Figure 2

In vivo NGF response in WT and NPC1-/- mice. A and B. WT and NPC1^-/- mice respond similarly to axotomy of the fimbria-fornix. Brain sections from 8-week-old axotomized (unilateral fimbria-fornix axotomy) WT and NPC1^-/- mice were stained with an antibody against ChAT (A). Quantification shows an evident and similar reduction in the number of neurons labeled with ChAT (B). The differences between the phenotypes are not significant, p = 0.755. C. Brain sections from 8-week-old axotomized WT and NPC1^-/- mice were immunostained for ChAT, and the images were acquired by confocal microscopy. In WT mice, axotomized septal neurons (IL, ipsilateral to the axotomy) exhibit lower levels of ChAT than contralateral neurons (CL). In NPC1^-/- mice, ChAT staining in the axotomized septal neurons is similar to that in the contralateral neurons. D. The quantification of ChAT intensity in septal neurons (90-92 neurons for contralateral side and 40-42 neurons for ipsilateral) from three WT and three NPC1^-/- mice demonstrates a significant reduction in the ChAT levels in WT axotomized neurons, while NPC1^-/- axotomized neurons appear protected, *p < 0.005, unpaired one-way ANOVA. The increased staining of ChAT-labeled cells in C and D is not due to increased cell size because the total fluorescence or immunostaining intensity of each cell was normalized by cell area. E. Upper panel: ChAT levels were analyzed by western blot using homogenates of the MS from three 8-week-old WT and three NPC1^-/- mice. Bands corresponding to ChAT from three different WT and NPC1^-/- mice are shown. Lower panel: quantification of the western blot indicates that ChAT levels are significantly higher in NPC1^-/- mice than in WT mice. *p < 0.01, unpaired Student's t-test. n = 3 animals per phenotype. ChAT was normalized against total Akt. F-H. Increased ChAT staining in response to NGF infusion in NPC1^-/- septal cholinergic neurons. Brain sections from 8-week-old axotomized WT and NPC1^-/- mice infused with NGF for one week (F). Black arrows indicate cholinergic neurons with increased ChAT staining. Quantification shows that NGF protected septal cholinergic neurons equally in WT and NPC1^-/- mice (G). Quantification of ChAT staining in cholinergic cells (as indicated by the arrows in F). AU, arbitrary units. The differences are statistically significant, *p < 0.0001. Two sections through the medial septum of four WT and four NPC1^-/- age-matched mice were used for quantification. A total of approximately 180 cells were considered for each phenotype (H).