Vascular endothelial growth factor improves the therapeutic effects of cyclodextrin in Niemann-Pick type C mice

Abstract

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative disorder caused by a deficiency in the function of the NPC1 gene. Malfunction of this gene/protein leads to progressive accumulation of unesterified cholesterol and sphingolipids in many organs, including the brain. To date, drugs that target pivotal stages in the pathogenic cascade have been tested as monotherapies or in combination with a second agent, showing additive benefits. In this study, we have investigated the effects of combining centrally and systemically administered therapies in a mouse model of NP-C, i.e. overexpression of brain-specific vascular endothelial growth factor (VEGF) in combination with systemic administration of 2-hydroxypropyl-β-cyclodextrin (CD). We found that animals treated using a combination of VEGF and CD showed an improvement in pathophysiology compared to those treated with CD alone or brain VEGF overexpression alone, or non-treated NP-C mice. Combination therapy increased the time period over which NP-C mice maintained their body-weight and motor function, and decreased the abnormal accumulation of lipids. In addition, combination therapy delayed the onset of Purkinje cell loss and reduced neuroinflammation. Taken together, our results demonstrate that combination therapy using VEGF and CD is a promising therapeutic modality for treating NP-C, and suggest that it represents a potential strategy for the treatment of diseases that cause both visceral and brain pathologies.

Keywords: 2-hydroxypropyl-β-cyclodextrin; Niemann-Pick type C disease mouse; combination therapy; neuroinflammation; vascular endothelial growth factor.

Figure 1.

Effect of combination therapy on survival, body weight, and motor function in NP-C mice. (A) Experimental design to determine the effect of combination therapy on NP-C. (B) Survival curve of WT and NP-C mice in each treatment group (n = 10–12 per group). (C) Average body-weights of WT and NP-C mice in each treatment group according to age (n = 10–12 per group). (D) Rotarod scores of WT and NP-C mice in each treatment group (n = 10–12 per group). (E) Beam test of WT and NP-C mice in each treatment group. Left, 12-mm square beam. Right, 6-mm square beam (n = 10–12 per group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01. All error bars indicate s.e.m.

Figure 2.

Effect of combination therapy on PN survival and neuroinflammation. (A) Confocal images and quantification of cerebellar PNs in the cerebellum of mice from each treatment group (n = 3 per group). Scale bar: 50 μm. (B) Representative fluorescence images and quantification of GFAP in the cerebellum of mice from each treatment group (n = 3 per group). Scale bars: 50 μm. One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01. All error bars indicate s.e.m.

Figure 3.

Effect of combination therapy on sphingosine and sphingomyelin accumulation. Sphingosine and sphingomyelin levels in cerebellum, liver, lung, kidney and spleen of each treatment group (n = 3 per group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.005. All error bars indicate s.e.m.

Figure 4.

Effect of combination therapy on cholesterol accumulation using Amplex red assay. Unesterified cholesterol levels in cerebellum, liver, lung, kidney and spleen of each treatment group (n = 4 per group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.005. All error bars indicate s.e.m.

Figure 5.

Effect of combination therapy on cholesterol accumulation using filipin staining. Representative images and quantification of unesterified cholesterol using filipin staining in mice (n = 3 per group). One-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.005. Scale bars (cerebellum): 100 μm. Scale bars (liver, lung, kidney and spleen): 50 μm. All error bars indicate s.e.m.