Neural cell alignment by patterning gradients of the extracellular matrix protein laminin.

Anisotropic orientation and accurate positioning of neural cells is achieved by patterning stripes of the extracellular matrix protein laminin on the surface of polystyrene tissue culture dishes by micromoulding in capillaries (MIMICs). Laminin concentration decreases from the entrance of the channels in contact with the reservoir towards the end. Immunofluorescence analysis of laminin shows a decreasing gradient of concentration along the longitudinal direction of the stripes. The explanation is the superposition of diffusion and convection of the solute, the former dominating at length scales near the entrance (characteristic length around 50 µm), the latter further away (length scale in excess of 900 µm). These length scales are independent of the channel width explored from about 15 to 45 µm. Neural cells are randomly seeded and selectively adhere to the pattern, leaving the unpatterned areas depleted even upon 6 days of incubation. Cell alignment was assessed by the orientation of the long axis of the 4',6-diamidino-2-phenylindole-stained nuclei. Samples on patterned the laminin area exhibit a large orientational order parameter. As control, cells on the unpatterned laminin film exhibit no preferential orientation. This implies that the anisotropy of laminin stripes is an effective chemical stimulus for cell recruiting and alignment.

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.

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

BACKGROUND: Amyloid-related diseases (such as Alzheimer's 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.

Anxiolytic properties of a 2-phenylindolglyoxylamide TSPO ligand: Stimulation of in vitro neurosteroid production affecting GABAA receptor activity.

A number of neurosteroids have been demonstrated to exert anxiolytic properties by means of a positive modulation of inhibitory GABAergic neurotransmission. The observation that neurosteroid synthesis can be pharmacologically regulated by ligands to the mitochondrial translocator protein (TSPO) has prompted the search for new, more selective TSPO ligands able to stimulate steroidogenesis with great efficacy. In the present study, the potential anxiolytic activity of a selective TSPO ligand, N,N-di-n-propyl-2-(4-methylphenyl)indol-3-ylglyoxylamide (MPIGA), was tested by means of the elevated plus maze paradigm. Moreover, the in vitro effects on synaptoneurosomal GABA(A) receptor (GABA(A)R) activity exerted by the conditioned salt medium from MPIGA-treated ADF human glial cells were investigated. MPIGA (30mg/kg) was found to affect rats' performance in the elevated plus maze test significantly, leading to an increase in both entries and time spent in the open arms. This same dose of MPIGA had no effect on rats' spontaneous exploratory activity. The conditioned salt medium from MPIGA-treated ADF cells potentiated the (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The exposure of ADF cells to MPIGA stimulated the production of pregnelonone derivatives including allopregnanolone, one of the major positive GABA(A)R allosteric modulator. In conclusion, the TSPO ligand MPIGA is a promising anxiolytic drug. The mechanism of action by which MPIGA exerts its anxiolytic activity was identified in the stimulation of endogenous neurosteroid production, which in turn determined a positive modulation of GABA(A)R activity, thus opening the way to the potential use of this TSPO ligand in anxiety and depressive disorders.

Reductions in platelet 18-kDa translocator protein density are associated with adult separation anxiety in patients with bipolar disorder.

BACKGROUND: Recent studies indicate that adult separation anxiety disorder is a discrete diagnostic entity and worthy of attention. Previously, we found a significant association between platelet expression of the 18-kDa translocator protein (TSPO) and adult separation anxiety in patients with panic disorder or major depression. The aim of this study was to explore whether adult separation anxiety might be a factor differentiating TSPO expression in a sample of patients with bipolar disorder. METHODS: The equilibrium binding parameters of the specific TSPO ligand [(3)H]PK 11195 were estimated on the platelet membranes of 24 adult outpatients with a DSM-IV diagnosis of bipolar disorder (with or without separation anxiety disorder) and 14 healthy controls. Patients were assessed by SCID-I, HAM-D, YMRS, 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 in mean platelet TSPO density was found in bipolar patients with respect to controls. However, the lower density was only evident in the subgroup of bipolar patients who also fulfilled DSM-IV criteria for adult separation anxiety disorder. Individual TSPO density values correlated significantly and negatively with both SCI-SAS-A and ASA-27 total scores. CONCLUSIONS: TSPO expression may be a useful biological marker of adult separation anxiety co-occurring with other anxiety and mood disorders, including bipolar disorder.

