Marina Montali

GABAA/Bz Receptor Subtypes as Targets for Selective Drugs

The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory neuronal receptors in the mammalian brain. Their activation by GABA opens the intrinsic ion channel, enabling chloride flux into the cell with subsequent hyperpolarization. Several GABA(A) receptor subunit isoforms have been cloned, the major isoform containing alpha, beta, and gamma subunits, and a regional heterogeneity associated with distinct physiological effects has been suggested. As a variety of allosteric ligands can modulate GABA-gated conductance changes through binding to distinct sites, the development of subtype-selective ligands may lead to the selective treatment of GABA system-associated pathology. In particular, the best characterized binding site is the benzodiazepine site (BzR), localized at the alpha/gamma subunit interface, in which the alpha subunit is the main determinant of BzR ligand action selectivity. The alpha1-containing BzR have been proposed to be responsible for the sedative action; the alpha2 and/or the alpha3 subtypes have been suggested to mediate the anxiolytic activity and the myorelaxation effects, and the alpha5 subtype has been associated with cognition processes. The discovery of alpha-selective subtype ligands may help in the specific treatment of anxiety, sleep disorders, convulsions and memory deficits with fewer side effects. Selectivity may be achieved by two approaches: selective affinity or selective efficacy. Selective affinity needs a compound to bind with a higher affinity to one receptor subtype compared with another, whereas subtype-selective efficacy relies on a compound binding to all subtypes, but having different efficacies at various subtypes. The status of BzR ligands, subdivided on the basis of their main chemical structural features, is reviewed in relation to structure-activity relationships which determine their affinity or efficacy selectivity for a certain BzR subtype.

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Identification and purification of mesodermal progenitor cells from human adult bone marrow.

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Peripheral-type benzodiazepine receptor binding sites in platelets of patients with panic disorder associated to separation anxiety symptoms.

RationaleAlthough it is still a matter of debate whether panic disorder (PD) and separation anxiety (SA) are associated or causally linked disorders, some investigators have suggested that SA may be a specific subtype of panic-agoraphobic spectrum. Several psychiatric disorders, including PD, are associated with lower levels of peripheral-type benzodiazepine receptor (PBR).ObjectivesThe aim of the present study was to evaluate the kinetic binding parameters of the specific PBR ligand, PK 11195, in platelets from patients with PD in relation to the presence and severity of adulthood SA.MethodsUsing the specific radioligand, [3H] PK 11195, the kinetic binding parameters of PBR were determined on platelet membranes of 27 adult outpatients with a DSM-IV diagnosis of PD and 18 healthy controls. Patients were assessed with the SCID-I, the Panic Disorder Severity Scale, the Structured Clinical Interview for Separation Anxiety Symptoms and the Adult Separation Anxiety Checklist.ResultsPD patients had significantly lower PBR density than controls. However, the lower density was only evident in the subgroup of PD patients who also fulfilled the DSM-IV criteria for adult separation anxiety disorder. PBR density was negatively correlated with each of the two SA scales total scores.ConclusionsPatients with SA symptoms had significantly lower densities of PBRs. PBR expression might become a useful biological marker of these two associated conditions.

Selective Culture of Mesodermal Progenitor Cells

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Regulation of A1 adenosine receptor functioning induced by P2Y1 purinergic receptor activation in human astroglial cells

In the rat brain, a heteromeric association between adenosine A1 and purinergic P2Y1 receptors has been demonstrated. It is suggested that this association plays an important role in the control of purine‐mediated responses during pathophysiological conditions. Recently, we have demonstrated that these receptors colocalize on glutamatergic synaptic and astroglial membranes in rat hippocampus and reciprocally interact, thus modulating their functional responses at the G protein coupling level. In the present work, by means of immunoprecipitation studies, we demonstrated that A1 and P2Y1 receptors are present in human astroglial cells (ADF) and aggregate to form a multimeric complex. P2Y1 receptor activation by its agonist, 2‐methylthio‐adenosine 5′‐diphosphate (MeSADP), induced a time‐dependent reduction in agonist‐mediated A1 receptor functional responses, causing a drop in A1 receptor agonist potency to promote receptor–G protein coupling and to inhibit the adenylate cyclase pathway. These effects appeared to be selectively mediated by P2Y1 receptor activation and probably occurred as a consequence of a direct receptor–receptor interaction at the plasma membrane level. These results indicated that P2Y1 receptor activation induces A1 receptor heterologous desensitization. The interaction between A1 and P2Y1 receptors may play an important role in the purinergic signaling cascade in astrocytes, which are involved in cell‐to‐cell communication and in control of synaptic transmission, particularly during pathological conditions, when large amounts of purines are released.

