Chronic Cyclodextrin Treatment of Murine Niemann-Pick C Disease Ameliorates Neuronal Cholesterol and Glycosphingolipid Storage and Disease Progression (original) (raw)
Figure 5
Chronic CD treatment study in Npc1−/− mice.
(A) Filipin labeling of unesterified cholesterol in the neocortex of untreated (end-stage, 78 days old) and CD-treated (start at P21; end-stage, 197 days old) Npc1−/− mice showed reduced cholesterol accumulation in a CD-treated mouse. (B) Higher magnification of neocortex in same CD-treated Npc1−/− animal as previous panel, showed presence of neurons with cholesterol accumulation while neighboring cells lacked this storage. (C) Confocal microscopy further revealed that gangliosides and cholesterol appeared to always co-sequester within neurons in an untreated Npc1−/− mouse (end-stage, 78 days old; upper panel). However, some neocortical neurons in a CD-treated Npc1−/− mouse (start at P7; end-stage, 182 days old; lower panel) had little to no detectable cholesterol accumulation, yet still exhibited ganglioside storage. Cholesterol (red, visualized with BC Theta), GM2 (blue), and GM3 (green); n denotes nucleus of single neuron shown in each image. (D) IHC of untreated and CD-treated Npc1−/− mice (same mice as A), revealed less GM2 storage (also GM3, not shown) in the neocortex of a CD-treated mouse. (E) Biochemical analysis of ganglioside levels further corroborated the reduction in GM2 and GM3 seen with IHC. Data from WT and untreated mutant mice represent mean ± SD. (F) Ultrastructural analysis of neocortical neurons in untreated and CD-treated Npc1−/− mice (same mice as C) revealed presence of PCBs in both groups, but CD-treated mice appeared to have fewer of these storage bodies. (G) Western blot analysis of LC3-II in 85 day old untreated and CD-treated (start at P21) Npc1−/− and WT mice revealed a reduction in LC3-II levels in the CD-treated Npc1−/− mouse. Images taken at 20X (A), 40X (B), 63X (C), and 10X (D); scale bars 20 µm (A, B), 2 µm (C), 50 µm (D), 1 µm (F).