Lipid Trafficking Defects Increase Beclin-1 and Activate Autophagy in Niemann-Pick Type C Disease

Niemann-Pick type C disease (NPC) is a sphingolipid storage disorder characterized by progressive neurodegeneration that typically shows juvenile onset. Mutations in the Npc1 gene cause ~95% of NPC cases. NPC1 is a multipass transmembrane protein involved in lipid and cholesterol trafficking. Loss of function mutations in Npc1 lead to the accumulation of sphingolipids and cholesterol in late endosomes and lysosomes. In our study, we demonstrated that NPC1 deficiency results in increased basal autophagy in human fibroblasts and in mice. We further demonstrated that NPC1 deficiency activates basal autophagy through increased expression of Beclin-1, a highly conserved member of the class III PI3K complex that is critical for the formation of autophagosomes. In contrast, enhanced basal autophagy was not associated with activation of the Akt–mTOR–p70 S6K signaling pathway. Increased Beclin-1 levels and elevated autophagy were also observed in other sphingolipid storage diseases characterized by disrupted cholesterol and sphingolipid trafficking. We propose a model in which the disordered cholesterol trafficking that occurs in many sphingolipid storages diseases results in upregulation of Beclin-1 and enhanced levels of autophagy.

Addendum to:

Autophagy in Niemann-Pick Type C is Beclin-1 Dependent and Responsive to Lipid Trafficking Defects

C.D. Pacheco, R. Kunkle and A.P. Lieberman

Human Mol Genet 2007; 16:1495-503     

Lipid homeostasis and lipoprotein secretion in Niemann-Pick C1-deficient hepatocytes

Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1(-/-) hepatocytes was 5-fold higher than that of Npc1(+/+) hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1(-/-) hepatocytes was 50% lower than of Npc1(+/+) hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis.     

Pre-Symptomatic Activation of Antioxidant Responses and Alterations in Glucose and Pyruvate Metabolism in Niemann-Pick Type C1-Deficient Murine Brain

Niemann-Pick Type C (NPC) disease is an autosomal recessive neurodegenerative disorder caused in most cases by mutations in the NPC1 gene. NPC1-deficiency is characterized by late endosomal accumulation of cholesterol, impaired cholesterol homeostasis, and a broad range of other cellular abnormalities. Although neuronal abnormalities and glial activation are observed in nearly all areas of the brain, the most severe consequence of NPC1-deficiency is a near complete loss of Purkinje neurons in the cerebellum. The link between cholesterol trafficking and NPC pathogenesis is not yet clear; however, increased oxidative stress in symptomatic NPC disease, increases in mitochondrial cholesterol, and alterations in autophagy/mitophagy suggest that mitochondria play a role in NPC disease pathology. Alterations in mitochondrial function affect energy and neurotransmitter metabolism, and are particularly harmful to the central nervous system. To investigate early metabolic alterations that could affect NPC disease progression, we performed metabolomics analyses of different brain regions from age-matched wildtype and Npc1 ^-/- mice at pre-symptomatic, early symptomatic and late stage disease by ¹H-NMR spectroscopy. Metabolic profiling revealed markedly increased lactate and decreased acetate/acetyl-CoA levels in Npc1 ^-/- cerebellum and cerebral cortex at all ages. Protein and gene expression analyses indicated a pre-symptomatic deficiency in the oxidative decarboxylation of pyruvate to acetyl-CoA, and an upregulation of glycolytic gene expression at the early symptomatic stage. We also observed a pre-symptomatic increase in several indicators of oxidative stress and antioxidant response systems in Npc1 ^-/- cerebellum. Our findings suggest that energy metabolism and oxidative stress may present additional therapeutic targets in NPC disease, especially if intervention can be started at an early stage of the disease.     

Altered vitamin E status in Niemann-Pick type C disease

Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in many species. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene, which regulates lipid transport through the endocytic pathway. NPC disease is characterized by massive intracellular accumulation of unesterified cholesterol and other lipids in lysosomal vesicles. We examined the roles that NPC1/2 proteins play in the intracellular trafficking of tocopherol. Reduction of NPC1 or NPC2 expression or function in cultured cells caused a marked lysosomal accumulation of vitamin E in cultured cells. In vivo, tocopherol significantly accumulated in murine Npc1-null and Npc2-null livers, Npc2-null cerebella, and Npc1-null cerebral cortices. Plasma tocopherol levels were within the normal range in Npc1-null and Npc2-null mice, and in plasma samples from human NPC patients. The binding affinity of tocopherol to the purified sterol-binding domain of NPC1 and to purified NPC2 was significantly weaker than that of cholesterol (measurements kindly performed by R. Infante, University of Texas Southwestern Medical Center, Dallas, TX). Taken together, our observations indicate that functionality of NPC1/2 proteins is necessary for proper bioavailability of vitamin E and that the NPC pathology might involve tissue-specific perturbations of vitamin E status.     

