u-18666a and Niemann-Pick-Disease--Type-C

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has resulted in the death of more than 328,000 persons worldwide in the first 5 months of 2020. Herculean efforts to rapidly design and produce vaccines and other antiviral interventions are ongoing. However, newly evolving viral mutations, the prospect of only temporary immunity, and a long path to regulatory approval pose significant challenges and call for a common, readily available, and inexpensive treatment. Strategic drug repurposing combined with rapid testing of established molecular targets could provide a pause in disease progression. SARS-CoV-2 shares extensive structural and functional conservation with SARS-CoV-1, including engagement of the same host cell receptor (angiotensin-converting enzyme 2) localized in cholesterol-rich microdomains. These lipid-enveloped viruses encounter the endosomal/lysosomal host compartment in a critical step of infection and maturation. Niemann-Pick type C (NP-C) disease is a rare monogenic neurodegenerative disease caused by deficient efflux of lipids from the late endosome/lysosome (LE/L). The NP-C disease-causing gene (NPC1) has been strongly associated with viral infection, both as a filovirus receptor (e.g., Ebola) and through LE/L lipid trafficking. This suggests that NPC1 inhibitors or NP-C disease mimetics could serve as anti-SARS-CoV-2 agents. Fortunately, there are such clinically approved molecules that elicit antiviral activity in preclinical studies, without causing NP-C disease. Inhibition of NPC1 may impair viral SARS-CoV-2 infectivity via several lipid-dependent mechanisms, which disturb the microenvironment optimum for viral infectivity. We suggest that known mechanistic information on NPC1 could be utilized to identify existing and future drugs to treat COVID-19.

Topics: Androstenes; Angiotensin-Converting Enzyme 2; Anticholesteremic Agents; Antiviral Agents; Betacoronavirus; Cholesterol; Coronavirus Infections; COVID-19; Drug Repositioning; Humans; Hydroxychloroquine; Intracellular Signaling Peptides and Proteins; Lysosomes; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Pandemics; Peptidyl-Dipeptidase A; Pneumonia, Viral; Protein Binding; Receptors, Virus; SARS-CoV-2; Spike Glycoprotein, Coronavirus

Neuronal cell death can occur by means of either necrosis or apoptosis. Both necrosis and apoptosis are generally believed to be distinct mechanisms of cell death with different characteristic features distinguished on the basis of their morphological and biochemical properties. The brain is the most cholesterol-rich organ in the body but not much is known about the mechanisms that regulate cholesterol homeostasis in the brain. Recently, several clinical and biochemical studies suggest that cholesterol imbalance in the brain may be a risk factor related to the development of neurological disorders such as Niemann-Pick disease type C (NPC) and Alzheimer's disease (AD). NPC is a fatal juvenile neurodegenerative disorder characterized by premature neuronal death and somatically altered cholesterol metabolism. The main biochemical manifestation in NPC is elevated intracellular accumulation of free cholesterol caused by a genetic deficit in cholesterol trafficking. The pharmacological agent, U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), is a well-known class-2 amphiphile which inhibits cholesterol transport. Cells treated with this agent accumulate intracellular cholesterol to massive levels, similar to that observed in cells from NPC patients. NPC and AD have some pathological similarities which may share a common underlying cause. AD is one of the most common types of dementia affecting the elderly. However, the molecular mechanisms of neurodegeneration in NPC and AD are largely unknown. This review provides a consolidation of work done using U18666A in the past half century and focuses on the implications of our research findings on the mechanism of U18666A-mediated neuronal apoptosis in primary cortical neurons, which may provide an insight to elucidate the mechanisms of neurodegenerative diseases, particularly NPC and AD, where apoptosis might occur through a similar mechanism.

Topics: Alzheimer Disease; Androstenes; Animals; Anticholesteremic Agents; Apoptosis; Cerebral Cortex; Cholesterol; Humans; Neurons; Niemann-Pick Disease, Type C

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

Topics: Androstenes; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Cell Line, Tumor; Cholesterol; Fibroblasts; Genistein; HeLa Cells; Humans; Lysosomal Storage Diseases; Lysosomes; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C

Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.

Topics: Androstenes; Animals; Cytosol; Endosomes; Fibroblasts; Glucosylceramides; Humans; Lysosomes; Mice; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C

Niemann-Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease-causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), a cholesterol transport-inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A-treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A-treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A-treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.

