Cyclodextrin alleviates neuronal storage of cholesterol in Niemann-Pick C disease without evidence of detectable blood-brain barrier permeability

Abstract

Niemann-Pick type C disease is an inherited autosomal recessive neurodegenerative disorder characterised by the accumulation of unesterified cholesterol and sphingolipids within the endosomal/lysosomal compartments. It has been observed that the administration of hydroxypropyl-β-cyclodextrin (HPBCD) delays onset of clinical symptoms and reduces accumulation of cholesterol and gangliosides within neuronal cells. It was assumed that HPBCD exerts its action by readily entering the CNS and directly interacting with neurones and other brain cells to facilitate removal of stored cholesterol from the late endosomal/lysosomal compartment. Here, we present evidence that refutes this hypothesis. We use two well established techniques for accurately measuring brain uptake of solutes from blood and show that there is no significant crossing of HPBCD into the brain. The two techniques are brain in situ perfusion and intraperitoneal injection followed by multi-time-point regression analysis. Neither study demonstrates significant, time-dependent uptake of HPBCD in either adult or neonatal mice. However, the volume of distribution available to HPBCD (0.113 ± 0.010 ml/g) exceeds the accepted values for plasma and vascular volume of the brain. In fact, it is nearly three times larger than that for sucrose (0.039 ± 0.006 ml/g). We propose that this indicates cell surface binding of HPBCD to the endothelium of the cerebral vasculature and may provide a mechanism for the mobilisation and clearance of cholesterol from the CNS.

Figures

Figure 1. Data from in situ brain perfusion studies

A. From left to right; volumes of distribution (Vd) for sucrose, 2 min (0.039±0.006 ml/g); Vd for cyclodextrin 2 minutes perfusion (0.113±0.010 ml/g); Vd for cyclodextrin 4 minutes perfusion (0.106±0.005ml/g) Vd for cyclodextrin 2 minutes in the presence of 2mM cyclodextrin (0.082±0.006 ml/g); Vd of sucrose 2 minutes in the presence of 2mM cyclodextrin (0.044±0.009ml/g). All data Npc1+/+. (n=4–6 for each column ± SEM, significance 1-way anova) B. From left to right; volumes of distribution (Vd) for cyclodextrin 2 minutes Npc1+/+ (0.113±0.010ml/g); Vd for cyclodextrin 2 minutes Npc1−/− (0.071±0.002ml/g); Vd for cyclodextrin 2 minutes in the presence of 2mM cyclodextrin Npc1−/− (0.083±0.003ml/g); Vd for sucrose, 2 min Npc1+/+ (0.039±0.006ml/g); Vd for sucrose, 2 min Npc1−/− (0.047±0.005ml/g). (n=4–6 for each column ± SEM, significance 1-way anova)

Figure 2

Plasma radioactivity (cpm/50μl) with time after intraperitoneal injection of tracer quantities of [14C]-cyclodextrin or at high dose (same radioactive quantity mixed with 4000mg cyclodextrin/kg, in Npc1+/+ and Npc1−/− mice. (n=3 for each time point)

Figure 3

Volumes of distribution (Vd).of cyclodextrin after intraperitoneal injection of tracer levels with time in whole brain, cortex, medulla, olfactory bulb, cerebellum and midbrain in Npc1+/+ and Npc1−/− mice. (n=3 for each time point)

Figure 4

Volumes of distribution (Vd) of cyclodextrin after intraperitoneal injection of high dose with time in whole brain, cortex, medulla, olfactory bulb, cerebellum and midbrain in Npc1+/+ and Npc1−/− mice. (n=3 for each time point)

Figure 5

Volumes of distribution (Vd)of cyclodextrin after intraperitoneal injection of tracer levels with time in whole brain, hind brain and left and right hemispheres in Npc1+/+ at 7 days of age. (n=3 for each time point)