Autophagy links inflammasomes to atherosclerotic progression

We investigated the role of autophagy in atherosclerosis. During plaque formation in mice, autophagic markers colocalized predominantly with macrophages (mφ). Atherosclerotic aortas had elevated levels of p62, suggesting that dysfunctional autophagy is characteristic of plaques. To determine whether autophagy directly influences atherogenesis, we characterized Beclin-1 heterozygous-null and mφ-specific ATG5-null (ATG5-mφKO) mice, commonly used models of autophagy haploinsufficiency and deficiency, respectively. Haploinsufficent Beclin-1 mice had no atherosclerotic phenotype, but ATG5-mφKO mice had increased plaques, suggesting an essential role for basal levels of autophagy in atheroprotection. Defective autophagy is associated with proatherogenic inflammasome activation. Classic inflammasome markers were robustly induced in ATG5-null mφ, especially when coincubated with cholesterol crystals. Moreover, cholesterol crystals appear to be increased in ATG5-mφKO plaques, suggesting a potentially vicious cycle of crystal formation and inflammasome activation in autophagy-deficient plaques. These results show that autophagy becomes dysfunctional in atherosclerosis and its deficiency promotes atherosclerosis in part through inflammasome hyperactivation.     

Chronic Cyclodextrin Treatment of Murine Niemann-Pick C Disease Ameliorates Neuronal Cholesterol and Glycosphingolipid Storage and Disease Progression

Background

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused most commonly by a defect in the NPC1 protein and characterized by widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids (GSLs). While current treatment therapies are limited, a few drugs tested in Npc1^−/− mice have shown partial benefit. During a combination treatment trial using two such compounds, N-butyldeoxynojirimycin (NB-DNJ) and allopregnanolone, we noted increased lifespan for Npc1^−/− mice receiving only 2-hydroxypropyl-β-cyclodextrin (CD), the vehicle for allopregnanolone. This finding suggested that administration of CD alone, but with greater frequency, might provide additional benefit.

Methodology/Principal Findings

Administration of CD to Npc1^−/− mice beginning at either P7 or P21 and continuing every other day delayed clinical onset, reduced intraneuronal cholesterol and GSL storage as well as free sphingosine accumulation, reduced markers of neurodegeneration, and led to longer survival than any previous treatment regime. We reasoned that other lysosomal diseases characterized by cholesterol and GSL accumulation, including NPC disease due to NPC2 deficiency, GM1 gangliosidosis and mucopolysaccharidosis (MPS) type IIIA, might likewise benefit from CD treatment. Treated Npc2^−/− mice showed benefits similar to NPC1 disease, however, mice with GM1 gangliosidosis or MPS IIIA failed to show reduction in storage.

Conclusions/Significance

Treatment with CD delayed clinical disease onset, reduced intraneuronal storage and secondary markers of neurodegeneration, and significantly increased lifespan of both Npc1^−/− and Npc2^−/− mice. In contrast, CD failed to ameliorate cholesterol or glycosphingolipid storage in GM1 gangliosidosis and MPS IIIA disease. Understanding the mechanism(s) by which CD leads to reduced neuronal storage may provide important new opportunities for treatment of NPC and related neurodegenerative diseases characterized by cholesterol dyshomeostasis.     

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1(-/-) mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1(+/+) and Npc1(-/-) microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1(-/-) microglia is ~60% lower than in Npc1(+/+) microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1(-/-) and Npc1(+/+) neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1(-/-) microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.     

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative disorder caused in 95% of cases by loss of function of NPC1, a ubiquitous endosomal transmembrane protein. A biochemical hallmark of NPC deficiency is cholesterol accumulation in the endocytic pathway. Although cholesterol trafficking defects are observed in all cell types, neurons are the most vulnerable to NPC1 deficiency, suggesting a specialized function for NPC1 in neurons. We investigated the subcellular localization of NPC1 in neurons to gain insight into the mechanism of action of NPC1 in neuronal metabolism. We show that NPC1 is abundant in axons of sympathetic neurons and is present in recycling endosomes in presynaptic nerve terminals. NPC1 deficiency causes morphological and biochemical changes in the presynaptic nerve terminal. Synaptic vesicles from Npc1(-/-) mice have normal cholesterol content but altered protein composition. We propose that NPC1 plays a previously unrecognized role in the presynaptic nerve terminal and that NPC1 deficiency at this site might contribute to the progressive neurological impairment in NPC disease.     

Autophagic-lysosomal dysfunction and neurodegeneration in Niemann-Pick Type C mice: lipid starvation or indigestion?