Topics: Acetylation; Adolescent; Adult; Androstenes; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Separation; Cells, Cultured; Cholesterol; Cilia; Cytoplasm; Down-Regulation; Female; Fibroblasts; Filipin; Fluorescent Antibody Technique; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Microscopy, Fluorescence; Middle Aged; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Patched-1 Receptor; Primary Cell Culture; Tubulin; Young Adult

Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

Topics: Androstenes; Biological Transport; Cells, Cultured; Chikungunya virus; Cholesterol; Fibroblasts; Humans; Imipramine; Niemann-Pick Disease, Type C; Skin; Virus Internalization; Virus Replication

While lysosomes are degradative compartments and one of the defenses against invading pathogens, they are also hubs of metabolic activity. Late endocytic compartments accumulate around

Topics: Androstenes; Animals; Cholesterol; Endosomes; Liver; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 1; Lysosomal-Associated Membrane Protein 2; Lysosomes; Membrane Proteins; Niemann-Pick Disease, Type C; Parasites; Plasmodium berghei

Cathepsin D is an aspartyl protease that plays a crucial role in normal cellular functions and in a variety of neurodegenerative disorders, including Niemann-Pick type C (NPC) disease, which is characterized by intracellular accumulation of cholesterol and glycosphingolipids in many tissues, including the brain. There is evidence that the level and activity of cathepsin D increased markedly in vulnerable neurons in NPC pathology, but its involvement in neurodegeneration remains unclear. In the present study, using mouse hippocampal cultured neurons, we evaluated the significance of cathepsin D in toxicity induced by U18666A, a class II amphiphile, which triggers cell death by impairing the trafficking of cholesterol, as observed in NPC pathology. Our results showed that U18666A-mediated toxicity is accompanied by an increase in cathepsin D mRNA and enzyme activity but a decrease in the total peptide content. The cytosolic level of cathepsin D, on the other hand, was increased along with cytochrome c and activated caspase-3 in U18666A-treated neurons. The cathepsin D inhibitor, pepstatin A, partially protected neurons against toxicity by attenuating these signaling mechanisms. Additionally, down-regulation of cathepsin D level prevented, whereas overexpression of the protease increased, vulnerability of cultured N2a cells to U18666A-induced toxicity. We also showed that extracellular cathepsin D from U18666A-treated neurons or application of exogenous enzyme can induce neurotoxicity by activating the autophagic pathway. These results suggest that increased release/activation of cathepsin D can trigger neurodegeneration and possibly development of NPC pathology. Thus, targeting cathepsin D level/activity may provide a new therapeutic opportunity for the treatment of NPC pathology.

Topics: Androstenes; Animals; Autophagy-Related Protein 5; Biomarkers; Caspase 3; Cathepsin D; Cell Death; Cell Survival; Cells, Cultured; Cholesterol; Cytochromes c; Extracellular Space; Fibroblasts; Hippocampus; Humans; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Neurons; Niemann-Pick Disease, Type C; Protease Inhibitors; Staurosporine

Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) α (cPLA(2) α) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) α knockdown L929 cells. These results suggest that cPLA(2) α-AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.

Topics: Androstenes; Animals; Arachidonic Acid; Carrier Proteins; Cell Cycle Checkpoints; Cell Death; CHO Cells; Cholesterol; Cricetinae; Cyclooxygenase 1; Cyclooxygenase 2; Cytosol; Dinoprostone; Fibrosarcoma; Group IV Phospholipases A2; Membrane Glycoproteins; Mice; Niemann-Pick Disease, Type C; Reactive Oxygen Species; Tumor Cells, Cultured

Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.

Topics: Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Androstenes; Animals; CHO Cells; Cholesterol; Cricetinae; Endocytosis; Endosomes; Hippocampus; Immunohistochemistry; Microscopy, Confocal; Neurons; Niemann-Pick Disease, Type C; Rats; Transfection

Niemann-Pick type C disease (NPC) is a lysosomal storage disorder causing accumulation of unesterified cholesterol in lysosomal storage organelles. Recent studies have shown that hydroxypropyl-beta-cyclodextrin injections in npc1(-/-) mice are partially effective in treating this disease. Using cultured fibroblasts, we have investigated the cellular mechanisms responsible for reduction of cholesterol accumulation. We show that decreased levels of cholesterol accumulation are maintained for several days after removal of cyclodextrin from the culture medium. This suggests that endocytosed cyclodextrin can reduce the cholesterol storage by acting from inside endocytic organelles rather than by removing cholesterol from the plasma membrane. To test this further, we incubated both NPC1 and NPC2 mutant cells with cholesterol-loaded cyclodextrin for 1 h, followed by chase in serum-containing medium. Although the cholesterol content of the treated cells increased after the 1-h incubation, the cholesterol levels in the storage organelles were later reduced significantly. We covalently coupled cyclodextrin to fluorescent dextran polymers. These cyclodextrin-dextran conjugates were delivered to cholesterol-enriched lysosomal storage organelles and were effective at reducing the cholesterol accumulation. We demonstrate that methyl-beta-cyclodextrin is more potent than hydroxypropyl-beta-cyclodextrin in reducing both cholesterol and bis(monoacylglycerol) phosphate accumulation in NPC mutant fibroblasts. Brief treatment of cells with cyclodextrins causes an increase in cholesterol esterification by acyl CoA:cholesterol acyl transferase, indicating increased cholesterol delivery to the endoplasmic reticulum. These findings suggest that cyclodextrin-mediated enhanced cholesterol transport from the endocytic system can reduce cholesterol accumulation in cells with defects in either NPC1 or NPC2.