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.

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.

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.

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 individual's 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.

New fluorescent 2-phenylindolglyoxylamide derivatives as probes targeting the peripheral-type benzodiazepine receptor: design, synthesis, and biological evaluation.

Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent, selective class of PBR ligands previously described by us. All the new ligands are moderately to highly potent at the PBR, with a complete selectivity over the central benzodiazepine receptor. Results from fluorescence microscopy showed that these probes specifically labeled the PBR at the mitochondrial level in C6 glioma cells.

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 [(3)H]4'-chloro derivative of diazepam [(3)H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [(3)H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (K(d)) of 1.77 +/- 0.30 and 2.20 +/- 0.20 microM, 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 K(i) >1.0 x 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.

PIGA (N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a new mitochondrial benzodiazepine-receptor ligand, induces apoptosis in C6 glioma cells.

Mitochondrial benzodiazepine-receptor (mBzR) ligands constitute a heterogeneous class of compounds that show a pleiotropic spectrum of effects within the cells, including the modulation of apoptosis. In this paper, a novel synthetic 2-phenylindol-3-ylglyoxylamide derivative, N,N-di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide (PIGA), which shows high affinity and selectivity for the mBzR, is demonstrated to induce apoptosis in rat C6 glioma cells. PIGA was able to dissipate mitochondrial transmembrane potential (DeltaPsim) and to cause a significant cytosolic accumulation of cytochrome c. Moreover, typical features of apoptotic cell death, such as caspase-3 activation and DNA fragmentation, were also detected in PIGA-treated cells. Our data expand the knowledge on mBzR ligand-mediated apoptosis and suggest PIGA as a novel proapoptotic compound with therapeutic potential against glial tumours, in which apoptosis resistance has been reported to be involved in carcinogenesis.

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

RATIONALE: Although 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). OBJECTIVES: The 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. METHODS: Using the specific radioligand, [(3)H] 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. RESULTS: PD 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. CONCLUSIONS: Patients with SA symptoms had significantly lower densities of PBRs. PBR expression might become a useful biological marker of these two associated conditions.

Peripheral benzodiazepine receptor ligands: mitochondrial transmembrane potential depolarization and apoptosis induction in rat C6 glioma cells.

The peripheral benzodiazepine receptor (PBR) is a component of a multiprotein complex, located at the contact site between the inner and outer mitochondrial membranes, which constitutes the mitochondrial permeability transition (MPT)-pore. The opening of the MPT-pore, leading to the transmembrane mitochondrial potential (DeltaPsi(m)) dissipation, is a critical event in the mechanism of apoptosis. In the present work, we investigated the ability of the specific PBR ligands, PK 11195 or Ro5-4864, to affect mitochondrial potential and to induce apoptotic cell death in rat C6 glioma cells. Both specific ligands inhibited cell survival in a dose- and time-dependent manner, as assessed by MTS conversion assay, whereas the non-site selective ligand Diazepam or the low-affinity benzodiazepine Clonazepam showed no significant effects. After cell exposure to PK 11195 or Ro5-4864 we evidenced typical alterations of apoptotic cell death such as DNA fragmentation and chromatin condensation assessed by flow cytometric and transmission electron microscopy (TEM) analysis, respectively. Activation of the "effector" caspase-3 confirmed the ability of specific PBR ligands to induce apoptosis. Moreover, PK 11195 and Ro5-4864 induced a decrease of DeltaPsi(m), as evidenced by JC-1 flow cytometry analysis. Our data demonstrate the pro-apoptotic effects of specific PBR ligands on rat C6 glioma cells.

N,N-dialkyl-2-phenylindol-3-ylglyoxylamides. A new class of potent and selective ligands at the peripheral benzodiazepine receptor.

