Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease

Abstract

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Keywords: Brain delivery; Cholesterol; Cognitive decline; Huntington's disease; Nanoparticles.

Graphical abstract

Fig. 1

Cognitive and motor abilities of zQ175DN mice after “early treatment” and “late treatment”. A Experimental paradigm of the “early treatment”: zQ175DN mice (N = 9–18) were treated with g7-hybrid-NPs-chol (0.12 mg NPs/gr body weight) from 5 to 9 weeks of age with 2 ip injection/week; wt (N = 10–13) and zQ175DN (N = 10–11) littermates were treated with saline solution as controls. Novel Object Recognition (NOR), Paw Clasping (PC), Grip Strength (GS), and Activity Cage (AC) tests were performed at 20–29–35–45 weeks of age, Rotarod (RR) at 46 weeks of age, Fear Conditioning (FC) at 48 weeks of age. Mice were sacrificed at 50 weeks of age. B-E Behavioral tests. Discrimination index (DI; %) in NOR (B); PC score (C); GS (gr, D); latency to fall (sec) in RR (E). F Experimental paradigm of the “late treatment”: zQ175DN mice (N = 13–14) were treated with g7-hybrid-NPs-chol (0.12 mg NPs/gr body weight) from 21 weeks of age to 25 weeks of age with 2 ip injection/week; wt (N = 13–14) and zQ175DN (N = 13–14) littermates were treated with saline solution as controls. NOR, PC, GS, and AC were performed at 40–49 weeks of age, RR at 50 weeks of age, and mice were sacrificed at 51 weeks of age. G-J Behavioral tests. Discrimination index (DI; %) in NOR (G); PC score (H); GS (gr, I); latency to fall (sec) in RR (J). (C and H): wt mice develop clasping with age; the mean of combined PC data in wt mice at 40, 45 and 49w (C and H) is higher than the mean of PC data in wt mice at 20, 29, 35w (C) (1.034 +/- 0117 and 0,2892 +/- 0.05 respectively; p < 0.0001 Welch’ t test). Data information: data in B-E are from three independent trials and shown as scatterplot graphs with mean±SEM. Data in G–J are from two independent trials and shown as scatterplot graphs with mean±SEM. Each dot (B, D, E, G, I, J) corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Tuckey post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 2

Cognitive and motor abilities of zQ175DN mice after “2-cycle treatment”. A Experimental paradigm of the “2-cycle treatment”: zQ175DN mice (N = 9–17) were treated with g7-hybrid-NPs-chol (0.12 mg NPs/gr body weight) from 5 weeks of age to 9 weeks of age and from 21 to 25 weeks of age with 2 ip injection/week/cycle; wt (N = 15–21) and zQ175DN (N = 11–21) littermates were treated with saline solution as controls. NOR, PC, GS, and AC were performed at 20–29–35–45 weeks of age, Y-maze (YM) test at 30–48 weeks of age, RR at 46 weeks of age, FC at 48 weeks of age. Mice were sacrificed at 51 weeks of age. B-H Behavioral tests. Discrimination index (DI; %) in NOR (B); n° of freezing episodes in FC (cued paradigm, C); PC score (D); GS (gr, E); latency to fall (sec) in RR (F); global activity (G) and distance (cm, H) in AC. Data information: data in B–H are from three independent trials and shown as scatterplot graphs with mean±SEM. Each dot (B, D–H) corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Tuckey post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 3

Segregation of the behavioral tasks in cholesterol treated HD mice with wt mice versus HD mice. A Heat maps summarizing the behavior related to NOR, PC, GS, and AC tests of all treatment using conditional formatting (excel). Light blue: best performance; dark blue: worst performance. B-C Principal component analysis (PCA) by combining all the values related to motor and cognitive tasks from all mice at 29 weeks (B) and 45–49 weeks of age (C). The convex hull of the set of points belonging to each group of mice was also visualized. D Data-visualization of the overall cognitive performance assessed by NOR test among wt, HD and HD cholesterol-treated mice, comprehensive of data collected from five different studies. Data were visualized using a dotplot also showing their distribution. The differences between groups have been assessed using a pairwise t-test, multiple testing correction was performed using the Bonferroni method.

Fig. 4

Sterols in the striatum of zQ175DN mice after “early treatment”, “late treatment” and “2-cycle treatment”. A-B Cholesterol (A) and lathosterol (B) content quantified by mass spectrometry in the striatum of animals (N = 4/group) from the “early treatment” (29- and 50-week), “late treatment” (51-week) and “2-cycle treatment” (50-week). Data information: data in A-B are shown as scatterplot graphs with mean±SEM. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Tuckey post-hoc test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 5

