scholarly journals Endocytic trafficking of glycosphingolipids in sphingolipid storage diseases

2003 ◽  
Vol 358 (1433) ◽  
pp. 885-891 ◽  
Author(s):  
Richard E. Pagano
Keyword(s):  
Cell Types ◽  
Membrane Lipid ◽  
Dominant Negative ◽  
Cholesterol Homeostasis ◽  
Wild Type ◽  
Lipid Trafficking ◽  
Trafficking Defects ◽  
Niemann Pick ◽  
Phosphoinositide 3 Kinase ◽  
Future Therapy

In this review, recent studies of membrane lipid transport in sphingolipid (SL) storage disease (SLSD) fibroblasts are summarized. Several fluorescent glycosphingolipid (GSL) analogues are internalized from the plasma membrane via caveolae and are subsequently transported to the Golgi complex of normal fibroblasts, while in 10 different SLSD cell types, these lipids accumulate in endosomes and lysosomes. Additional studies have shown that cholesterol homeostasis is perturbed in multiple SLSDs secondary to accumulation of endogenous SLs, and that mis–targeting of the GSLs is regulated by cellular cholesterol. Golgi targeting of GSLs internalized via caveolae is dependent on microtubules and phosphoinositide 3–kinase(s) and is inhibited by expression of dominant–negative rab7 and rab9 constructs. Overexpression of wild–type rab7 or rab9 (but not rab11) in Niemann–Pick C fibroblasts results in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM1 ganglioside (monitored by the transport of fluorescent cholera toxin), and a dramatic reduction in accumulation of intracellular cholesterol. These results suggest an approach for restoring normal lipid trafficking in this, and perhaps other, SLSD cell types, and may provide a basis for future therapy of these diseases.

Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis

2021 ◽  
Author(s):  
Sara Awan ◽  
Magalie Lambert ◽  
Ali Imtiaz ◽  
Fabien Alpy ◽  
Catherine Tomasetto ◽  
...  
Keyword(s):  
Dietary Cholesterol ◽  
Cell Types ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Cholesterol Trafficking ◽  
Atherosclerotic Lesions ◽  
Crucial Component ◽  
Multiple Cell ◽  
Niemann Pick

Background: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and two lysosomal proteins, the Niemann-Pick C1 (NPC1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. Methods: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells (VSMCs). To establish whether Wnt5a also protects against cholesterol accumulation in human VSMCs, we used a CRISPR/Cas9 guided nuclease approach to generate human VSMCs knockout for Wnt5a. Results: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum (ER). Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the ER, and promoted atherosclerosis. Conclusions: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

scholarly journals The pathogenesis of Niemann–Pick type C disease: a role for autophagy?

2008 ◽  
Vol 10 ◽  
Author(s):  
Chris D. Pacheco ◽  
Andrew P. Lieberman
Keyword(s):  
Neuronal Loss ◽  
Neurological Impairment ◽  
Cell Stress ◽  
Model Systems ◽  
Class Iii ◽  
Lipid Trafficking ◽  
Promote Cell Survival ◽  
Type C ◽  
Niemann Pick ◽  
Phosphoinositide 3 Kinase

Niemann–Pick type C disease (NPC) is a sphingolipid-storage disorder that results from inherited deficiencies of intracellular lipid-trafficking proteins, and is characterised by an accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. Patients with this disorder develop progressive neurological impairment that often begins in childhood, is ultimately fatal and is currently untreatable. How impaired lipid trafficking leads to neurodegeneration is largely unknown. Here we review NPC clinical features and biochemical defects, and discuss model systems used to study this disorder. Recent studies have established that NPC is associated with an induction of autophagy, a regulated and evolutionarily conserved process by which cytoplasmic proteins are sequestered within autophagosomes and targeted for degradation. This pathway enables recycling of limited or damaged macromolecules to promote cell survival. However, in other instances, robust activation of autophagy leads to cell stress and programmed cell death. We summarise evidence showing that autophagy induction and flux are increased in NPC by signalling through a complex of the class III phosphoinositide 3-kinase and beclin-1. We propose that an imbalance between induction and flux through the autophagic pathway contributes to cell stress and neuronal loss in NPC and related sphingolipid-storage disorders, and discuss potential therapeutic strategies for modulating activity of this pathway.

