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

Highlights

• NPC1-deficient mice have increased lung mass starting during neonatal development.

• Lung content of cholesterol and phospholipid in Npc1^−/− mice increases with age.

• Lungs of Npc1^−/− mice show increase in synthesis of cholesterol but not fatty acids.

• Lungs of Npc1^−/− mice show an increase in lipid-laden macrophages in the alveoli.

• Effects of NPC2 deficiency on lung lipid metabolism resemble those in Npc1^−/− mice.

Abstract

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 70 days of age). Similar measurements were made in Npc2^−/− mice at 70 days. 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 70 days 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 28 days. 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.

Introduction

In Niemann–Pick type C (NPC) disease unesterified cholesterol (UC), gangliosides, and glycosphingolipids continually accumulate in the late endosomal/lysosomal (E/L) compartment of all cells in every organ. It is an autosomal recessive disorder primarily affecting children and is characterized by neurodegeneration, hepatic and pulmonary dysfunction, and premature death [1]. A lot of what we currently know about NPC1 and NPC2 function, and also the etiology and pathogenesis of NPC disease has come from the study of animal models with NPC1 or NPC2 deficiency, primarily in the mouse [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], and also from various in vitro systems as well as techniques such as X-ray crystallography [15], [16], [17]. Unequivocally, it is the sequestration of UC in the E/L compartment that is the fundamental cause of NPC disease [18]. This accumulation of UC is due to a loss of function mutation in genes encoding either NPC1 or NPC2, both of which are essential for facilitating cholesterol transport out of the late E/L compartment [17]. Disruption of this egress and the resultant buildup of UC, together with that of other types of lipids, leads to cell death and multi-organ damage. While neurodegeneration is a major cause of morbidity and mortality in NPC disease, there are cases where death occurs within about the first six months of life from either liver failure or severe pulmonary dysfunction [1].

Although there is currently no effective treatment for NPC disease, a variety of agents has been evaluated primarily in animal or in vitro models, with 2-hydroxypropyl-ß-cyclodextrin (2HPßCD) showing the best efficacy thus far [19], [20], [21], [22], [23], [24]. In a particularly definitive study it was demonstrated that continuous infusion of 2HPßCD into the ventricular system of Npc1^−/− animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration [25]. Earlier studies established that weekly subcutaneous administration of 2HPßCD to Npc1^−/− mice, starting at 7 days of age, nearly normalized hepatic and whole animal cholesterol pools and prevented development of liver disease [26]. While there was also a slowing of cerebellar neurodegeneration and an increase in lifespan, systemic 2HPßCD treatment had little to no effect on the development of progressive pulmonary disease [26]. Subsequent studies further demonstrated lack of impact of systemically administered 2HPßCD on a number of parameters of lung function and pathology in the Npc1^−/− mouse [27]. There is nothing intrinsically different about UC sequestration in the lungs in NPC1 or NPC2 deficiency because NPC2 replacement therapy in a mouse model of NPC2 disease resulted in a striking reduction in the cholesterol content of several organs including the lungs [28]. Rather, the inaction of 2HPßCD in the NPC lung may simply reflect lack of penetrance at a cellular level. Irrespective of whether this is the case, those studies revealed the paucity of published information about how NPC1 or NPC2 deficiency impacts lung cholesterol metabolism, particularly in early stage disease. In one of our initial studies in the Npc^nih mouse model we found that even in 1-day old Npc1^−/− pups the cholesterol concentration in most organs, including the lungs, was elevated [29]. More recently, a murine model of infantile NPC1 deficiency has been described but lungs were not included in the tissues examined [30].

The present studies represent the first systematic evaluation, in quantitative terms, of the ontogenic changes in lung mass, lipid composition, rates of cholesterol and fatty acid synthesis, and also histology in mice with NPC1 or NPC2 deficiency.