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1.
Brain Res ; 1642: 298-307, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27018294

ABSTRACT

Neurodegenerative diseases are frequently associated with hypoxic conditions. During hypoxia the neuronal cytoskeleton is rapidly reorganized and such abnormalities are directly linked to adverse outcomes. Besides their roles as master regulators of the cytoskeleton, the Rho GTPases are also involved in cellular processes stimulated by hypoxic stress. We investigated the contribution of Rac1-mediated signaling to hypoxic responses of mature neurons using primary cortical cells cultured for 17 days in vitro. We show Rac1 is both upregulated and activated during hypoxia. Pharmacological inhibition of Rac1, but not RhoA, completely abrogated hypoxic HIF-1α stabilization and expression of the HIF-1 targets VEGF and GLUT1. Furthermore activity of JNK and GSK3ß were also highly dependent on Rac1 activity and biphasic effects were observed after 6 and 24h of exposure. Notably, inhibition of either pathway suppressed HIF-1α accumulation. Although inhibition of Rac1 did not affect neuronal viability during acute exposure cell death was strongly induced after 24h revealing a time-dependent effect of Rac1 signaling on survival. Thus hypoxia-activated Rac1 is critical for neuronal HIF-1α stabilization and survival during oxygen deprivation via integration of complex signaling cascades.


Subject(s)
Cerebral Cortex/metabolism , Hypoxia, Brain/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neurons/metabolism , Neuropeptides/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Cell Hypoxia , Cells, Cultured , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Up-Regulation
2.
Neuropharmacology ; 110(Pt A): 211-222, 2016 11.
Article in English | MEDLINE | ID: mdl-27016021

ABSTRACT

1-Deoxysphingolipids (1-deoxySL) are atypical and neurotoxic sphingolipids formed by alternate substrate usage of the enzyme serine-palmitoyltransferase. Pathologically increased 1-deoxySL formation causes hereditary sensory and autosomal neuropathy type 1 (HSAN1) - a progressive peripheral axonopathy. However, the underlying molecular mechanisms by which 1-deoxySL acts are unknown. Herein we studied the effect of 1-deoxysphinganine (1-deoxySA) and its canonical counterpart sphinganine (SA) in aged cultured neurons comparing their outcome on cell survival and cytoskeleton integrity. 1-deoxySA caused rapid neuronal cytoskeleton disruption and modulated important cytoskeletal regulatory and associated components including Rac1, Ezrin and insulin receptor substrate 53. We show that 1-deoxySA is internalized and metabolized downstream to 1-deoxydihydroceramide since inhibition of ceramide synthase protected neurons from 1-deoxySA-mediated cell death. In addition, 1-deoxySA reduced protein levels of N-methyl-d-aspartate receptor (NMDAR) subunit GluN2B, the postsynaptic density protein 95 and induced cleavage of p35 to p25. Notably, blocking NMDAR activation by MK-801 or memantine significantly prevented 1-deoxySA neurotoxicity. Functional studies of differentiating primary neurons via the patch-clamp technique demonstrated that 1-deoxySA irreversibly depolarizes the neuronal membrane potential in an age-dependent manner. Notably, only neuronal cells that displayed functional NMDAR- and NMDA-induced whole-cell currents responded to 1-deoxySA treatment. Furthermore, pre-exposure to the non-competitive antagonist MK-801 blocked the current response of NMDA and glycine, as well as 1-deoxySA. We conclude that 1-deoxySA-induced neurotoxicity compromises cytoskeletal stability and targets NMDAR signaling in an age-dependent manner. Thus stabilization of cytoskeletal structures and/or inhibition of glutamate receptors could be a potential therapeutic approach to prevent 1-deoxySA-induced neurodegeneration.


Subject(s)
Neurons/drug effects , Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Sphingolipids/toxicity , Animals , Cell Line, Tumor , Cerebral Cortex , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Disks Large Homolog 4 Protein/metabolism , Dizocilpine Maleate/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Glycine/administration & dosage , Glycine/metabolism , Humans , Memantine/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice, Inbred C57BL , N-Methylaspartate/administration & dosage , N-Methylaspartate/metabolism , Neurons/pathology , Neuroprotective Agents/pharmacology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Signal Transduction/drug effects , Sphingosine/analogs & derivatives , Sphingosine/toxicity , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolism
3.
Diabetes ; 63(4): 1326-39, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24379346

ABSTRACT

Irreversible failure of pancreatic ß-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic ß-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.


Subject(s)
Insulin-Secreting Cells/drug effects , Sphingosine/analogs & derivatives , Animals , Apoptosis/drug effects , Biomarkers , Blood Glucose/metabolism , Cell Survival/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Cytoskeleton/drug effects , Humans , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Lipids , Mice , Rats , Signal Transduction , Sphingosine/metabolism , Sphingosine/pharmacology , Sphingosine/toxicity
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