Unesterified cholesterol accumulation in late endosomes/lysosomes causes neurodegeneration and is prevented by driving cholesterol export from this compartment

Abstract

While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase, which hydrolyzes C esters, and NPC1 (Niemann-Pick type C1). In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1(-/-) mouse, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a t(½) of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of sterol regulatory-binding protein 2 (SREBP2) target genes, and activation of liver X receptor-controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED(50) for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted >7 d. Continuous infusion of HP-β-CD into the ventricular system of npc1(-/-) animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.

Figure 1.

Comparison of cholesterol trafficking through cells of systemic tissues, represented here by liver, and neurons of the CNS. A, The pericellular fluid surrounding cells of systemic organs may contain various lipoproteins including LDL and the remnants of chylomicrons (CMr), and very-low-density lipoproteins (VLDLr). These particles are taken up into cells by receptor-mediated and bulk-phase endocytosis, and delivered into the late E/L compartment of cells where the CE is metabolized to C by LAL (a). This C then interacts sequentially with NPC2 (b) and NPC1 (c) before reaching the limiting membrane and exiting the E/L compartment (d) to join newly synthesized C (e) in a metabolically active pool in the cytoplasmic compartment. The size of this pool is closely regulated by the sterol-sensing transcript factors, SREBP2 and LXR (i), and, depending on the cell type, this C may be re-esterified (f), used for turnover and remodeling of the plasma membrane (g), or hydroxylated to an oxysterol (h) and, in the liver, to bile acid (BA). The magnitude of sterol flow through each systemic organ is determined, in part, by the ability of the various lipoprotein fractions to penetrate the endothelial barrier, described by the reflection coefficient, ∂, separating the blood from the pericellular fluid. B, The situation in the CNS is quite different, however, since the endothelial barrier is impermeable to plasma lipoproteins. In this compartment it is postulated that C is synthesized by glia, particularly by astrocytes, and carried as a complex with apoE to neurons. Uptake of this C presumably bypasses the need for LAL so that the sterol interacts directly with NPC2 and NPC1 to move into the metabolically active pool. There it may also be re-esterified (k), used for membrane remodeling and formation/maintenance of dendrites and synapses (l), or metabolized to the oxysterol, 24(S)-hydroxycholesterol (m), for excretion. It should be noted that in both systemic cells and neurons, sudden expansion of the metabolically active pool of C in the cytosolic compartment is associated with suppression of cholesterol synthesis, an increase in the CE concentration, suppression of various SREBP2 target genes, and activation of some genes controlled by LXR. In the young adult mouse, the flow of C through all systemic cells is ∼140 mg · kg⁻¹ · d⁻¹, while flow across the CNS is ∼1.4 mg · kg⁻¹ · d⁻¹ (Dietschy and Turley, 2004).

Figure 2.

Comparison of various anatomical, metabolic, inflammatory, and clinical parameters in lal^−/− and npc1^−/− mice. A–F, Utilizing 49-d-old lal^+/+, lal^−/−, npc1^+/+, and npc1^−/− animals, whole-body weights (A) and total cholesterol pools (C), along with plasma total cholesterol concentrations (B), were assessed, as were the various liver function tests, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP) (D–F). G–L, Relative liver (G) and brain (H) weights were determined, and whole-organ total cholesterol contents (I, J) and synthesis rates (K, L) were measured. M–R, Inflammation in these two organs was assessed by measuring mRNA expression for the markers CD68 (M, N), CD11c (O, P), and TNFα (Q, R). These mRNA values are all expressed relative to the values found in the control lal^+/+ and npc1^+/+ mice. Each column shows the mean ± 1 SEM (n = 6 animals in each group). Significant differences (p < 0.05) among the groups are indicated by different letters.

Figure 3.

Representative histological sections of basal pontine neurons and the anterior-superior cerebellar vermis of control, npc1^+/+/lal^+/+ mice, and of lal^−/− and npc1^−/− animals. A, D, G, Typical appearance of basal pontine neurons in these three groups of mice. B, E, H, Calbindin immunoreactivity in sections of the anterior-superior cerebellar vermis to illustrate the morphology of the Purkinje cell network. C, F, I, GFAP immunoreactivity in the anterior-superior vermis illustrating variation in the degree of activation of Bergmann glia in these same animals. The arrow in G points to vesicular cytoplasmic lipid collections, while the arrows in E and H point to perikarya of Purkinje cells in the cerebellum. The molecular (m) and granular (g) layers of the cerebellum are identified. H&E represents hematoxylin and eosin staining.

Figure 4.