Mutant HTT aggregates in the striatum and cortex of zQ175DN mice after “early treatment”, “late treatment” and “2-cycle treatment”. A–G Experimental scheme of the “early treatment” (A) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (B) and cortex (E) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 29 and 50 weeks of age (N = 3/group); relative quantification of aggregates n° and size (C, D, F, G). H-N Experimental scheme of the “late treatment” (H) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (I) and cortex (L) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 51 weeks of age (N = 3/group); relative quantification of aggregates n° and size (J, K, M, N). O-U Experimental scheme of the “2-cycle treatment” (O) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (P) and cortex (S) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 50 weeks of age (N = 3/group); relative quantification of aggregates n° and size (Q, R, T, U). V Meta-analysis and data-visualization representing the mean of the number of muHTT aggregates in the striatum of HD mice after cholesterol-raising strategies. The normalized data were represented using a circular barplot to show the mean number of aggregates in the different treatments. Data information: Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 20 µm. 10 images/animal were analyzed from 9 sections throughout the entire striatum and cortex. Data in C-D, F-G, J-K, M-N, Q-R, T-U are shown as scatterplot graphs with mean±SEM. Each dot corresponds to the value obtained from each image. Statistics: Student’s t-test (**p < 0.01; ***p < 0.001; ****p < 0.0001). Statistics in V: Student’s t-test (*p<0.05;***p<0.001; ****p<0.0001). R6/2 minipumps: HD treated vs HD untreated (****); R6/2 SREBP2 gene therapy: HD treated vs HD untreated (****); R6/2 g7-PLGA-NPs-chol: HD treated vs HD untreated (ns); R6/2 g7-hybrid-NPs-chol: HD treated vs HD untreated (ns); zQ175DN g7-hybrid-NPs-chol (early): HD treated vs HD untreated (*); zQ175DN g7-hybrid-NPs-chol (late): HD treated vs HD untreated (***); zQ175DN g7-hybrid-NPs-chol (2-cycle): HD treated vs HD untreated (****).

Fig. 5

Mutant HTT aggregates in the striatum and cortex of zQ175DN mice after “early treatment”, “late treatment” and “2-cycle treatment”. A–G Experimental scheme of the “early treatment” (A) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (B) and cortex (E) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 29 and 50 weeks of age (N = 3/group); relative quantification of aggregates n° and size (C, D, F, G). H-N Experimental scheme of the “late treatment” (H) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (I) and cortex (L) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 51 weeks of age (N = 3/group); relative quantification of aggregates n° and size (J, K, M, N). O-U Experimental scheme of the “2-cycle treatment” (O) and immunolabeling of muHTT aggregates (showing muHTT aggregates positive for EM48 antibody in green) in striatum (P) and cortex (S) in brain coronal slices of zQ175DN and zQ175DN+chol mice at 50 weeks of age (N = 3/group); relative quantification of aggregates n° and size (Q, R, T, U). V Meta-analysis and data-visualization representing the mean of the number of muHTT aggregates in the striatum of HD mice after cholesterol-raising strategies. The normalized data were represented using a circular barplot to show the mean number of aggregates in the different treatments. Data information: Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 20 µm. 10 images/animal were analyzed from 9 sections throughout the entire striatum and cortex. Data in C-D, F-G, J-K, M-N, Q-R, T-U are shown as scatterplot graphs with mean±SEM. Each dot corresponds to the value obtained from each image. Statistics: Student’s t-test (**p < 0.01; ***p < 0.001; ****p < 0.0001). Statistics in V: Student’s t-test (*p<0.05;***p<0.001; ****p<0.0001). R6/2 minipumps: HD treated vs HD untreated (****); R6/2 SREBP2 gene therapy: HD treated vs HD untreated (****); R6/2 g7-PLGA-NPs-chol: HD treated vs HD untreated (ns); R6/2 g7-hybrid-NPs-chol: HD treated vs HD untreated (ns); zQ175DN g7-hybrid-NPs-chol (early): HD treated vs HD untreated (*); zQ175DN g7-hybrid-NPs-chol (late): HD treated vs HD untreated (***); zQ175DN g7-hybrid-NPs-chol (2-cycle): HD treated vs HD untreated (****).

Fig. 6

Electrophysiological analysis in MSNs of zQ175DN mice after “2-cycle treatment”. A–C Membrane Capacitance (Cm, A), Resting Potential (mV; B) and Input Resistance (Rin, C) recorded from MSNs of wt (6 cells from N = 3 mice), zQ175DN (8 cells from N = 4 mice) and zQ175DN+chol mice (5 cells from N = 3 mice) from the “2-cycle treatment”. D Representative traces of spontaneous EPSCs (sEPSCs) at a holding potential of − 70 mV. E–F Mean frequency (E) and cumulative inter‐event histogram (F) of spontaneous EPSCs (sEPSCs) recorded from MSNs of wt, zQ175DN and zQ175DN+chol mice. G–H Mean frequency (G) and cumulative inter‐event histogram (H) of miniature EPSCs (mEPSCs) recorded from MSNs of wt, zQ175DN and zQ175DN+chol mice. Data information: data in A, B, C, E, and F are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each cell. Statistics (A-H): one-way ANOVA with Tuckey post-hoc test (*p < 0.05; **p < 0.01; ***P < 0001).