Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies

Stimulation of salivary secretion in vivo by CFTR potentiators in Cftr(+/+) and Cftr(-/-) mice

International audiencePhysiologically, salivary secretion is controlled by cholinergic and adrenergic pathways but the role of ionic channels in this process is not yet clearly understood. In cystic fibrosis (CF), most exocrine glands failed to response to β-adrenergic agonists. Methods To determine the implication of CFTR in this process, we measured in vivo the salivary secretion of Cftr+/+ and Cftr−/− mice in the presence of 2 water-soluble benzo[c]quinolizinium derivatives; MPB-07 a potentiator of CFTR Cl− channel and MPB-05 an inactive analogue. We also used genistein and its vehicle ethanol to confirm the implication of CFTR in salivary secretion. Results We showed that subcutaneous injection of MPB-07 in the mice cheek enhanced in a dose dependent manner the isoprenaline-induced salivary secretion in Cftr+/+ but not in Cftr−/− mice. By contrast, MPB-05 did not activate the salivary secretion in Cftr+/+ mice. The CFTR activator genistein (50 μM) significantly potentiated the secretory response of Cftr+/+ mice whereas its vehicle, ethanol, had no effect. Conclusions These results show for the first time in vivo pharmacological stimulation of salivary secretion by a water-soluble CFTR potentiator, MPB-07 and by the isoflavone, ethanol-soluble genistein and suggest that this chloride channel plays an important role in salivary gland physiology

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity