Deregulation of calcium homeostasis mediates secreted aesynuclein - induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinson's disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinson's disease

Etude de la differenciation des cellules erytroleucemiques de souris : methylation du DNA, regulation de l'expression de l'ornithine decarboxylase

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

3D-QSAR using pharmacophore-based alignment and virtual screening for discovery of novel MCF-7 cell line inhibitors

The development of a novel approach for the prediction of antiestrogenic activity is described, bringing up to date a previous pharmacophore study. Software Phase has been used to derive a 3D-QSAR model based, as alignment rule, on a pharmacophore built on three compounds highly active against MCF-7 cell line. Five features comprised the pharmacophore: two hydrogen-bond acceptors, one hydrogen-bond donor, and two aromatic rings. The sequential 3D-QSAR yielded a test set q(2) equal to 0.73 and proved to be predictive with respect to an external test set of 21 compounds (r(2) = 0.69). The model was used to detect new MCF-7 inhibitors through 3D-database searching and identified fourteen compounds that were subsequently tested in vitro against the MCF-7 human breast adenocarcinoma cell line. Eleven out of the fourteen compounds exhibited inhibitory activity with IC50 values ranging between 30 and 186 μM. The results of the study confirmed the fundamental validity of the chosen approach as a hit discovery tool

Neuroprotective effects of steroid analogues on P19-N neurons

Naturally occurring neurosteroids are potent allosteric modulators of gamma-aminobutyric acid(A) receptor and through augmentation of gamma-aminobutyric acid(A) receptor function, can protect neuronal cells against N-methyl-D-aspartate receptor over-activation, ischemia and traumatic brain injury. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons and were subjected to excitotoxicity in the presence of N-methyl-D-aspartate. Novel synthetic analogues of the endogenous neurosteroids allopregnanolone and dehydroepiandrostrone, inhibited excitotoxic cell death of P19-N neurons, by directly maintaining the activation of PKB/Akt kinase, and interfering with the intrinsic mitochondrial apoptotic pathway, preserving cytochrome c in the mitochondria and Bax in the cytoplasm. The efficiency and the potency of these neurosteroids were similar to those of allopregnanolone and dehydroepiandrostrone. Their effects were gamma-aminobutyric acid(A) receptor mediated, since they were abolished in the presence of bicuculline, an antagonist of receptor’s function. In addition, the synthetic compounds retained the ability to alter gamma-aminobutyric acid(A) receptor subunit gene expression, but their effects on transcriptional activity were less pronounced than those of allopregnanolone and dehydroepiandrostrone. These results suggest that synthetic neurosteroids may serve as potent, membrane acting, neuroprotectants against N-methyl-D-aspartate receptor neurotoxicity on neuronal cells. (c) 2007 Elsevier Ltd. All rights reserved

Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca2+ Signaling Pathways

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling

Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca²⁺ Signaling Pathways

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling

Synthesis and biological evaluation of benzopyran analogues bearing class III antiarrhythmic pharmacophores

We have synthesized a series of compounds combining the hydroxy-benzopyran ring of vitamin E with the methylsulfonylaminophenyl group of class III antiarrhythmic drugs, connected through tertiary amine moieties. Evaluation of the antiarrhythmic and antioxidant activity of the new compounds was carried out on isolated rat heart preparations using the non-recirculating Langendorff mode. The new analogues were present, at 10 mu M concentration, during ischemia and reperfusion. Selected compounds were further studied by a conventional microelectrode method in order to get insight into their cellular mode of action. The most active compound, N-[4-[2-[[2-(3,4-dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5 -yl)ethyl] methylamine]ethyl]phenyl]methanesulfonamide (19a), reduces premature beats, prolongs QT and QRS intervals during ischemia and reperfusion, and reduces MDA content, leading to a fast recovery of the heart. In addition, it exhibits moderate class III antiarrhythmic action. (c) 2006 Elsevier Ltd. All rights reserved