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Background Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4negCD105+CD90bright and MSCA-1+), in the primary cultures, resulted lower than 2%. Methodology/Principal Finding We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50 = 0.5 µM, p<0.01), while endothelial differentiation was unaffected. Conclusion The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

New Fluoro Derivatives of the Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide System: Evaluation of Fluorine Binding Properties in the Benzodiazepine Site on γ-Aminobutyrric Acid Type A (GABAA) Receptor. Design, Synthesis, Biological, and Molecular Modeling Investigation

In the search for potent ligands at the benzodiazepine site on the GABA(A) receptor, new fluoro derivatives of the pyrazolo[5,1-c][1,2,4]benzotriazine system were synthesized to evaluate the importance of the introduction of a fluorine atom in this system. Biological and pharmacological studies indicate that the substitution at position 8 with a trifluoromethyl group confers pharmacological activity due to potential metabolic stability in comparison to inactive 8-methyl substituted analogues. In particular, the compound 3-(2-methoxybenzyloxycarbonyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (21) emerges because of its selective anxiolytic profile without side effects. An analysis of all the newly synthesized compounds in our pharmacophoric map confirms the essential interaction points for binding recognition and the important areas for affinity modulation. The fluorine atom was able to form a hydrogen bond interaction only when it is not in position 3.

Short‐term TNF‐Alpha treatment induced A2B adenosine receptor desensitization in human astroglial cells

Long‐term glial cell treatment with the proinflammatory cytokine TNF‐alpha has been demonstrated to increase the functional responsiveness of A2B adenosine receptors (A2B ARs), which in turn synergize with the cytokine inducing chronic astrogliosis. In the present study, we investigated the short‐term effects of TNF‐alpha on A2B AR functional responses in human astroglial cells (ADF), thus simulating the acute phase of cerebral damage which is characterized by both cytokine and adenosine high level release. Short‐term TNF‐alpha cell treatment caused A2B AR phosphorylation inducing, in turn, impairment in A2B AR‐G protein coupling and cAMP production. These effects occurred in a time‐dependent manner with a maximum following 3‐h cell exposure. Moreover, we showed PKC intracellular kinase is mainly involved in the TNF‐alpha‐mediated regulation of A2B AR functional responses. The results may indicate the A2B AR functional impairment as a cell defense mechanism to counteract the A2B receptor‐mediated effects during the acute phase of brain damage, underlying A2B AR as a target to modulate early inflammatory responses. J. Cell. Biochem. 104: 150–161, 2008.

Synthesis, in Vivo Evaluation, and Molecular Modeling Studies of New Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide Derivatives. Identification of a Bifunctional Hydrogen Bond Area Related to the Inverse Agonism

A new series of pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 8-alkyloxy-/aryloxy-/arylalkyloxy and 8-aryl-/arylalkylderivatives variously substituted at the 3-position were synthesized and binding studies at the benzodiazepine site on GABA(A) receptor were carried out. The pharmacological profile was identified for compounds 10, 11, 16(+), 16(-), and 17 by considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motility and explorative activity, potential anxiolytic-like effects, mouse learning and memory modulation, and finally, ethanol-potentiating action. Compound 17 stands out as the compound that improves mouse memory processes selectively, safely, and in a statistically significant manner. From a ligand-based pharmacophoric model, we identified a hydrogen bond interaction area HBp-3 near the lipophilic area. This new pharmacophoric model allowed us to identify four structural compound typologies and thus to rationalize the affinity data of all compounds.