Charina M. Ramirez

Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid

A mutation in NPC1 leads to sequestration of unesterified cholesterol in the late endosomal/lysosomal compartment of every cell culminating in the development of pulmonary, hepatic, and neurodegenerative disease. Acute administration of 2-hydroxypropyl-β-cyclodextrin (CYCLO) rapidly overcomes this transport defect in both the 7-day-old pup and 49-day-old mature npc1−/− mouse, even though this compound is cleared from the body and plasma six times faster in the mature mouse than in the neonatal animal. The liberated cholesterol flows into the cytosolic ester pool, suppresses sterol synthesis, down-regulates SREBP2 and its target genes, and reduces expression of macrophage-associated inflammatory genes. These effects are seen in the liver and brain, as well as in peripheral organs like the spleen and kidney. Only the lung appears to be resistant to these effects. Forty-eight h after CYCLO administration to the 49-day-old animals, fecal acidic, but not neutral, sterol output increases, whole-animal cholesterol burden is reduced, and the hepatic and neurological inflammation is ameliorated. However, lifespan is extended only when the CYCLO is administered to the 7-day-old animals. These studies demonstrate that CYCLO administration acutely reverses the cholesterol transport defect seen in the NPC1 mouse at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid.

Unesterified Cholesterol Accumulation in Late Endosomes/Lysosomes Causes Neurodegeneration and Is Prevented by Driving Cholesterol Export from This Compartment

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 t½ 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 ED50 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.

Weekly Cyclodextrin Administration Normalizes Cholesterol Metabolism in Nearly Every Organ of the Niemann-Pick Type C1 Mouse and Markedly Prolongs Life

Niemann-Pick type C1 (NPC1) disease arises from a mutation inactivating NPC1 protein that normally moves unesterified cholesterol from the late endosomal/lysosomal complex of cells to the cytosolic compartment for processing. As a result, cholesterol accumulates in every tissue of the body causing liver, lung, and CNS disease. Treatment of the murine model of this disease, the npc1−/− mouse, s.c. with β-cyclodextrin (4000 mg/kg) one time each week normalized cellular cholesterol metabolism in the liver and most other organs. At the same time, the hepatic dysfunction seen in the untreated npc1−/− mouse was prevented. The severity of cerebellar neurodegeneration also was ameliorated, although not entirely prevented, and the median lifespan of the animals was doubled. However, in contrast to these other organs, lung showed progressive macrophage infiltration with development of lipoid pneumonitis. These studies demonstrated that weekly cyclodextrin administration overcomes the lysosomal transport defect associated with the NPC1 mutation, nearly normalizes hepatic and whole animal cholesterol pools, and prevents the development of liver disease. Furthermore, this treatment slows cerebellar neurodegeneration but has little or no effect on the development of progressive pulmonary disease.

Frequency of the cholesteryl ester storage disease common LIPA E8SJM mutation (c.894G>A) in various racial and ethnic groups

Cholesteryl ester storage disease (CESD) and Wolman disease are autosomal recessive later‐onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ∼3%‐5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African‐American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African‐American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ∼1 in 300, respectively. No African‐American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% confidence interval [CI]: 51%‐69%) of reported mutations among multiethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ∼0.8 per 100,000 (∼1 in 130,000; 95% CI: ∼1 in 90,000 to 1 in 170,000). Conclusion: These data indicate that CESD may be underdiagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full‐gene LIPA sequencing to determine heterozygote frequencies, since c.894G>A is not common in these racial groups. (HEPATOLOGY 2013;53:958–965)

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED50 values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2−/− and npc1−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.

Persistence of herpes simplex virus DNA in cerebrospinal fluid of neonates with herpes simplex virus encephalitis

Objective:The significance of detecting herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of infants with HSV encephalitis after receipt of prolonged therapy with high-dose (60 mg kg−1 day−1) acyclovir is unknown. We report the clinical and laboratory characteristics, neuroimaging studies and outcomes of four neonates with HSV encephalitis who had persistence of CSF HSV DNA, by polymerase chain reaction (PCR) after 15 to 21 days of high-dose acyclovir therapy.Study Design:Retrospective chart review.Results:All four infants had abnormal neuroimaging studies and subsequently experienced severe developmental delay or death.Conclusion:A persistently positive CSF HSV PCR in neonates may be another risk factor for worse neurodevelopmental outcome. Prospective studies are needed to document how often HSV DNA persists in CSF, elucidate whether it represents an initially high CSF viral load, ongoing viral replication or viral resistance, and determine its possible association with neurodevelopmental impairment.

