Fig 2: CD40L reverse signaling restores the expression of PSD-95, RhoA/B/C, and Cdc42 and resembles the spine morphology in Cd40–/– cultured neurons. (A) Illustrative Western blots of the expression of PSD-95, synaptophysin, RhoA/B/C, Cdc42, and Rac1/2/3, using anti-ßIII tubulin and anti-GAPDH as loading controls in Cd40–/– cultured neurons treated with Fc or CD40-Fc 24 h after seeding and cultured during the indicated days. (B) Quantification of the relative expression of these proteins normalized to ßIII tubulin from Western blots from at least three independent cultures. Graphs show mean ± s.e.m. One-way ANOVA with multiple Newman-Keuls statistical comparison. **p < 0.01 and *p < 0.05. (C) Immunocytochemical localization of DARPP-32 (green), PSD-95 (red), and merge (yellow) in 18 days cultures of Cd40–/– treated 24 h after seeding with Fc or CD40-Fc. In the DARPP-32 images, * indicates spines in 40 µm fragments. In the merge images, the arrows indicate the co-localization of PSD-95 and DARPP-32 in spines and the dashed arrows indicate co-localization of these proteins in the base of spines. Scale bar 5 µm. Quantification of total number of spines and of the type of spines (branched, mushroom, stubby, thin, and filopodia) per 50 µm dendrite fragment. The means ± s.e.m from 50 µm fragments obtained from at least three independent cultures of Cd40–/– treated with Fc (41, dark gray bars) and CD40-Fc (68, pale gray bars). The dots represent the total number of spines counted. T-test **p < 0.01 and *p < 0.05.

Fig 3: AAV5-miHTT may prevent striatal volume loss and normalize striatal DARPP-32 levels in Hu128/21 mice. Stereological evaluation of (A) striatal (unpaired t-test between Hu21 and Hu128/21 saline treated P = 0.0517; one-way ANOVA on Hu128/21 treated animals P = 0.465. N = 12–14), (B) cortical (unpaired t-test between Hu21 and Hu128/21 saline treated P = 0.6380; one-way ANOVA on Hu128/21 treated animals P = 0.9741. N = 13–14) and (C) corpus callosum volumes (unpaired t-test between Hu21 and Hu128/21 saline treated P = 0.0623; one-way ANOVA on Hu128/21 treated animals P = 0.3810. N = 13–14) at 7 months post-injection. (D) DARPP-32 immunoreactivity was evaluated by integrated optical density (IOD) in a series of 4 mid-striatal sections at 2 months post-injection (unpaired t-test between Hu21 and Hu128/21 saline treated **P = 0.0316; one-way ANOVA on Hu128/21 treated animals P = 0.0002. Tukey multiple comparison test ###P< 0.001 Hu128/21 saline versus high, medium and low doses. N = 3) and 7 months post-injection (unpaired t-test between Hu21 and Hu128/21 saline treated P = 0.5222; one-way ANOVA on Hu128/21 treated animals P = 0.7315. N = 13–14). Values were normalized to the mean value for saline treated Hu21 animals at each timepoint.

Fig 4: AAV5-miHTT treatment may induce striatal atrophy in Hu21 control mice. Stereological evaluation of (A) striatal (one-way ANOVA P = 0.533. N = 13–15), (B) cortical (one-way ANOVA P = 0.5855. N = 13–15) and (C) corpus callosum (one-way ANOVA P = 0.1176. N = 13–15) volumes was performed at 7 months post-injection. (D) IOD of DARPP-32 immunoreactivity was evaluated in a series of 4 mid-striatal sections at 2 months post-injection (one-way ANOVA P = 0.0351. N = 3) and 7 months post-injection (one-way ANOVA P = 0.5430. N = 13–15). Values were normalized to the mean value for saline treated Hu21 animals at each timepoint.

Fig 5: Neuronal survival is compromised in YAC128 1:3 CS co-cultures. DIV21 WT and YAC128 co-cultures were fixed at DIV21 and stained for MAP2 and DARPP32 (D32). (a) Sample fields of view at 20X objective (scale bar = 100 µm). The numbers of (Bi) MAP2+ and (Bii) DARPP32+ neurons per field of view are reduced in YAC128 1:3 co-cultures. (Biii) The proportion of DARPP32+ neurons (# DARPP32+ divided by # MAP2+) surviving at DIV21 is also significantly lower in YAC128 1:3 co-cultures [n = 30 fields of view from three independent cultures; two-way ANOVA with Bonferroni post-hoc analysis; *p < 0.05, ***p < 0.001]