A novel pharmacological activity of caffeine in the cholinergic system.

Cholinergic deficit is regarded as an important factor responsible for Alzheimer's disease (AD) symptoms. Acetylcholinesterase (AChE) and nicotinic receptor (AChR) are two molecular targets for the treatment of this disease. We found here that methanolic extracts of Camellia sinensis exhibited anticholinesterase activity and induced AChR conformational changes. From bioguided fractionation we confirmed that caffeine was the active compound exerting such effects. It is well-known that caffeine acts as an inhibitor of AChE and here we explored the effect of caffeine on the AChR by combining single channel recordings and fluorescent measurements. From single channel recordings we observed that caffeine activated both muscle and α7 AChRs at low concentrations, and behaved as an open channel blocker which was evident at high concentrations. Fluorescent measurements were performed with the conformational sensitive probe crystal violet (CrV) and AChR rich membranes from Torpedo californica. Caffeine induced changes in the KD value of CrV in a concentration-dependent manner taking the AChR closer to a desentisized state. In the presence of α-bungarotoxin, an AChR competitive antagonist, high concentrations of caffeine increased the KD value of CrV, compatible with a competition with CrV molecules for the luminal channel. Our electrophysiological and fluorescent experiments show that caffeine has a dual effect on nicotinic receptors, behaving as an agonist and an ion channel blocker, probably through distinct AChR sites with quite different affinities. Thus, caffeine or its derivatives can be considered for the design of promising multitarget-directed drugs for AD treatment by modulation of different targets in the cholinergic pathway.

Phosphoinositides: Two-Path Signaling in Neuronal Response to Oligomeric Amyloid ß Peptide.

We have previously demonstrated that oligomeric amyloid ß peptide (oAß) together with iron overload generates synaptic injury and activation of several signaling cascades. In this work, we characterized hippocampal neuronal response to oAß. HT22 neurons exposed to 500 nM oAß showed neither increased lipid peroxidation nor altered mitochondrial function. In addition, biophysical studies showed that oAß did not perturb the lipid order of the membrane. Interestingly, although no neuronal damage could be demonstrated, oAß was found to trigger bifurcated phosphoinositide-dependent signaling in the neuron, on one hand, the phosphorylation of insulin receptor, the phosphatidylinositol 3-kinase (PI3K)-dependent activation of Akt, its translocation to the nucleus and the concomitant phosphorylation, inactivation, and nuclear exclusion of the transcription factor Forkhead Box O3a (FoxO3a), and on the other, phosphoinositide-phospholipase C (PI-PLC)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Pharmacological manipulation of the signaling cascades was used in order to better characterize the role of oAß-activated signals, and mitochondrial function was determined as a measure of neuronal viability. The inhibition of PI3K, PI-PLC, and general phosphoinositide metabolism impaired neuronal mitochondrial function. Furthermore, increased oAß-induced cell death was observed in the presence of phosphoinositide metabolism inhibition. Our results allow us to conclude that oAß triggers the activation of phosphoinositide-dependent signaling, which results in the subsequent activation of neuroprotective mechanisms that could be involved in the determination of neuronal fate.