Figure 3

C1q does not preferentially accumulate in brain regions with degenerating neurons in NPC mice. (A) A simplified diagram of a cerebellar lobule marks the location of activated microglia (MG), degenerating Purkinje neurons (PN), and their efferent axons (EA) that contact the deep cerbellar nuclei (DCN) at the center of the cerebellum. (B, top panel) In the cerebellar cortex from an Npc1 ^-/- mouse, abundant microglia (anti-CD68, red) and few remaining PNs (Nissl stain, green; arrows) can be seen. (B, bottom panel) Microglia are also present throughout the cerebellum, though visibly less swollen, near remaining DCN neuron bodies (Nissl stain, yellow; arrow). (C) C1qa mRNA can be detected throughout the cerebellum. (D) Nevertheless, C1q protein (red) is found concentrated at the DCN in Npc1 ^-/- mice. Hoechst nuclear stain (blue) demarcates the cerebellar lobes. (E) Similarly, C1qa mRNA (dark purple) can be detected throughout the hippocampus, in microglia and neurons in Npc1 ^-/- mice. (F, top panel) Microglia (anti-CD68, red) evenly decorate the CA3, CA2, and CA1 neuron fields (anti-D28K, cyan), (F, bottom panel) however C1q protein (red) concentrates at the CA2 region (bright cyan) opposite the stratum radiatum (sr) side of the CA3/CA2 field. (G) Analysis of the C1q immunofluorescence intensity shows a peak difference between CA1 and CA2. Graph depicts the mean log fold change and 95% confidence limit of fluorescent intensity between Npc1 ^-/- and C1qa ^-/-; Npc1 ^-/- mice compared to Npc1 ^+/- and C1qa ^-/-; Npc1 ^-/- mice. The average log fold change of C1q immunofluorescence in C1qa ^-/-; Npc1 ^-/- hippocampal neuron regions over tissue autofluorescence is 0.15 with a standard deviation of 0.15. All images are representative of data observed in at least six mice at various ages >P50. Scale bars are 100μm.