Critical role for glycosphingolipids in Niemann-Pick disease type C

Niemann-Pick type C (NPC) disease is a cholesterol lipidosis caused by mutations in NPC1 and NPC2 gene loci. Most human cases are caused by defects in NPC1, as are the spontaneously occurring NPC diseases in mice and cats. NPC1 protein possesses a sterol-sensing domain and has been localized to vesicles that are believed to facilitate the recycling of unesterified cholesterol from late endosomes/lysosomes to the ER and Golgi. In addition to accumulating cholesterol, NPC1-deficient cells also accumulate gangliosides and other glycosphingolipids (GSLs), and neuropathological abnormalities in NPC disease closely resemble those seen in primary gangliosidoses. These findings led us to hypothesize that NPC1 may also function in GSL homeostasis. To evaluate this possibility, we treated murine and feline NPC models with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glucosylceramide synthase, a pivotal enzyme in the early GSL synthetic pathway. Treated animals showed delayed onset of neurological dysfunction, increased average life span (in mice), and reduced ganglioside accumulation and accompanying neuropathological changes. These results are consistent with our hypothesis and with GSLs being centrally involved in the pathogenesis of NPC disease, and they suggest that drugs inhibiting GSL synthesis could have a similar ameliorating effect on the human disorder.     

The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1

Niemann-Pick C1-like 1 (NPC1L1) is a polytopic transmembrane protein that plays a critical role in cholesterol absorption. Ezetimibe, a hypocholesterolemic drug, has been reported to bind NPC1L1 and block cholesterol absorption. However, the molecular mechanism of NPC1L1-mediated cholesterol uptake and how ezetimibe inhibits this process are poorly defined. Here we find that cholesterol specifically promotes the internalization of NPC1L1 and that this process requires microfilaments and the clathrin/AP2 complex. Blocking NPC1L1 endocytosis dramatically decreases cholesterol internalization, indicating that NPC1L1 mediates cholesterol uptake via its vesicular endocytosis. Ezetimibe prevents NPC1L1 from incorporating into clathrin-coated vesicles and thus inhibits cholesterol uptake. Together, our data suggest a model wherein cholesterol is internalized into cells with NPC1L1 through clathrin/AP2-mediated endocytosis and ezetimibe inhibits cholesterol absorption by blocking the internalization of NPC1L1.     

Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.     

Knockdown of miR-214 Promotes Apoptosis and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma

MicroRNA-214 (MiR-214) is aberrantly expressed in several human tumors such as ovarian cancer and breast cancer. However, the role of miR-214 in nasopharyngeal carcinoma (NPC) is still unknown. In this study, we report that miR-214 was overexpressed in NPC cell lines and tissues. Silencing of miR-214 by LNA-antimiR-214 in NPC cells resulted in promoting apoptosis and suppressing cell proliferation in vitro, and suppressed tumor growth in nude mice in vivo. Luciferase reporter assay was performed to identify Bim as a direct target of miR-214. Furthermore, this study showed that low Bim expression in NPC tissues correlated with poor survival of NPC patients. Taken together, our findings suggest that miR-214 plays an important role in NPC carcinogenesis.     

Small molecule inhibitor TW-37 is tolerable and synergistic with chemotherapy in nasopharyngeal carcinoma

Chemotherapy is a crucial adjuvant therapy of advanced nasopharyngeal carcinoma (NPC). However, enhancing sensitivity and tolerance of chemotherapeutics in NPC treatment have been challenging. Both Bcl-2 and Mcl-1, 2 pro-survival proteins of Bcl-2 family, play essential roles on the chemotherapy tolerance of numerous cancers. In the present study, we explored the influences of TW-37, a small molecule inhibitor of Bcl-2 and Mcl-1, on the efficiency of chemotherapy for NPC. Oncomine cancer database shows that NPC tissues have higher expression of Bcl-2 and Mcl-1 than those of normal nasopharyngeal epithelial (NPE) tissues. And our results reveal that chemotherapeutics, Cisplatin (CDDP) and 5-Fluoracil (5-FU), result in the greater decrease of protein level of Bcl-2 and Mcl-1 in NPC cells than those in NPE cells. TW-37 does not have significant impact on the chemotherapeutics-treated NPE cell viability at a dosage that efficiently reduces chemotherapeutics-treated NPC cell viability. Moreover, impacts of TW-37 on the cell viability of chemotherapeutics-treated NPC cells are dependent on the expression of Bcl-2 and Mcl-1 in NPC cells. Further explorations suggest that TW-37 prominently promotes apoptosis in NPC cells under chemotherapeutics treatments but not in NPE cells. Meanwhile, TW-37 also remarkably reduces colony formation ability of chemotherapeutics-treated NPC cells. Importantly, in vivo models, TW-37 observably increases chemosensitivity of NPC tumors but has not markedly influence on the normal tissues in mice. In conclusion, our results point to TW-37 as a promising ancillary drug for the chemotherapy of NPC.     

