Retinoic Acid [BSA]

In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co-localizes with hyperphosphorylated tau (P-tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg-VLW, here we examined whether modulating phosphorylated tau levels by over-expressing wild-type human tau and glycogen synthase kinase-3 beta (GSK3 beta) influences AChE expression. In SH-SY5Y neuroblastoma cells expressing higher levels of P-tau, AChE activity and protein increased by (20% +/- 2%) and (440% +/- 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergicACHE-Tvariant, for which the protein and transcript levels increased similar to 60% and similar to 23%, respectively. Moreover, in SH-SY5Y cells differentiated into neurons by exposure to retinoic acid (10 mu M), over-expression of GSK3 beta and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 +/- 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3 beta inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 +/- 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib-treated patients. Moreover, CSF levels of P-tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P-tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD.

Currently, cardiomyocyte (CM) differentiation methods require a purification step after CM induction to ensure the high purity of the cell population. Here we show an improved human CM differentiation protocol with which high-purity ventricular-type CMs can be obtained and maintained without any CM purification process. We induced and collected a mesodermal cell population (platelet-derived growth factor receptor-alpha (PDGFR alpha)-positive cells) that can respond to CM differentiation cues, and then stimulated CM differentiation by means of Wnt inhibition. This method reproducibly generated CMs with purities above 95% in several human pluripotent stem cell lines. Furthermore, these CM populations were maintained in culture at such high purity without any further CM purification step for over 200 days. The majority of these CMs (>95%) exhibited a ventricular-like phenotype with a tendency to structural and electrophysiological maturation, including T-tubule-like structure formation and the ability to respond to QT prolongation drugs. This is a simple and valuable method to stably generate CM populations suitable for cardiac toxicology testing, disease modeling and regenerative medicine.