Beatrice Chelli

Peripheral-type benzodiazepine receptor ligands:: Mitochondrial permeability transition induction in rat cardiac tissue

Strong evidence is emerging that mitochondrial permeability transition (MPT) may be important in certain physiological conditions and, above all, in the processes of cell damage and death. Reversible MPT, triggered by inducing agents in the presence of calcium ions, has resulted in the opening of a dynamic multiprotein complex formed in the inner mitochondrial membrane and has caused large-amplitude mitochondrial swelling. In the present work, the exposure of de-energized rat cardiac mitochondria to peripheral benzodiazepine receptor (PBR) ligands (1-(2-chlorophenyl-N-methyl-1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864), and diazepam) produced a dose-dependent and cyclosporin A (CSP)-sensitive loss of absorbance, which was indicative of mitochondrial swelling. By contrast, the addition of a high-affinity central benzodiazepine receptor ligand (clonazepam) was ineffective, even at the highest concentration tested. The ultrastructural changes associated with swelling were similar in mitochondria exposed either to PK 11195 or to calcium. Supporting the apoptotic role of PK 11195-induced swelling, supernatants from mitochondria that had undergone permeability transition caused apoptotic changes in isolated cardiac nuclei. In addition, ultrastructural abnormalities were observed in rat cardiac tissue following in vivo PK 11195 administration, with these abnormalities being prevented by CSP co-administration. These data indicate that PBR ligands induce mitochondrial permeability transition and ultrastructural alterations in isolated cardiac mitochondria as well as in myocardiocytes, suggesting a novel strategy for studying the implication of PBR ligands as apoptosis inducers, through a probable effect on the MPT pore.

Structure-based optimization of pyrazolo[3,4-d]pyrimidines as Abl inhibitors and antiproliferative agents toward human leukemia cell lines.

Results from molecular docking calculations and Grid mapping laid the foundations for a structure-based optimization approach to improve the biological properties of pyrazolo-pyrimidine derivatives in terms of inhibition of Abl enzymatic activity and antiproliferative properties toward human leukemia cells. Insertion of halogen substituents with various substitution patterns, suggested by simulations, led to a significant improvement of leukemia cell growth inhibition and to an increase up to 1 order of magnitude of the affinity toward Abl.

Organic ultra-thin film transistors with a liquid gate for extracellular stimulation and recording of electric activity of stem cell-derived neuronal networks

Electronic transducers of neuronal cellular activity are important devices in neuroscience and neurology. Organic field-effect transistors (OFETs) offer tailored surface chemistry, mechanical flexibility, and high sensitivity to electrostatic potential changes at device interfaces. These properties make them attractive for interfacing electronics with neural cells and performing extracellular recordings and stimulation of neuronal network activity. In this work we operate pentacene ultra-thin film (9 nm thick) transistors with a liquid gate both as transducers and electrical stimulators of neuronal network activity. These devices are highly sensitive to small potential changes in cell medium and exhibit sufficient stability under standard cell culture conditions for nine days. We show that murine neural stem cells can be adhered on top of functional devices without the need for an additional layer of cell-adhesive molecules, and then differentiated into neuronal networks. OFET response is monitored during the different phases of the neuronal differentiation process up to nine days. Only when stem cells are differentiated into neurons, it is possible to measure electrical signals in the OFET current following the stimulation. Due to the large sensing area of our device, which accommodates from hundreds to thousands of interconnected neurons, the OFET electrical signals arise from the collective electrophysiological response of the neuronal population. The maximum extracellular potential change in the cleft region adjacent to the transistor surface amounts to 350 μV. This demonstrates that pentacene ultra-thin film OFETs enable good cellular adhesion and efficient coupling of the ionic currents at the biological-organic semiconductor interface with the OFET current.

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.

Translocator Protein Ligands as Promising Therapeutic Tools for Anxiety Disorders

The Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18 kDa mitochondrial protein primarily involved in steroid biosynthesis in both peripheral and glial cells. It has been extensively reported that TSPO regulates the rate-limiting translocation of cholesterol from the outer to the inner mitochondrial membrane before its transformation by cytochrome P450(scc) into pregnenolone, which is further converted into an array of different steroids. In the brain, neurosteroids such as allopregnanolone and pregnenolone, acting as positive modulators of gamma-aminobutyric type A (GABA(A)) receptors, exert anxiolytic activity. Specific ligands targeting TSPO increase neurosteroid production and for this reason they have been suggested to play an important role in anxiety modulation. Unlike benzodiazepines (Bzs), which represent the most common anti-anxiety drugs administered around the world, selective TSPO ligands have shown anxiolytic effects in animal models without any of the side effects associated with Bzs. Therefore, specific TSPO ligands that are able to promote neurosteroidogenesis may represent the future of therapeutic treatment of anxiety disorders. Furthermore, TSPO expression levels are altered in several different psychiatric disorders in which anxiety is the main symptom. This article reviews the primary and patent literature over the last decade concerning the development of novel TSPO ligands that have resulted effective in various models of anxiety, taking into special consideration their structure-activity relationships.

Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [3H]4′-chloro derivative of diazepam [3H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [3H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (Kd) of 1.77 ± 0.30 and 2.20 ± 0.20 μM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a Ki >1.0 × 10–4 M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 → threonine and His162 → arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells

Gliomas are one of the most malignant cancers. The molecular bases regulating the onset of such tumors are still poorly understood. The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is a mitochondrial permeability transition (MPT)-pore protein robustly expressed in gliomas and involved in the regulation of apoptosis and cell proliferation. TSPO expression levels have been correlated with tumor malignancy. Here we describe the production of C6 rat glioma cells engineered to over-express the TSPO protein with the aim of providing the first direct evidence of a correlation between TSPO expression level and glioma cell aggressiveness. We observed that TSPO potentiates proliferation, motility and transmigration capabilities as well as the ability to overcome contact-induced cell growth inhibition of glioma cells. On the whole, these data demonstrate that TSPO density influences metastatic potential of glioma cells. Since several data suggest that TSPO ligands may act as chemotherapeutic agents, in this paper we also demonstrate that TSPO ligand-induced cell death is dependent on TSPO density. These findings suggest that the use of TSPO ligands as chemotherapeutic agents could be effective on aggressive tumor cells with a high TSPO expression level.

PK 11195 differentially affects cell survival in human wild-type and 18 kDa translocator protein-silenced ADF astrocytoma cells†

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over‐expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro‐apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild‐type and TSPO‐silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO‐silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195‐induced anti‐proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific‐cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells. J. Cell. Biochem. 105: 712–723, 2008.

Platelet 18 kDa Translocator Protein density is reduced in depressed patients with adult separation anxiety.

RATIONALE Recent studies indicate that Adult Separation Anxiety Disorder (ASAD) may represent a discrete diagnostic entity worthy of attention. Adults with separation anxiety report extreme anxiety and fear about separations from major attachment figures (partner, children or parents). These symptoms affect individuals behavior, lead to severe impairment in social relationships and are not better accounted for by the presence of agoraphobia. In a previous study we found platelet expression reduction of the 18 kDa Translocator Protein (TSPO) (the new nomenclature for the peripheral-type benzodiazepine receptor) in patients with panic disorder who also fulfilled the diagnostic criteria for ASAD. OBJECTIVES To explore whether separation anxiety might be a factor differentiating TSPO expression in a sample of patients with major depression. METHODS The equilibrium binding parameters of the specific TSPO ligand [3H]PK 11195 were estimated on platelet membranes from 40 adult outpatients with DSM-IV diagnosis of MDD, with or without separation anxiety symptoms, and 20 healthy controls. Patients were assessed by SCID-I, HAM-D, the Structured Clinical Interview for Separation Anxiety Symptoms (SCI-SAS-A) and the Adult Separation Anxiety Self-report Checklist (ASA-27). RESULTS A significant reduction of platelet TSPO density mean value was found in depressed patients with associated ASAD symptoms, while no significant differences were found between depressed patients without ASAD and the control group. Individual TSPO density values were significantly and negatively correlated with both SCI-SAS-A and ASA-27 total scores, but not with HAM-D total score or HAM-D anxiety/somatization factor score. CONCLUSIONS The reduction of platelet TSPO density in our sample of patients with depression was specifically related to the presence of ASAD. These data suggest that TSPO expression evaluation is a useful biological marker of ASAD.

Structure-activity relationship of acridine derivatives to amyloid aggregation of lysozyme.

BACKGROUND Amyloid-related diseases (such as Alzheimers disease or diabetes type II) are associated with self-assembly of protein into amyloid aggregates. METHODS Spectroscopic and atomic force microscopy were used to determine the ability of acridines to affect amyloid aggregation of lysozyme. RESULTS We have studied the effect of acridine derivatives on the amyloid aggregation of lysozyme to investigate the acridine structure-activity relationship. The activity of the effective planar acridines was characterized by the half-maximum depolymerization concentration DC(50) and half-maximal inhibition concentration IC(50). For the most effective acridine derivatives we examined their interaction with DNA and their effect on cell viability in order to investigate their eventual influence on cells. We thus identified planar acridine derivatives with intensive anti-amyloid activity (IC(50) and DC(50) values in micromolar range), low cytotoxicity and weak ability to interfere with the processes in the cell. CONCLUSIONS Our findings indicate that both the planarity and the tautomerism of the 9-aminoacridine core together with the reactive nucleophilic thiosemicarbazide substitution play an important role in the anti-amyloid activities of studied derivatives. GENERAL SIGNIFICANCE The present findings favor the application of the selected active planar acridines in the treatment of amyloid-related diseases.