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

On the octagonal structure of the nuclear pore complex: insights from coarse-grained models

The basic structure of the nuclear pore complex (NPC), conserved across almost all organisms from yeast to humans, persists in featuring an octagonal symmetry involving the nucleoporins that constitute the NPC ring. In this article, we seek to understand and evaluate the potential biomechanical reasons for this eightfold symmetry. Our analytical investigation shows that the eightfold symmetry maximizes the bending stiffness of each of the eight NPC spokes while our computational analyses identify the most likely deformation modes, frequencies, and associated kinetic energies of the NPC. These modes have energies close to other published findings using membrane analysis of the nuclear membrane pore opening, and deformation states in agreement with experimental observations. A better understanding of NPC mechanics is essential for characterizing the nucleocytoplasmic transport, which has a central importance in cell biology.     

Proteomic analysis of the mammalian nuclear pore complex

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.     

Dopaminergic modulation of neurogenesis in the subventricular zone of the adult brain

Dopaminergic receptors are expressed on neural precursor cells (NPCs) in the subventricular zone (SVZ) and are known to regulate NPC proliferation and differentiation fate in this region. We now report that this optimally requires the simultaneous activation of both D1-like and D2-like dopaminergic receptors with the agonists Bromocriptine, SKF-38393 and 7-OH-pipat maleate (BSP) in vitro. This is consistent with our previous findings that dopamine stimulates NPC proliferation through an EGF paracrine mechanism within the SVZ. Furthermore this combined dopamine agonist therapy rescues NPC proliferation in the SVZ in the 6-OHDA animal model of PD and importantly significantly increases neuronal differentiation in the olfactory bulb to a greater extent than we showed previously with L-dopa. This result has implications for the use of dopaminergic therapies in PD and in the development of such therapies focusing on upregulating SVZ neurogenesis.     

Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.     

A sharper instrument for dissecting signalling events: a specific AGC kinase inhibitor

Dietary and biliary cholesterol are taken up by intestinal epithelial cells and transported to the endoplasmic reticulum. At the endoplasmic reticulum, cholesterol is esterified, packaged into chylomicrons and secreted into the lymph for delivery to the bloodstream. NPC1L1 (Niemann-Pick C1-like 1) is a protein on the enterocyte brush-border membrane that facilitates cholesterol absorption. Cholesterol's itinerary as it moves to the endoplasmic reticulum is unknown, as is the identity of any cellular proteins that facilitate the movement. Two proteins that play an important role in intracellular cholesterol transport and could potentially influence NPC1L1-mediated cholesterol uptake are NPC1 and NPC2 (Niemann-Pick type C disease proteins 1 and 2). In this issue of the Biochemical Journal, Dixit and colleagues show that the absence or presence of NPC1 and NPC2 has no effect on intestinal cholesterol absorption in the mouse. Thus neither protein fills the gap in our knowledge of intra-enterocyte cholesterol transport. Furthermore, the NPC1/NPC2 pathway would not be a good target for limiting the uptake of dietary cholesterol.     

NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

Aggregation,Phase Separation and Spatial Morphologies of the Assemblies of FG Nucleoporins

Nuclear pore complex (NPC) is a biomolecular “nanomachine” that controls nucleocytoplasmic transport in eukaryotic cells. The key component of the functional architecture of the NPC is the assembly of intrinsically disordered proteins that line its passageway and play a central role in the NPC transport mechanism. Due to paucity of experimental methods capable to directly probe the morphology of this assembly in intact NPCs, much of our knowledge about its properties derives from in vitro experiments augmented by theoretical and computational modeling. I review the major insights into the biophysics of the assemblies of the intrinsically disordered proteins of the NPC arising from the theoretical analysis of the recent in vitro experimental results, with the emphasis on the phase separation and aggregation phenomena.     

Effects of neonatal neural progenitor cell implantation on adult neuroanatomy and cognition in the Ts65Dn model of Down syndrome

As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model.     

