Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease.
ABSTRACT: Niemann-Pick type C2 (NPC2) disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2(-/-) mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2(-/-) mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2(-/-) and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2(-/-) mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2(-/-) animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the treatment of the visceral manifestations in NPC2 disease and further suggest that neurodegeneration is not secondary to visceral dysfunction.
Project description:Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies (LB, mean area 2-fold larger), while NPC2-mutant cells had predominantly smaller lamellar bodies (mean area 50% of normal) than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities.
Project description:Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt(-/-)) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt(-/-) mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with (131)I-labeled 6?-iodocholesterol demonstrated that Gnmt(-/-) mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt(-/-) mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81-105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171-295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.
Project description:Backround: Steatohepatitis (SH)-associated liver carcinogenesis is an increasingly important issue in clinical medicine. SH is morphologically characterized by steatosis, hepatocyte injury, ballooning, hepatocytic cytoplasmic inclusions termed Mallory-Denk bodies (MDBs), inflammation and fibrosis.17-20-months-old Krt18-/- and Krt18+/- mice in contrast to wt mice spontaneously developed liver lesions closely resembling the morphological spectrum of human SH as well as liver tumors. The pathologic alterations were more pronounced in Krt18-/- than in Krt18+/- mice. The frequency of liver tumors with male predominance was significantly higher in Krt18-/- compared to age-matched Krt18+/- and wt mice. Krt18-deficient tumors in contrast to wt animals displayed SH features and often pleomorphic morphology. aCGH analysis of tumors revealed chromosomal aberrations in Krt18-/- liver tumors, affecting loci of oncogenes and tumor suppressor genes.Livers of 3-, 6-, 12- and 17-20-months-old aged wild type (wt), Krt18+/- and Krt18-/- (129P2/OlaHsd background) mice were analyzed by light and immunofluorescence microscopy as well as immunohistochemistry. Liver tumors arising in aged mice were analyzed by array comparative genomic hybridization (aCGH).Our findings show that K18 deficiency of hepatocytes leads to steatosis, increasing with age, and finally to SH. K18 deficiency and age promote liver tumor development in mice, frequently on the basis of chromosomal instability, resembling human HCC with stemness features.
Project description:Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH, suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the sterol response element-binding protein-2 (SREBP-2) transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP-2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine-specific overexpression of active SREBP-2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and their wild-type littermates were fed a regular chow diet or a high-fat, high-cholesterol (HFHC) diet (15% fat, 1% cholesterol) for 15 wk. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice compared with wild-type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice compared with wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver.NEW & NOTEWORTHY The current study highlights the role of overactivation of intestinal SREBP-2 transcription factor in the progression of hepatic fibrosis associated with diet-induced NASH. Mice with intestine-specific overexpression of SREBP-2 demonstrated more inflammation and severe fibrosis in the liver in response to 15 wk of being fed a high-cholesterol, high-fat diet as compared with their wild-type littermates. These data demonstrate a novel link between intestinal regulatory processes of cholesterol metabolism and the pathogenesis of fatty liver diseases.
Project description:Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death.
Project description:Niemann-Pick C disease (NPC) is a neurodegenerative lysosomal disorder characterized by storage of cholesterol and other lipids caused by defects in NPC1, a transmembrane protein involved in cholesterol export from the lysosome, or NPC2, an intralysosomal cholesterol transport protein. Alterations in lysosomal activities have been implicated in NPC pathogenesis therefore the aim of this study was to conduct a proteomic analysis of lysosomal proteins in mice deficient in either NPC1 or NPC2 to identify secondary changes that might be associated with disease. Lysosomal proteins containing the specific mannose 6-phosphate modification were purified from wild-type and Npc1(-/-) and Npc2(-/-) mutant mouse brains at different stages of disease progression and identified by bottom-up LC-MS/MS and quantified by spectral counting. Levels of a number of lysosomal proteins involved in lipid catabolism including prosaposin and the two subunits of ?-hexosaminidase were increased in both forms of NPC, possibly representing a compensatory cellular response to the accumulation of glycosphingolipids. Several other lysosomal proteins were significantly altered, including proteases and glycosidases. Changes in lysosomal protein levels corresponded with similar alterations in activities and transcript levels. Understanding the rationale for such changes may provide insights into the pathophysiology of NPC.
Project description:NPC (Niemann-Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, we have developed a method for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified ('free') fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, we show that the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. In addition, we show that, although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups.
Project description:The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.
Project description:TAF15, an RNA binding protein was recently implicated in Amyotrophic Lateral Sclerosis (ALS). ALS is a fatal neurodegenerative disease. We report the identification of the conserved neuronal RNA targets of TAF15 and the assessment of the impact of TAF15 depletion on the neuronal transcriptome. Our study uncovers regulation of splicing of sets of neuronal RNAs encoding proteins with essential roles in synaptic activities including glutamergic receptors such as zeta-1 subunit of the glutamate N-methyl-D-aspartate (NMDA) receptor (Grin1). Identification of TAF15 neuronal targets using normal human brain samples and mouse neurons. Mouse background: E14Tg2a.4 wildtype cells derived from 129P2/OlaHsd.
Project description:Export of LDL-derived cholesterol from lysosomes requires the cooperation of the integral membrane protein Niemann-Pick C1 (NPC1) and a soluble protein, Niemann-Pick C2 (NPC2). Mutations in the genes encoding these proteins lead to Niemann-Pick disease type C (NPC). NPC2 binds to NPC1's second (middle), lumenally oriented domain (MLD) and transfers cholesterol to NPC1's N-terminal domain (NTD). Here, we report the 2.4-Å resolution crystal structure of a complex of human NPC1-MLD and NPC2 bearing bound cholesterol-3-O-sulfate. NPC1-MLD uses two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. Docking of the NPC1-NPC2 complex onto the full-length NPC1 structure reveals a direct cholesterol transfer tunnel between NPC2 and NTD cholesterol binding pockets, supporting the "hydrophobic hand-off" cholesterol transfer model.