 000 in 0.3% BSA-PBS-T-EDTA, added in duplicate to ELISA wells, incubated overnight at 4°C, washed and bound C3 fragments detected using in-house HRP-labelled rabbit anti-human C3 (cross-reactive with mouse), 1:500 in 0.3% BSA-PBS-T-EDTA for 1.5 h at RT.

Techniques: Recombinant, Control, Inhibition, Activation Assay, Two Tailed Test, Sandwich ELISA, Immunostaining

Journal: Brain

Article Title: Brain-penetrant complement inhibition mitigates neurodegeneration in an Alzheimer’s disease mouse model

doi: 10.1093/brain/awae278

Figure Lengend Snippet: Impact of 3 months of treatment with Nb62-r-mAb on brain parameters in APP NL-G-F mice. APP NL-G-F mice were treated with anti-C7 mAb plus either Nb62-r-mAb ( n = 12) or control-r-mAb ( n = 12) over 3 months; systemic complement activity, complement activation in brain, brain inflammation, neurodegeneration and cognition were assessed. ( A ) Classical pathway haemolytic assays (CH50) demonstrated that systemic complement was inhibited in the Nb62-r-mAb and control-r-mAb-treated mice over the 3-month time course. ( B and C ) Levels of C3 fragments (C3b/iC3b/C3c; B ) and terminal complement complex (TCC; C ) in the total brain homogenate (TBH) were significantly reduced at end point in Nb62-r-mAb-treated APP NL-G-F mice compared to controls. ( D and E ) Levels of cytokines: IL-1α and IL1-β in TBH at end point were significantly decreased in Nb62-r-mAb-treated APP NL-G-F mice compared to controls. ( F ) Levels of amyloid-β (Aβ) in tissue bound protein (TBP) were significantly decreased at end point in Nb62-r-mAb-treated APP NL-G-F mice compared to controls. ( G – I ) Aβ plaques in hippocampus and cortex were either stained with the plaque stain X34 ( G ) or immunostained with anti-Aβ antibodies 6E10 ( H ) and 4G8 ( I ); analysis revealed no significant differences in plaque coverage with any of these stains at end point between the Nb62-r-mAb- and control-r-mAb-treated APP NL-G-F mice. ( J ) Representative confocal images of DiOIistics labelled CA1 hippocampal dendritic segments from APP NL-G-F mice treated with Nb62-r-mAb or control-r-mAb. Scale bar = 5 µm. ( K and L ) Quantification of DiOIistics-labelled dendritic spines in prefixed coronal brain slices. ( K ) APP NL-G-F mice treated with Nb62-r-mAb showed significantly increased overall spine density compared to control-r-mAb-treated mice. Analysis of spine subtypes showed that the numbers of thin and mushroom spines were significantly increased in Nb62-r-mAb-treated groups, most significantly for thin spines. ( M ) Representative images of Bassoon (green) and PSD95 (red) immunoreactive synaptic puncta in the stratum radium of Nb62-r-mAb- and control-r-mAb-treated APP NL-G-F mice at end point; scale bar = 5 µm. ( N ) Synaptic puncta stained with Bassoon or PSD95 were quantified (region of interest, 20 µm × 20 µm, 12 per mouse) using Imaris Spot function; puncta were increased in Nb62-r-mAb-treated mice compared with controls for both stains but significantly only for PSD95. ( O – Q ) Comparison of Nb62-r-mAb-treated and control-r-mAb-treated APP NL-G-F mice in behavioural tests. ( O ) In the burrowing test, Nb62-r-mAb-treated mice burrowed significantly more of the gravel compared to controls. ( P ) In the Open Field (OF) test, Nb62-r-mAb mice spent significantly more time exploring the central area of the box. ( Q ) In the Novel Object Recognition (NOR) test, Nb62-r-mAb-treated mice spent significantly more time exploring the novel object. Each point represents one animal in these analyses. For all quantitative analyses, an unpaired two-tailed t -test was used to compare the two groups. Error bars correspond to standard error of the mean, and P -values are included where appropriate.

Article Snippet: To measure mouse C3b/iC3b/C3c, plates were coated with 2/11 mAb anti-mouse C3b/iC3b/C3c (5 µg/ml, HM1065, Hycult Biotech), blocked in 3% BSA-PBS-T and washed in PBS-T. TBH or TBP were diluted 1:800, sera 1:20 000 in 0.3% BSA-PBS-T-EDTA, added in duplicate to ELISA wells, incubated overnight at 4°C, washed and bound C3 fragments detected using in-house HRP-labelled rabbit anti-human C3 (cross-reactive with mouse), 1:500 in 0.3% BSA-PBS-T-EDTA for 1.5 h at RT.

Techniques: Control, Activity Assay, Activation Assay, Staining, Comparison, Two Tailed Test