Increased production of nitric oxide correlates with tumor growth in Algerian patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is thought to arise because of chronic inflammation. The correlation between nitric oxide (NO) production, a biomarker of inflammation and NPC development remains unexplored. To investigate this question, we performed a profile analysis on plasma collected from untreated, treated, remissive, cured and relapsing patients. Nitrites were measured to assess NO activity. We observed that increased nitrites concentrations in untreated and relapsing patients associated with tumor development. Moreover, nitrites levels were similar in remissive, cured and healthy individuals. Altogether, our results suggest that NO might be an interesting blood biomarker to monitor tumor growth in NPC patients.     

Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

The accumulation of an intratumoral CD4⁺ interleukin-17-producing subset (Th17) of tumor-infiltrating lymphocytes (TILs) is a general characteristic in many cancers. The relationship between the percentage of Th17 cells and clinical prognosis differs among cancers. The mechanism responsible for the increasing percentage of such cells in NPC is still unknown, as is their biological function. Here, our data showed an increase of Th17 cells in tumor tissues relative to their numbers in normal nasopharynx tissues or in the matched peripheral blood of NPC patients. Th17 cells in tumor tissue produced more IFNγ than did those in the peripheral blood of matched NPC patients and healthy controls. We observed high levels of CD154, G-CSF, CXCL1, IL-6, IL-8, and macrophage inhibitory factor (MIF) out of 36 cytokines examined in tumor tissue cultures. MIF promoted the generation and recruitment of Th17 cells mediated by NPC tumor cells in vitro; this promoting effect was mainly dependent on the mammalian target of rapamycin pathway and was mediated by the MIF-CXCR4 axis. Finally, the expression level of MIF in tumor cells and in TILs was positively correlated in NPC tumor tissues, and the frequency of MIF-positive TILs was positively correlated with NPC patient clinical outcomes. Taken together, our findings illustrate that tumor-derived MIF can affect patient prognosis, which might be related to the increase of Th17 cells in the NPC tumor microenvironment.     

Nuclear transport of baculovirus: revealing the nuclear pore complex passage

Baculoviruses are one of the largest viruses that replicate in the nucleus of their host cells. During an infection the capsid, containing the DNA viral genome, is released into the cytoplasm and delivers the genome into the nucleus by a mechanism that is largely unknown. Here, we used capsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus in combination with electron microscopy and discovered this capsid crosses the NPC and enters into the nucleus intact, where it releases its genome. To better illustrate the existence of this capsid through the NPC in its native conformation, we reconstructed the nuclear import event using electron tomography. In addition, using different experimental conditions, we were able to visualize the intact capsid interacting with NPC cytoplasmic filaments, as an initial docking site, and midway through the NPC. Our data suggests the NPC central channel undergoes large-scale rearrangements to allow translocation of the intact 250-nm long baculovirus capsid. We discuss our results in the light of the hypothetical models of NPC function.     

Excessive microglial activation aggravates olfactory dysfunction by impeding the survival of newborn neurons in the olfactory bulb of Niemann–Pick disease type C1 mice

Progressive olfactory impairment is one of the earliest markers of neurodegeneration. However, the underlying mechanism for this dysfunction remains unclear. The present study investigated the possible role of microgliosis in olfactory deficits using a mouse model of Niemann–Pick disease type C1 (NPC1), which is an incurable neurodegenerative disorder with disrupted lipid trafficking. At 7 weeks of age, NPC1 mutants showed a distinct olfactory impairment in an olfactory test compared with age-matched wild-type controls (WT). The marked loss of olfactory sensory neurons within the NPC1 affected olfactory bulb (NPC1-OB) suggests that NPC1 dysfunction impairs olfactory structure. Furthermore, the pool of neuroblasts in the OB was diminished in NPC1 mice despite the intact proliferative capacity of neural stem/progenitor cells in the subventricular zone. Instead, pro-inflammatory proliferating microglia accumulated extensively in the NPC1-OB as the disease progressed. To evaluate the impact of abnormal microglial activation on olfaction in NPC1 mice, a microglial inhibition study was performed using the anti-inflammatory agent Cyclosporin A (CsA). Importantly, long-term CsA treatment in NPC1 mice reduced reactive microgliosis, restored the survival of newly generated neurons in the OB and improved overall performance on the olfactory test. Therefore, our study highlights the possible role of microglia in the regulation of neuronal turnover in the OB and provides insight into the possible therapeutic applications of microglial inhibition in the attenuation or reversal of olfactory impairment.