A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades

2014 ◽  
Vol 306 (9) ◽  
pp. F1018-F1025 ◽  
Author(s):  
Cory Wilson ◽  
Stuart E. Dryer
Keyword(s):  
Transient Receptor Potential ◽  
Muscle Tone ◽  
Protein Complexes ◽  
Receptor Potential ◽  
Cell Types ◽  
Dominant Negative ◽  
Mutant Form ◽  
Wild Type ◽  
Transient Receptor ◽  
In Cells

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive.

scholarly journals Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells

2000 ◽  
Vol 20 (19) ◽  
pp. 7378-7387 ◽  
Author(s):  
Hironori Katoh ◽  
Hidekazu Yasui ◽  
Yoshiaki Yamaguchi ◽  
Junko Aoki ◽  
Hirotada Fujita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Morphological Changes ◽  
Cell Types ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Wild Type ◽  
Rho Family ◽  
Constitutively Active

ABSTRACT The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia

2001 ◽  
Vol 358 (2) ◽  
pp. 481-487 ◽  
Author(s):  
Toshihiko AKI ◽  
Yoichi MIZUKAMI ◽  
Yoshitomo OKA ◽  
Kazuhito YAMAGUCHI ◽  
Koichi UEMURA ◽  
...  
Keyword(s):  
Cell Death ◽  
Metabolic Acidosis ◽  
Glucose Metabolism ◽  
Dominant Negative ◽  
Hypoxic Cell ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoting Effect ◽  
Rat Cardiomyocytes ◽  
Phosphoinositide 3 Kinase

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.

scholarly journals Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia

2019 ◽  
Author(s):  
Alessio Colombo ◽  
Lina Dinkel ◽  
Stephan A. Müller ◽  
Laura Sebastian Monasor ◽  
Martina Schifferer ◽  
...  
Keyword(s):  
Neurodegenerative Disorder ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Pathological Feature ◽  
Clinical Tool ◽  
Lipid Trafficking ◽  
Trafficking Defects ◽  
Niemann Pick ◽  
Disease Signatures ◽  
Phagocytic Uptake

AbstractNiemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in Npc1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, consequences of NPC1 loss on microglial function remain uncharacterized. Here, we provide an in-depth characterization of microglial proteomic signatures and phenotypes in a NPC1-deficient (Npc1-/-) murine model and patient blood-derived macrophages. We demonstrate enhanced phagocytic uptake and impaired lipid trafficking in Npc1-/- microglia that precede neuronal death. Loss of NPC1 compromises microglial developmental functions as revealed by increased synaptic pruning and deficient myelin turnover. Undigested myelin accumulates within multi-vesicular bodies of Npc1-/- microglia while lysosomal degradation remains preserved. To translate our findings to human disease, we generated novel ex vivo assays using patient macrophages that displayed similar proteomic disease signatures and lipid trafficking defects as murine Npc1-/- microglia. Thus, peripheral macrophages provide a novel promising clinical tool for monitoring disease progression and therapeutic efficacy in NPC patients. Our study underscores an essential role for NPC1 in immune cells and implies microglial therapeutic potential.

scholarly journals Wild-type and mutant ferroportins do not form oligomers in transfected cells

2006 ◽  
Vol 396 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Ana Sofia Gonçalves ◽  
Françoise Muzeau ◽  
Rand Blaybel ◽  
Gilles Hetet ◽  
Fathi Driss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Kidney ◽  
Cell Types ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Form ◽  
Hek 293 ◽  
C Terminus ◽  
293 Cells ◽  
Iron Export

Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.

scholarly journals Autophagy in Niemann–Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects

2007 ◽  
Vol 16 (12) ◽  
pp. 1495-1503 ◽  
Author(s):  
Chris D. Pacheco ◽  
Robin Kunkel ◽  
Andrew P. Lieberman
Keyword(s):  
Beclin 1 ◽  
Lipid Trafficking ◽  
Trafficking Defects ◽  
Niemann Pick

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

Autophagy ◽  
2007 ◽  
Vol 3 (5) ◽  
pp. 487-489 ◽  
Author(s):  
Christopher D. Pacheco ◽  
Andrew P. Lieberman
Keyword(s):  
Beclin 1 ◽  
Lipid Trafficking ◽  
Type C ◽  
Trafficking Defects ◽  
Niemann Pick

scholarly journals Apoptotic Activities of Wild-Type and Alzheimer's Disease-Related Mutant Presenilins in Drosophila melanogaster

1999 ◽  
Vol 146 (6) ◽  
pp. 1351-1364 ◽  
Author(s):  
Yihong Ye ◽  
Mark E. Fortini
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Drosophila Melanogaster ◽  
Genetic Model ◽  
Cell Types ◽  
Notch Receptor ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Wild Type

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. We show that loss of presenilin function in Drosophila melanogaster increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact ∼300-kD precursor and its ∼120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. We describe a genetic model in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and we find that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin.

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