We report the synthesis and the affinity data at both the peripheral (PBR) and the central benzodiazepine receptors of a series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamide derivatives III, designed as conformationally constrained analogues of 2-phenylindole-3-acetamides II such as FGIN-1-27. Most of the new compounds showed a high specificity and affinity for PBR, with K(i) in the nanomolar to subnanomolar range. The most potent ligands (4-7, 9, 13-27) stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classical ligands. The SARs of this new class of compounds are discussed.

A(2A) adenosine receptor ligands and proinflammatory cytokines induce PC 12 cell death through apoptosis.

A(2A) adenosine receptor-mediated signaling affects a variety of important processes in the central nervous system both in physiological and pathological conditions, and has been indicated as possible novel therapeutic target in several nervous system diseases. In the present work, cell death induction was investigated after neuronal PC 12 cell treatment with proinflammatory cytokines and adenosine receptor ligands. Interleukin-1-beta (IL-1-beta, 500 U/mL), tumor necrosis factor-alpha (TNF-alpha, 1000 U/mL) and the non selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), caused a significant reduction of cell viability with a maximal effect within 3-48 hr. Moreover, an addictive effect was detected when the cells were simultaneously treated with Interleukin-1-beta and NECA for 3 hr. To investigate the adenosine receptor subtypes involved in PC 12 cell death, the effects of several adenosine receptor agonists/antagonists were evaluated. The endogenous nucleoside, adenosine, and the selective A(2A) adenosine receptor agonist, 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide (CGS21680) reduced PC 12 cell viability. This effect was counteracted by the selective A(2A) adenosine receptor antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5c]pyrimidine (SCH58261), but not by selective A(2B) adenosine receptor antagonist N-(4-acethylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706), suggesting the specific involvement of A(2A) adenosine receptor subtype in adenosine-mediated cytotoxicity. Moreover, the selective A(1) adenosine receptor agonist, N(6)-cyclohexyladenosine (CHA), did not induce any significant effect on cell viability. By ELISA immunoassay cell death detection and transmission electron microscopy (TEM) we demonstrated that A(2A) adenosine receptor ligands and cytokines induced cell death through an apoptotic pathway. In conclusion, our results showed that A(2A) adenosine receptors are involved in the control of PC 12 cell survival/death and may contribute to modulate cellular activity in response to tissue damage associated with inflammatory mediator production.

Peripheral benzodiazepine binding sites in platelets of patients affected by mitochondrial diseases and large scale mitochondrial DNA rearrangements.

BACKGROUND: The peripheral-type benzodiazepine receptors (PBR) are localized on the outer mitochondrial membrane, as a constituent of mitochondrial permeability transition (MPT)-pore. Among its hypothesized functions, the regulation of the mitochondrial respiratory chain and apoptosis have been suggested; in addition alterations of PBR site density have been shown in some neuropathologic conditions with putative mitochondrial involvement. The aim of this work has been to evaluate PBR kinetic binding parameters in platelets from patients affected by mitochondrial disorders (MD) with large-scale mitochondrial DNA deletions and reduced cytochrome c oxidase activity. MATERIALS AND METHODS: Using the specific PBR radioligand [(3) H] PK 11195, the kinetic binding parameters of PBR sites were determined in platelet membrane of 15 healthy subjects and 11 patients affected by different form of MD. RESULTS: Significant changes of dissociation constant (K(d)) and maximal number of binding sites (B(max)) values were evidenced in platelets of patients versus controls. In all patients the B(max) values were decreased (2,387.0 +/- 305.6 fmol/ mg proteins versus 4889.0 +/- 357.8 fmol/mg proteins, p< 0.05), whereas the K(d) values were higher in patients than controls (13.18 +/- 2.06 nM versus 5.63 +/- 0.46 nM, p< 0.05). CONCLUSIONS: These data suggest that the kinetic binding parameters of PBR are altered in MD and that the observed changes might be related to the mitochondrial dysfunction associated with MD.