Mechanistic convergence and shared therapeutic targets in Niemann-Pick disease

Abstract

Niemann-Pick disease type C (NPC) and Tangier disease are genetically and clinically distinct rare inborn errors of metabolism. NPC is caused by defects in either NPC1 or NPC2; whereas Tangier disease is caused by a defect in ABCA1. Tangier disease is currently without therapy, whereas NPC can be treated with miglustat, a small molecule inhibitor of glycosphingolipid biosynthesis that slows the neurological course of the disease. When a Tangier disease patient was misdiagnosed with NPC and treated with miglustat, her symptoms improved. This prompted us to consider whether there is mechanistic convergence between these two apparently unrelated rare inherited metabolic diseases. In this study, we found that when ABCA1 is defective (Tangier disease) there is secondary inhibition of the NPC disease pathway, linking these two diseases at the level of cellular pathophysiology. In addition, this study further supports the hypothesis that miglustat, as well as other substrate reduction therapies, may be potential therapeutic agents for treating Tangier disease as fibroblasts from multiple Tangier patients were corrected by miglustat treatment.

Keywords: ABCA1; Niemann-Pick disease type C; Tangier disease; lysosome; substrate reduction therapy.

Figure 1

Tangier patient fibroblasts have enlarged lysosomal compartment, and cellular phenotypes consistent with lysosomal storage disorders. A, Summaries of Tangier patient details, including mutations, age of diagnosis, and phenotypes. B, ABCA1 gene schematic indicating mutation placement. C, Relative lysosomal volume by LysoTracker staining showed significant increase in volume in the four Tangier patient cells with (D) representative traces. E, BODIPY‐LacCer trafficking and cholesterol accumulation were observed in Tangier cells 1 and 2 and resembled the phenotype observed in NPC1 patient cells

Figure 2

Tangier patient fibroblasts accumulate glycosphingolipids, cholesterol and free fatty acids. A, Representative glycosphingolipid (GSLs) traces from control, NPC1, and Tangier patient fibroblasts lines and B, the mean ± SEM for total GSLs and main storage species Gb3; 3 control, 3 NPC1, 4 Tangier, ***P < .001 vs control, **P < .01 vs control, *P < .05 vs control, calculated by a two‐way ANOVA test. C, Free cholesterol was measure from the patient cells and data presented as the mean ± SEM, ****P < .0001 vs control. D, Free fatty acids were measure from individual patient cell lines and presented as the mean, n = 3, ****P < .0001 vs control

Figure 3

Tangier patient fibroblasts have reduced acidic store calcium and significant sphingosine and sphinganine storage, and altered NPC1 and NPC2 expression. A, Representative traces showing intracellular [Ca²⁺] changes monitored in single fura‐2‐loaded fibroblasts in response to 200 μM GPN. B, Maximal peak fluorescence changes were determined as the difference between basal and the maximum fluorescence (Δ350/380). Data are presented as the mean ± SEM; 66 control, 81 NPC1, 78 Tangier patient 1, and 72 Tangier patient 2 cells, *** P < .001 vscontrol, calculated by an one‐way ANOVA test. C, Sphingosine (C18:1) and D, Sphinganine (C18:0) levels were measured in patient fibroblasts by HPLC. Date presented as mean ± SEM, n = 3, *P < .05, **P < .01, calculated by a one‐way ANOVA test. E, Western blot analysis of three NPC1 patient and four Tangier patient fibroblast lines. The NPC1 patients all had little/no level of NPC1, and normal/high levels of NPC2. The Tangier patients had variable levels of the proteins, with patients 1 and 2 having low levels of NPC1 and patients 3 and 4 having a high expression level. Similarly, Tangier patients 1 and 2 had normal/low levels of NPC2 whereas tangier patient 3 and 4 had reduced levels of NPC2 expression. (F) Data presented as a fold‐change of the relative amount of protein to the control for NPC1 protein levels and G, NPC2

Figure 4

Tangier patient cells respond to substrate reduction therapy (miglustat and eliglustat) treatment, acetyl‐d‐leucine (ADLL) treatment but not HPβCD. A, Relative lysosomal volume by LysoTracker staining showed significant reduction in volume in the four Tangier cell lines following 50 μM miglustat treatment. B, Total levels of glycosphingolipids following miglustat treatment were not significantly elevated from control levels. C, Similar to the levels of Gb3, which following miglustat treatment were not significantly different in the NPC1 and Tangier fibroblasts. D, Significant reduction in relative lysosomal volume in Tangier patient 3 and 4 cell lines was observed following 100 nM eliglustat treatment. E, 250 μM HPβCD treatment did not have any significant effect on relative lysosomal volume in any of the four Tangier patient cells. F, Treatment with 1 mM ADLL significantly reduced the relative lysosomal volume by LysoTracker staining in Tangier patient 2 and 3 cells