Systemic administration of 2-hydroxypropyl-β-cyclodextrin to symptomatic Npc1-deficient mice slows cholesterol sequestration in the major organs and improves liver function

In Niemann–Pick type C (NPC) disease, loss‐of‐function mutations in either NPC1 or NPC2 result in progressive accumulation of unesterified cholesterol (UC) and glycosphingolipids in all organs, leading to neurodegeneration, pulmonary dysfunction and sometimes liver failure. There is no cure for this disorder. Studies using primarily NPC mouse models have shown that systemic administration of 2‐hydroxypropyl‐β‐cyclodextrin (2HPβCD), starting in early neonatal life, diminishes UC accumulation in most organs, slows disease progression and extends lifespan. The key question now is whether delaying the start of 2HPβCD treatment until early adulthood, when the amount of entrapped UC throughout the body is markedly elevated, has any of the benefits found when treatment begins at 7 days of age. In the present study, Npc1−/− and Npc1+/+ mice were given saline or 2HPβCD subcutaneously at 49, 56, 63 and 70 days of age, with measurements of organ weights, liver function tests and tissue cholesterol levels performed at 77 days. In Npc1−/− mice, treatment with 2HPβCD from 49 days reduced whole‐liver cholesterol content at 77 days from 33.0 ± 1.0 to 9.1 ± 0.5 mg/organ. Comparable improvements were seen in other organs, such as the spleen, and in the animal as a whole. There was a transient increase in biliary cholesterol concentration in Npc1−/− mice after 2HPβCD. Plasma alanine aminotransferase and aspartate aminotransferase activities in 77‐day‐old 2HPβCD‐treated Npc1−/− mice were reduced compared with saline‐treated controls. The lifespan of Npc1−/− mice given 2HPβCD marginally exceeded that of the saline‐treated controls (99 ± 1.1 vs 94 ± 1.4 days, respectively; P < 0.05). Thus, 2HPβCD is effective in mobilizing entrapped cholesterol in late‐stage NPC disease leading to improved liver function.

Ontogenic changes in lung cholesterol metabolism, lipid content, and histology in mice with Niemann-Pick type C disease.

Niemann-Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1(-/-) mice (Npc1(nih)) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70days of age). Similar measurements were made in Npc2(-/-) mice at 70days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1(-/-) mice and remained so at 70days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1(-/-) mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1(-/-) mice starting as early as 28days. Similar metabolic and histologic changes were evident in the lungs of the Npc2(-/-) mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1(-/-)), and subsequently in Cav-1(-/-) mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) (Cav-1(-/-):Npc1(-/-)). In 50-day-old Cav-1(-/-) mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1(+/+) controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1(-/-):Npc1(-/-) mice (0.356±0.022) markedly exceeded that in their Cav-1(+/+):Npc1(+/+) controls (0.137±0.009), as well as in their Cav-1(-/-):Npc1(+/+) (0.191±0.013) and Cav-1(+/+):Npc1(-/-) (0.213±0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74±0.17, 0.71±0.05, 0.96±0.05 and 3.12±0.43, respectively, with the extra cholesterol in the Cav-1(-/-):Npc1(-/-) and Cav-1(+/+):Npc1(-/-) mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1(-/-):Npc1(-/-) mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.

565 Acute Reversal of the Lysosomal Transport Defect in the Liver of the Niemann Pick Type C Mouse

Nonalcoholic fatty liver disease (NAFLD) affects up to 30% of adults in the United States and is associated with obesity, metabolic syndrome, type II diabetes and hepatocellular carcinoma (HCC). Here we report spontaneous insulin resistance, NAFLD and HCC with parent-specific inheritance in AxB mice. Helicobacter-free F1 offspring (n=59) from wildtype C57BL/6 and A/JCr parents were maintained on a standard chow diet and evaluated at 3, 9 and 15 months. ABF1 (A/J dam x B6 sire) but not BAF1 males (B6 dam x A/J sire) developed progressively severe mixed microvesicular and macrovesicular hepatic steatosis resulting in sporadic HCC. Females were unaffected. NAFLD in males was a component of metabolic syndrome characterized by hyperinsulinemia, hypercholesterolemia and increased body mass (all P<0.05, unpaired t-test). Liver microarray confirmed widely dysregulated metabolic signaling and immune activation. A comprehensive evaluation of uniparentally expressed transcripts including mitochondrial, imprinted and sex-linked genes by qRT-PCR revealed a disease association with X-linked thyroxine-binding globulin. In a follow-up study using full-length chromosome substitution (consomic) C57BL/6J-ChrX/Y_A/NaJ mice on a standard or high-fat diet, we found an additive contribution from both sex chromosomes on disease phenotype. Our results demonstrate that parent-specific inheritance of insulin resistance in AxB mice is polygenic and regulated in part at the chromosomal level. This is a promising new mouse model to study genetic determinants of insulin resistance, metabolic syndrome and NAFLD.