Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

Abstract

Astrocytes secrete cholesterol in lipoprotein particles.Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1 ؊/؊ ) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1 ؊/؊ astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors. The brain is the most cholesterol-rich organ in the body, containing roughly 25% of the unesterified cholesterol present in the whole individual. The input of cholesterol into the brain comes entirely, or almost entirely, from in situ synthesis because blood lipoproteins do not cross the blood-brain-barrier (1). Glial cells are thought to be responsible for a large part of this biosynthetic activity. Most of the sterol in the brain is acquired during myelination in the early stages of development and is produced by oligodendrocytes (2).Astrocytes, the most abundant glial cells, are intimately associated with neuronal synapses and secrete cholesterol in lipoprotein particles. Astrocytes have been proposed to provide cholesterol for synapse formation (3) as well as to participate in the recycling of cholesterol after injury (4). Nascent lipoproteins isolated from neonatal mouse astrocytes are a heterogeneous population of particles in the size range of plasma high density lipoproteins and appear to be composed of two separate classes that contain either apolipoprotein E or J (5).Niemann-Pick type C disease (NPC) 1 is an inherited, fatal neurodegenerative disorder in which large amounts of cholesterol and sphingolipids accumulate intracellularly within late endocytic organelles. The disease is caused by mutations in either the NPC1 or NPC2 gene (6). The NPC1 protein is a late endosomal membrane protein harboring a sterol-sensing domain, whereas NPC2 is a secretory protein that has cholesterol binding properties and uses the mannose 6-phosphate marker for late endosomal targeting (7). The exact functions of the proteins remain unknown, but genetic evidence suggests that they function in concert to facilitate lysosomal lipid egress (8).In the NPC1 Ϫ/Ϫ mouse astrocytes are considered to contribute to neurodegeneration (9, 10). Interestingly, NPC1 Ϫ/Ϫ astrocytes were shown t...

Significance of Sterol Structural Specificity

“…Accordingly, also in model membranes, lathosterol formed rafts that were at least as detergent-resistant as, and even more thermally stable than, cholesterol-containing rafts (13). Moreover, 7-dehydrocholesterol was found to be more strongly domain-promoting than cholesterol (17).In contrast to many relatives of cholesterol, desmosterol is an abundant structural membrane component in mammalian cells, such as spermatozoa and astrocytes (18,19). Inability to convert desmosterol to cholesterol (lack of functional 24-dehydrocholesterol reductase) leads to the human disorder desmosterolosis (20).…”

“…In contrast to many relatives of cholesterol, desmosterol is an abundant structural membrane component in mammalian cells, such as spermatozoa and astrocytes (18,19). Inability to convert desmosterol to cholesterol (lack of functional 24-dehydrocholesterol reductase) leads to the human disorder desmosterolosis (20).…”

Niemann‐Pick C2 Protein Expression Regulates Lithogenic Diet‐Induced Gallstone Formation and Dietary Cholesterol Metabolism in Mice

“…Npc1 levels were decreased in the liver of NPC2 (h/h) mice fed a lithogenic diet. Interestingly, a reciprocal regulation in the levels of NPC1 in the NPC2 (h/h) mice was not found, whereas in various models NPC2 increases in the absence of NPC1 [13,15]. Further studies are required to elucidate the molecular mechanisms involved in this regulation.…”

“…However, it is not known whether NPC2 present in plasma is functional at neutral pH or whether it is found free or as a component of a plasma lipoprotein complex. Studies in primary astrocytes have shown that most sterols are secreted separately from NPC2 into the surrounding medium, suggesting that secreted NPC2 is not primarily associated with cholesterolcontaining particles [13]. Further studies are required to determine how NPC2 deficiency affects cholesterol levels in plasma.…”

“…NPC2 expression has been detected in several cellular types, including fibroblasts, hepatocytes, neurons, and astrocytes [4,[12][13][14]. We have previously reported that NPC2 is expressed in the liver and is secreted into the plasma and bile, suggesting that NPC2 may have a global function in cholesterol homeostasis [15].…”