NPC1 mRNA Research Articles

Determinants of NPC1 Expression and Action: Key Promoter Regions, Posttranscriptional Control, and the Importance of a “Cysteine-Rich” Loop

Mutations in the NPC1 gene cause Niemann–Pick type C disease, which is characterized by the accumulation of free cholesterol and other lipids in lysosomes. The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containing the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs −111 and −37 play an important role in controlling NPC1 transcription. Treatment of proliferating granulosa cells with 30 μM progesterone, which induces a reversible phenocopy of the cholesterol trafficking defect of Niemann–Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cycloheximide, also increased NPC1 mRNA levels, augmenting the progesterone-induced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPC1 promoter activity. Cysteine residues in a “cysteine-rich” loop predicted to reside in the intralumenal compartment of vesicles containing NPC1 were mutated, resulting in proteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPC1 gene is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann–Pick type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of NPC1 in progesterone-treated cells. A recombinant protein representing the cysteine-rich loop was shown to bind to a zinc–NTA agarose column. We conclude: (1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPC1 gene; (2) that NPC1 expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralumenal cysteine-rich loop with zinc-binding activity is critical to NPC1's ability to unload lysosomal cargo.

NPC1-Containing Compartment of Human Granulosa-Lutein Cells: A Role in the Intracellular Trafficking of Cholesterol Supporting Steroidogenesis

Steroidogenic cells represent unique systems for the exploration of intracellular cholesterol trafficking. We employed cytochemical and biochemical methods to explore the expression, regulation, and function of the Niemann-Pick C1 protein (NPC1) in human granulosa-lutein cells. NPC1 was localized in a subset of lysosome-associated membrane glycoprotein 2 (LAMP-2)-positive vesicles. By analyzing the sensitivity of NPC1 N-linked oligosaccharide chains to glycosidases and neuraminidase, evidence was obtained for movement of nascent NPC1 from the endoplasmic reticulum through the medial and trans compartments of the Golgi apparatus prior to its appearance in cytoplasmic vesicles. NPC1 protein content and the morphology and cellular distribution of NPC1-containing vesicles were not affected by treatment of the granulosa-lutein cells with 8-Br-cAMP, which stimulates cholesterol metabolism into progesterone. In contrast, steroidogenic acute regulatory (StAR) protein levels were increased by 8-Br-cAMP. Incubation of granulosa-lutein cells with low-density lipoprotein (LDL) in the presence of the hydrophobic amine, U18666A, caused accumulation of free cholesterol in granules, identified by filipin staining, that contained LAMP-2 and NPC1. These granules also stained for neutral lipid with Nile red, reflecting accumulation of LDL-derived cholesterol esters. LDL-stimulated progesterone synthesis was completely blocked by U18666A, leaving steroid output at levels similar to those of cells incubated in the absence of LDL. The hydrophobic amine also blocked the LDL augmentation of 8-Br-cAMP-stimulated progesterone synthesis, reducing steroid production to levels seen in cells stimulated with 8-Br-cAMP in the absence of LDL. Steroidogenesis recovered after U18666A was removed from the culture medium. U18666A treatment caused a 2-fold or more increase in NPC1 protein and mRNA levels, suggesting that disruption of NPC1's function activates a compensatory mechanism resulting in increased NPC1 synthesis. We conclude that the NPC1 compartment plays an important role in the trafficking of LDL-derived substrate in steroidogenic cells; that NPC1 expression is up-regulated when NPC1 action is blocked; and that the NPC1 compartment can be functionally separated from other intracellular pathways contributing substrate for steroidogenesis.

Expression of Niemann–Pick type C transcript in rodent cerebellum in vivo and in vitro

This study assesses the developmental expression of the Niemann–Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human ( NPC1) and mouse ( Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.