Extended Data Fig. 3: Complement proteins associate with specific synaptic connections in HD mouse models.
(a) Bar charts showing quantification of C1q puncta in different brain regions of 13 mo BACHD mice and WT littermates. In disease affected regions (dorsolateral striatum and motor cortex) of BACHD mice but not the less affected dentate gyrus there is a significant increase in the levels of C1q relative to that seen in WT littermates, striatum n = 3 WT and 4 BACHD mice (2 F and 1 M for WT and 2 F and 2 M for BACHD), motor cortex n = 3 WT and 3 BACHD mice (2 F and 1 M for both genotypes) and hippocampus (DG) n = 3 WT and 3 BACHD mice (2 F and 1 M for both genotypes). Unpaired two-tailed t-test for comparisons of WT and BACHD in each brain region, striatum p = 0.017, motor cortex p = 0.008, hippocampus (DG) p = 0.973. (b) Bar charts showing quantification of C3 puncta in different brain regions of 13 mo BACHD mice and WT littermates, striatum n = 4 WT and 4 BACHD mice (2 F and 2 M for both genotypes), motor cortex n = 3 WT and 4 BACHD mice (2 F and 1 M for WT and 2 F and 2 M for BACHD) and hippocampus (DG) n = 4 WT and 4 BACHD mice (2 F and 2 M for both genotypes). Unpaired two-tailed t-test for comparisons of WT and BACHD in each brain region, striatum p = 0.011, motor cortex p = 0.001, hippocampus (DG) p = 0.045. (c) Representative confocal images of the dorsolateral striatum of 7 mo WT and BACHD mice co-stained with C1q and VGLUT1 and 2 or C3 and VGLUT1 and 2. Note the increased association of both complement proteins with these synaptic markers in the BACHD tissue. Insets show examples of complement proteins co-localized with the presynaptic markers VGLUT1 and 2. Scale bar = 5 μm. Bar charts show quantification of the percentage of VGLUT 1 and 2 puncta colocalizing with C1q or C3 in the disease affected striatum and less disease affected hippocampus (DG), for C1q n = 3 WT and 3 BACHD mice (1 F and 2 M for WT and 2 F and 1 M for BACHD) for C3 n = 3 WT and 4 BACHD mice (2 F and 1 M for WT and 2 F and 2 M for BACHD). Unpaired two-tailed t-test for comparisons of WT and BACHD, Striatum C1q p = 0.004, Hippocampus (DG) C1q p = 0.635, Striatum C3 p = 0.004, Hippocampus (DG) C3 p = 0.364 (d) Representative pictographs of Imaris processed super-resolution images from the dorsolateral striatum of 7 mo WT and zQ175 mice co-stained with C3 and VGLUT1 in which the spheres function has been used to reflect immunoreactive puncta. The top two panels show all spheres generated from the super-resolution images and the bottom panels only show C3 and VGLUT1 spheres which are colocalized (defined as a distance of 0.3 μm or less between the center of each sphere). Scale bar = 5 μm. Note that there are more colocalized spheres in the 3 mo zQ175 striatum than in the 3 mo WT striatum. Quantification of these images is shown in the bar charts in Fig. 3g. (e) Confocal images of the dorsolateral striatum of 7 mo WT and zQ175 mice co-stained with C1q and VGLUT1 and 2 or C3 and VGLUT1 and 2. Bar charts show quantification of the percentage of VGLUT1 and 2 puncta colocalizing with C1q or C3 in disease affected regions (striatum) or less affected regions (dentate gyrus of the hippocampus), for C1q n = 3 WT and 3 zQ175 mice (3 F and 3 M for both genotypes), for C3 striatum n = 3 WT and 3 zQ175 mice (3 F and 3 M for both genotypes) and for C3 hippocampus (DG) n = 4 WT and 4 zQ175 mice (2 F and 2 M for both genotypes). Unpaired two-tailed t-test for comparisons of WT and zQ175, Striatum C1q p = 0.036, Hippocampus (DG) C1q p = 0.273, Striatum C3 p = 0.010, Hippocampus (DG) C3 p = 0.579. (f) Bar chart comparing the fold enrichment of C3 at VGLUT1 puncta in 3 mo zQ175 mice (taken from Fig. 3g) with that same analysis carried out after rotating the C3 channel 90 degrees, n = 4 WT and 4 zQ175 mice (2 F and 2 M for both genotypes). Unpaired two-tailed t-test p = 0.008 (g) Representative confocal images of the dorsolateral striatum of 3 mo zQ175 and WT mice co-stained with antibodies to C1q and VGLUT1 or C1q and VGLUT2. Scale bar = 5 μm. Bar charts show quantification of the % of VGLUT1 or VGLUT2 puncta colocalizing with C1q in both genotypes, for VGLUT1 n = 4 WT and 4 zQ175 mice (2 F and 2 M for both genotypes), for VGLUT2 n = 5 WT and 4 zQ175 mice (3 F and 2 M for WT and 2 F and 2 M for zQ175). Unpaired two-tailed t-test for comparisons of WT and zQ175, VGLUT1 p = 0.004, VGLUT2 p = 0.377 (h) Representative in situ hybridization (ISH) staining of C1q and NSE together with IHC for Iba1 in the dorsal striatum of 7 mo zQ175 mice. Inset shows a magnification of a selected area in the field. Scale bar = 20 μm. This experiment was repeated four times. (i) Representative ISH staining of C3 and Acta2 in the wall of the lateral ventricle. Inset shows a magnification of a selected area in the field Scale bar = 50 μm. This experiment was repeated three times. For bar charts, bars depict the mean. All error bars represent SEM. Stars depict level of significance with *=p < 0.05, **p = <0.01 and ***p < 0.001.
