Fig. 5: Astrocytes and microglia eliminate excitatory and inhibitory synapses in P301S mice in a complement-dependent manner.
a, Representative images of a confocal z-stack and Imaris 3D reconstructions of mouse brain sections immunostained for GFAP (blue), Iba1 (white), Lamp1 (green), Homer1 (yellow) and gephyrin (red). LAMP1+ lysosomes within GFAP+ or Iba1+ volumes were classified as astrocytic or microglial lysosomes, respectively. Scale bar in the raw image, 10 µm; scale bar in the Imaris 3D-rendered image, 2 µm. b,c, Fraction of Homer1 puncta identified inside astrocytic or microglial lysosomes across genotypes. d,e, Fraction of total gephyrin puncta identified inside astrocytic or microglial lysosomes across genotypes. f, Normalized number of Homer1 puncta engulfed by astrocytes or microglia, respectively (left). Ratio of Homer1 puncta within astrocytic/microglial lysosomes (right). g, Normalized number of gephyrin puncta engulfed by astrocytes or microglia, respectively (left) and ratio of gephyrin puncta within astrocytic/microglial lysosomes (right). Dotted line in f and g at a ratio of 1 indicates that astrocytic and microglial lysosomes contained the same number of synaptic puncta, ratio of >1 means that more synaptic puncta were localized within astrocytic lysosomes and <1 indicates that microglial lysosomes contained more synaptic puncta. Connected dots in the left of f and g show astrocytic and microglial Homer1 or gephyrin engulfment from the same mouse. h, Excitatory and inhibitory synapse density across genotypes as measured by number of identified Homer1 and gephyrin puncta per field of view (FOV). i, Representative confocal images of immunostained Homer1 (green) and C3 (red) in the CA1 region of WT, P301S and P301S;C1qKO brains. Colocalized Homer1 and C3 puncta are indicated by circles. Scale bar, 2 µm. j, Graph shows percentage of C3-labeled Homer1+ synapses. k, Total number of C3 puncta per FOV. Data were analyzed by one-way ANOVA with Dunnett’s post hoc test (b–e,h,j,k) and a two-tailed paired Student’s t-test (f,g). Each dot shows average data from one mouse; 7–10 mice per genotype were analyzed. All data are presented as mean ± s.e.m.