Increasing evidence shows that autophagy, particularly macroautophagy, plays a Dr. Jekyll and Mr. Hyde role in determining cell fate; autophagic activity can be protective under certain conditions, whereas it may lead to cell death under others. Niemann-Pick Type C (NPC) disease is an early onset autosomal recessive disorder characterized by accumulation of cholesterol and other lipids in late endosomes/lysosomes. About 95% of the cases are caused by mutations in the NPC1 gene, whereas the remaining 5% are due to mutations in the NPC2 gene. Severe neurodegeneration that accompanies NPC is likely the fatal cause in this disease, although the underlying mechanism remains unclear. Our study shows that autophagic activity is enhanced in Npc1-/- mice, as evidenced by increased levels of LC3-II and the number of autophagic vacuole-like structures. Interestingly, LC3 immunoreactivity co-localizes with filipin-labeled cholesterol clusters inside Purkinje cells. Furthermore, increases in autophagic activity are closely associated with alteration in lysosomal function and protein ubiquitination. In this article, these results are further discussed in the context of autophagic-lysosomal function and neuronal survival and degeneration.     

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.     

Autophagic-Lysosomal Dysfunction and Neurodegeneration in Niemann-Pick Type C Mice: Lipid Starvation or Indigestion?

Increasing evidence shows that autophagy, particularly macroautophagy, plays a Dr. Jekyll and Mr. Hyde role in determining cell fate; autophagic activity can be protective under certain conditions, whereas it may lead to cell death under others. Niemann-Pick Type C (NPC) disease is an early onset autosomal recessive disorder characterized by accumulation of cholesterol and other lipids in late endosomes/lysosomes. About 95% of the cases are caused by mutations in the NPC1 gene, whereas the remaining 5% are due to mutations in the NPC2 gene. Severe neurodegeneration that accompanies NPC is likely the fatal cause in this disease, although the underlying mechanism remains unclear. Our study shows that autophagic activity is enhanced in Npc1^-/- mice, as evidenced by increased levels of LC3-II and the number of autophagic vacuole-like structures. Interestingly, LC3 immunoreactivity co-localizes with filipin-labeled cholesterol clusters inside Purkinje cells. Furthermore, increases in autophagic activity are closely associated with alteration in lysosomal function and protein ubiquitination. In this article, these results are further discussed in the context of autophagic-lysosomal function and neuronal survival and degeneration.

Addendum to:

Cholesterol Accumulation is Associated with Lysosomal Dysfunction and Autophagic Stress in Npc1^-/- Mouse Brain

G. Liao, Y. Yao, J. Liu, Z. Yu, S. Cheung, A. Xie, X. Liang and X. Bi

Am J Pathol 2007; 171:962-75     

Increased copper levels in in vitro and in vivo models of Niemann-Pick C disease

Niemann-Pick type C disease (NPC) is a hereditary neurovisceral atypical lipid storage disorder produced by mutations in the NPC1 and NPC2 genes. The disease is characterized by unesterified cholesterol accumulation in late endosomal/lysosomal compartments and oxidative stress. The most affected tissues are the cerebellum and the liver. The lysotropic drug U18666A (U18) has been widely used as a pharmacological model to induce the NPC phenotype in several cell culture lines. It has already been reported that there is an increase in copper content in hepatoma Hu7 cells treated with U18. We confirmed this result with another human hepatoma cell line, HepG2, treated with U18 and supplemented with copper in the media. However, in mouse hippocampal primary cultures treated under similar conditions, we did not find alterations in copper content. We previously reported increased copper content in the liver of Npc1 (-/-) mice compared to control animals. Here, we extended the analysis to the copper content in the cerebella, the plasma and the bile of NPC1 deficient mice. We did not observe a significant change in copper content in the cerebella, whereas we found increased copper content in the plasma and decreased copper levels in the bile of Npc1(-/-) mice. Finally, we also evaluated the plasma content of ceruloplasmin, and we found an increase in this primary copper-binding protein in Npc1 (-/-) mice. These results indicate cell-type dependence of copper accumulation in NPC disease and suggest that copper transport imbalance may be relevant to the liver pathology observed in NPC disease.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

2-Hydroxypropyl-β-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) – comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG – shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.     

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1^−/− mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1^+/+ and Npc1^−/− microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1^−/− microglia is ~ 60% lower than in Npc1^+/+ microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1^−/− and Npc1^+/+ neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1^−/− microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.     

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy,improves lysosomal function,and reduces cholesterol storage

Niemann–Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1^I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.     

Ontogenic changes in lung cholesterol metabolism,lipid content,and histology in mice with Niemann–Pick type C disease

Niemann–Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1^−/− mice (Npc1^nih) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70 days of age). Similar measurements were made in Npc2^−/− mice at 70 days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1^−/− mice and remained so at 70 days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1^−/− mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1^−/− mice starting as early as 28 days. Similar metabolic and histologic changes were evident in the lungs of the Npc2^−/− mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.