Topics: Androstenes; Animals; beta-Cyclodextrins; Biological Transport, Active; Carrier Proteins; Cell Line; Cholesterol; Cholesterol Esters; Endocytosis; Fibroblasts; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Disease, Type C; Vesicular Transport Proteins

In human Niemann-Pick Type C (NPC) disease, endosomal trafficking defects lead to an accumulation of free cholesterol and other lipids in late endosome/lysosome (LE/LY) compartments, a subsequent block in cholesterol esterification and significantly reduced cholesterol efflux out of the cell. Here we report that nucleotide cycling or cellular knockdown of the small GTP-binding protein, ARF6, markedly impacts cholesterol homeostasis. Unregulated ARF6 activation attenuates the NPC phenotype at least in part by decreasing cholesterol accumulation and restoring normal sphingolipid trafficking. These effects depend on ARF6-stimulated cholesterol efflux out of the endosomal recycling compartment, a major cell repository for free cholesterol. We also show that fibroblasts derived from different NPC patients displayed varying levels of ARF6 that is GTP-bound, which correlate with their response to sustained ARF6 activation. These studies support emerging evidence that early endocytic defects impact NPC disease and suggest that such heterogeneity in NPC disease could result in diverse responses to therapeutic interventions aimed at modulating the trafficking of lipids.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Androstenes; Anticholesteremic Agents; Cholesterol; Endosomes; Filipin; Golgi Apparatus; HeLa Cells; Humans; Lipid Metabolism; Lysosomal Membrane Proteins; Niemann-Pick Disease, Type C; Recombinant Fusion Proteins; RNA, Small Interfering

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

Topics: Adaptor Proteins, Signal Transducing; Androstenes; Animals; Anticholesteremic Agents; Carrier Proteins; Cell Line; Cholesterol; Dynactin Complex; Endoplasmic Reticulum; Endosomes; Humans; Lovastatin; Microtubule-Associated Proteins; Models, Molecular; Niemann-Pick Disease, Type C; Protein Binding; Protein Conformation; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Receptors, Steroid; Recombinant Fusion Proteins; Vesicular Transport Proteins

Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with modified low density lipoprotein, eventually becoming foam cells that display altered immune responsiveness. We studied the effects of cholesterol accumulation on phagosome formation and maturation, using lipid transport antagonists and cholesterol transport-deficient mutants. In macrophages treated with U18666A, a transport antagonist that prevents cholesterol exit from late endosomes/lysosomes, the early stages of maturation proceeded normally; phagosomes acquired Rab5, phosphatidylinositol 3-phosphate, and EEA1 and merged with LAMP-containing vesicles. However, fusion with lysosomes was impaired. Rab7, which is required for phagolysosome formation, was acquired by phagosomes but remained inactive. Maturation was also studied in fibroblasts from Niemann-Pick type C individuals that have defective cholesterol transport. Transfection of FcgammaIIA receptors was used to confer phagocytic capability to these fibroblasts. Niemann-Pick type C phagosomes failed to fuse with lysosomes, whereas wild type fibroblasts formed normal phagolysosomes. These findings indicate that cholesterol accumulation can have a detrimental effect on phagosome maturation by impairing the activation of Rab7, sequestering it and its effectors in cholesterol-enriched multilamellar compartments.

Topics: Androstenes; Animals; Anticholesteremic Agents; Atherosclerosis; Cell Line; Cholesterol; Endosomes; Fibroblasts; Humans; Lysosomal Membrane Proteins; Lysosomes; Macrophages; Membrane Fusion; Mice; Niemann-Pick Disease, Type C; Phagosomes; Phosphatidylinositol Phosphates; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; rab7 GTP-Binding Proteins; Receptors, IgG; Vesicular Transport Proteins