Stem cell contributions to neurological disease modeling and personalized medicine.

Human induced pluripotent stem cells (iPSCs) represent a revolutionary tool for disease modeling and drug discovery. The generation of tissue-relevant cell types exhibiting a patient's genetic and molecular background offers the ability to develop individual and effective therapies. In this review, we present some major achievements in the neuroscience field using iPSCs and discuss promising perspectives in personalized medicine. In addition to disease modeling, the understanding of the cellular and molecular basis of neurological disorders is explored, including the discovery of new targets and potential drugs. Ultimately, we highlight how iPSC technology, together with genome editing approaches, may bring a deep impact on pre-clinical trials by reducing costs and increasing the success of treatments in a personalized fashion.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 mum. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Immobilized P2X2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening.

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM-cholate-P2X2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2'3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224-233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand-receptor interactions and screening of combinatorial pools for possible ligands.