Subject(s)
Phthalazines/chemistry , Receptors, Lysosphingolipid/agonists , Administration, Oral , Amino Acid Sequence , Animals , Binding Sites , Computer Simulation , Drug Design , Fingolimod Hydrochloride , Molecular Sequence Data , Myelin Basic Protein/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Phthalazines/chemical synthesis , Phthalazines/pharmacokinetics , Propylene Glycols/chemistry , Rats , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Sphingosine/chemistry , Structure-Activity RelationshipABSTRACT
Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.
Subject(s)
Astrocytes/drug effects , Cell Movement/drug effects , Immunosuppressive Agents/pharmacology , Propylene Glycols/pharmacology , Receptors, Lysosphingolipid/physiology , Sphingosine/analogs & derivatives , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Animals , Astrocytes/physiology , Calcium Signaling/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Embryo, Mammalian , Fingolimod Hydrochloride , Glial Fibrillary Acidic Protein/metabolism , Glutamic Acid/pharmacology , Hippocampus/cytology , Hippocampus/drug effects , Inositol Phosphates/metabolism , Organ Culture Techniques , Oxadiazoles/pharmacology , Rats , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/antagonists & inhibitors , Sphingosine/pharmacology , Thiophenes/pharmacology , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacologyABSTRACT
Sphingosine-1-phosphate receptors (S1P1-5) are activated by the endogenous agonist S1P and are expressed in the central nervous system. In astrocytes, activation of S1P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S1P receptors. We examined the effects of FTY720P on ERK phosphorylation and determined which S1P receptor subtype(s) mediated this signaling event. FTY720P augmented ERK phosphorylation in cortical cultures prepared from embryonic day 18 rat brains and was blocked by an MEK inhibitor or by pertussis toxin. Co-localisation of phosphorylated ERK occurred in glial fibrillary acidic protein (GFAP) positive astrocytes but not neurons or oligodendrocytes. Furthermore, FTY720P stimulated ERK phosphorylation in highly enriched astrocyte cultures made from postnatal day 2 rat cortices. The effects of FTY720P were mimicked by selective S1P1 receptor agonists and blocked by S1P1 receptor antagonists. Collectively, these results demonstrate that FTY720P mediates ERK phosphorylation in astrocytes via the activation of S1P1 receptors.
Subject(s)
Astrocytes/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Propylene Glycols/pharmacology , Receptors, Lysosphingolipid/drug effects , Sphingosine/analogs & derivatives , Animals , Astrocytes/drug effects , Blotting, Western , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Female , Fingolimod Hydrochloride , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Immunohistochemistry , Pertussis Toxin/pharmacology , Phosphorylation , Pregnancy , Rats , Rats, Sprague-Dawley , Sphingosine/pharmacologyABSTRACT
The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC50 = 1.33 microM) and the gamma-secretase potent inhibitors NVP-AHW700-NX (IC50 = 51 nM) and LY411575 (IC50 = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/genetics , Binding Sites/genetics , CHO Cells , Cell Line , Cricetinae , Cricetulus , Dipeptides/pharmacology , Endoplasmic Reticulum/metabolism , Extracellular Space/drug effects , Extracellular Space/metabolism , Hepacivirus/genetics , Hepacivirus/metabolism , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Mutation/genetics , Proteasome Endopeptidase Complex/metabolism , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Substrate Specificity , Transfection , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Mutations in the parkin gene are linked to autosomal-recessive juvenile parkinsonism (AR-JP). Parkin functions as a ubiquitin protein ligase in the degradation of several proteins, including the neuron-specific septin CDCrel-1. AR-JP-associated parkin mutations inhibit ubiquitination and degradation of CDCrel-1 and other parkin target proteins. Here we show that recombinant adeno-associated virus-mediated CDCrel-1 gene transfer to the substantia nigra of rats results in a rapid onset (6-10 days) of nigral and striatal CDCrel-1 expression that is followed by a progressive loss of nigral dopaminergic neurons and a decline of the striatal dopamine levels. In contrast, neurons of the globus pallidus are spared from CDCrel-1 toxicity. Furthermore, CDCrel-1 inhibits the release of dopamine from stably-transfected PC12 cells, and pharmacological inhibition of tyrosine hydroxylase and dopamine synthesis in rats prevents CDCrel-1-induced nigral neurodegeneration. These results show that CDCrel-1 overexpression exerts dopamine-dependent neurotoxicity and suggest that inhibition of dopamine secretion by CDCrel-1 may contribute to the development of AR-JP.