Time course for the clearance of HP-β-CD from the CNS, and its effects on rates of sterol synthesis and CE formation in the brain. A, B, The 49-d-old npc1^+/+ and npc1^−/− mice were injected at time 0 with either a subcutaneous (4000 mg/kg) (A) or an intracerebroventricular (40 mg/kg) (B) dose of ¹⁴C-labeled HP-β-CD. Animals were then killed at different intervals, and the total body content (n = 20) or total CNS content (n = 20) of HP-β-CD was determined. The calculated t_½ values for the whole body or whole CNS are shown. C, D, The 49-d-old npc1^−/− mice were administered varying doses of HP-β-CD either subcutaneously (C) or intracerebroventricularly (D), and 24 h later, rates of cholesterol synthesis were measured in the liver (n = 68) and brain (n = 12). Sigmoid curves were fitted to these experimental data to yield the log₁₀ ED₅₀ values, and these were converted to the dose of cyclodextrin administered, either systemically or into the brain, that resulted in 50% inhibition of synthesis. E, F, The 49-d-old npc1^+/+ and npc1^−/− animals (n = 4–6 in each group) were administered either a subcutaneous (4000 mg/kg) (E) or an intracerebroventricular (35 mg/kg) (F) dose of HP-β-CD, and 24 h later, the level of C and CE in the liver and brain of each animal was determined. G, H, Similarly treated groups of npc1^+/+ and npc1^−/− animals (n = 4–6 in each group) were used to measure rates of fatty acid synthesis 24 h after administration of the HP-β-CD. Significant differences (p < 0.05) among groups are indicated by different letters.

Figure 5.

Relative expression of mRNA levels for a variety of proteins in the CNS of npc1^+/+ and npc1^−/− mice treated with HP-β-CD. A single dose of HP-β-CD (35 mg/kg) or aCSF was administered into the left cerebral ventricle of 49-d-old npc1^+/+ and npc1^−/− mice. Twenty-four hours later, the brains were removed and mRNA was extracted. A–E, The mRNA levels for SREBP2 and its target genes, HMG CoA RED, HMG CoA SYN, LDLR, and PCSK9, that control the rates of C acquisition and synthesis. F–H, mRNA levels for the genes ABCA1, ABCG1, and MYLIP that are controlled by the nuclear receptor, LXR. I, J, NPC2 and SOAT1, genes involved in cholesterol transport and esterification. K, L, SREBP1c and FAS, two genes controlling fatty acid synthesis. M–O, Genes involved in the hydroxylation or transport of C in the brain. P–T, Genes reflecting activation, proliferation, and cytokine production by microglia in the CNS. Means ± 1 SEM (n = 4 in each group) are shown.

Figure 6.

Duration of action and regional effects of HP-β-CD within the CNS. A, B, The 49-d-old npc1^−/− mice were injected at time 0 with either a subcutaneous (4000 mg/kg) (A) or an intracerebroventricular (35 mg/kg) (B) dose of HP-β-CD, and rates of cholesterol synthesis were measured in groups (n = 2–5) of these animals at varying times thereafter. C, The 49-d-old npc1^−/− mice were administered either HP-β-CD (35 mg/kg) or aCSF into the ventricle of the left cerebrum, and rates of C synthesis were measured 24 h later in different regions of the brain (n = 2–5 in each group). D, Groups of 49-d-old npc1^−/− mice were given varying doses (0–40 mg/kg) of HP-β-CD by intracerebroventricular injection into the ventricle of the left cerebrum, and rates of C synthesis were measured 24 h later in the different regions of the brain (n = 2–5 in each group). E, The 49-d-old npc1^−/− animals were administered HP-β-CD (35 mg/kg) intracerebroventricularly, and rates of C synthesis were measured at varying times in the major regions of the brain (n = 2–5 in each group). Means ± 1 SEM are shown.

Figure 7.

Representative histological sections of basal pontine neurons and the anterior-superior cerebellar vermis of npc1^+/+ and npc1^−/− mice treated with chronic administration of HP-β-CD. These animals (n = 3 in each group) were given systemic, subcutaneous injections of either saline or HP-β-CD (4000 mg/kg) every 7 d (beginning at 7 d of age) along with the continuous intracerebroventricular infusion of either aCSF or HP-β-CD (23 mg · kg⁻¹ · d⁻¹) (between the ages of 21 and 49 d). All mice were killed for histological examination of the brain at 49 d of age. A, D, G, J, M, Typical appearance of basal pontine neurons in these five groups of mice. B, E, H, K, N, Calbindin immunoreactivity in sections of the anterior-superior cerebellar vermis illustrating the morphology of the Purkinje cell network in each of these groups. C, F, I, L, O, GFAP immunoreactivity in the anterior-superior vermis showing variation in the degree of activation of Bergmann glia in these same animals. The arrows in G and J point to vesicular cytoplasmic collections of lipids.

Figure 8.

Relative expression of mRNA for a variety of proteins in the CNS of npc1^+/+ and npc1^−/− mice treated chronically with HP-β-CD. Groups of both npc1^+/+ and npc1^−/− (n = 3 in each group) mice were given systemic, subcutaneous injections of either saline or HP-β-CD (4000 mg/kg) every 7 d (beginning at 7 d of age) along with the continuous intracerebroventricular infusion of either aCSF or HP-β-CD (27 mg · kg⁻¹ · d⁻¹) (between 21 and 49 d of age). All mice were killed for determination of mRNA levels at 49 d of age. The various mRNAs measured in this study correspond to those previously evaluated in mice receiving a single dose of HP-β-CD (Fig. 5).