Alessandra Falleni

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.

Severe ultrastructural mitochondrial changes in lymphoblasts homozygous for Huntington disease mutation

Mutated huntingtin is expressed in nervous and non nervous system included lymphoblasts. Eneregetic metabolism is impaired in Huntingtons disease (HD) and other neurodegenerative diseases. Human HD lymphoblasts have provided clear-cut data on mitochondnal disruption. Here we report morphological, morphometric and membrane potential differences in mitochondria from lymphoblasts obtained from patients homozygous and heterozygous for the CAG mutation, and controls. Homozygotes, who despite a similar age at onset show a more aggressive phenotype than heterozygotes, had giant mitochondria and a reduced membrane potential. We argue that early mitochondrial impairment at basal level may affect the severity of HD progression in patients.

Agonist‐Induced Internalization and Recycling of the Human A3 Adenosine Receptors

Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

Fine ultrastructure and biochemistry of PC12 cells: A comparative approach to understand neurotoxicity

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

Abnormal morphology of peripheral cell tissues from patients with Huntington disease

We investigated the genotype-dependency of morphological abnormalities in peripheral cells from Huntington disease (HD) patients. Cell cultures derived from skin and muscle biopsies showed a different set of abnormalities depending on the genotype (i.e. heterozygous and homozygous for CAG mutations) and the tissue (i.e. fibroblasts and myoblasts). In general, homozygotes’ cell lines showed massive ultrastructural damage of specific cell organelles compared with age matched control. These consist of vacuolization, deranged crests and matrix found within giant mitochondria. In addition, enlarged endoplasmic reticulum and the occurrence of numerous autophagic vacuoles, which were similar to those occurring in neurons within affected brain areas, were described. Despite a comparable dose-dependency on mitochondrial changes, this kind of alterations differ in fibroblasts compared with myoblasts. In fact, the internal mitochondrial structure was merely lost in myoblasts, while it shows pathological re-organization within fibroblasts, where altered crests appear as multilamellar circles. These data indicate that ultrastructural abnormalities from peripheral tissues of HD patients can be used as potential disease markers which are easier to get than autoptic brains. Moreover, the occurrence of ultrastructural cell pathology reminiscent of neuronal degeneration in HD, suggests the use of human peripheral cells as a tool to investigate the pathogenic cascade subsequent to huntingtin dysregulation.

Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin

The freshwater painters mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painters mussels for environmental quality assessment in freshwater.

Cellular responses in the cyprinid Leuciscus cephalus from a contaminated freshwater ecosystem

The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.

Nigrostriatal damage with 6-OHDA: validation of routinely applied procedures

Abstract:  The 6‐hydroxydopamine (6‐OHDA) model of Parkinsons disease in the rat represents a fundamental tool for investigating the pathophysiology of dopamine denervation. Nevertheless, 6‐OHDA can induce also noradrenergic lesions; therefore desmethylimipramine (DMI) is co‐administrated as a selective inhibitor of noradrenergic reuptake to protect noradrenaline (NA) fibers neighboring DA neurons and/or axons. The neurotoxin 6‐OHDA must be microinfused selectively into the substantia nigra pars compacta (SNpc) or into the medial forebrain bundle (MFB) to determine the nigrostriatal lesion. However, this experimental procedure is invasive and always produces a certain amount of mechanical damage that cannot be prevented by pharmacological approaches. For this reason, we have compared two types of experimental design in which we tested critical steps of the procedures, such as the flow rate. We microinfused rats in MFB with 8 μL of total volume of a solution containing the neurotoxin (infusion rate 2 μL/min in 4 min) according with general practice, and rats microinfused with an amount of 2μL of total volume with a slower rate (0.2 μL/min in 10 min) of infusion. Rats infused with a higher flow rate of infusion underwent striatal NA loss in spite of the administration of DMI. On the contrary, rats infused with a slow infusion flow rate had spared NA axons following DMI. These results suggest that the flow rate and the volume of 6‐OHDA infusion are critical to prevent the occurrence of nonspecific mechanical effects.

Mechanisms involved in the formation of dopamine-induced intracellular bodies within striatal neurons

Recent studies demonstrated that methamphetamine (METH) produces intracellular bodies which are reminiscent of those occurring during degenerative disorders. In vivo studies demonstrate the occurrence of these morphological alterations both in the dopamine (DA) neurons of the substantia nigra and striatal cells. These consist of neuronal bodies staining for a variety of antigens belonging to the ubiquitin–proteasome pathway. The formation of these intracellular bodies both in the substantia nigra and PC12 cells depends on the presence of endogenous DA. In the present study, we analyze the mechanisms which lead to METH‐induced intracellular bodies within non‐dopaminergic striatal neurons. We found that METH is no longer able to produce inclusions in vivo, in striatal cells, when striatal DA is lost. Similarly, in vitro, in primary striatal cell cultures which do not possess DA, METH administration does not produce inclusions. On the other hand, administration of DA to striatal cell cultures produces neuronal inclusions and cell death, which are both related to the inhibition of the ubiquitin–proteasome system and activation of specific‐DA receptors. In line with this, we produced subcellular alterations by administering dopamine agonists.

Upregulation of mitochondrial peripheral benzodiazepine receptor expression by cytokine‐induced damage of human pancreatic islets

Cytokines produced by immune system cells infiltrating pancreatic islets are candidate mediators of islet beta‐cell destruction in autoimmune insulin‐dependent diabetes mellitus. After 72 h exposure of human pancreatic islets to a cytotoxic cytokine combination of interleukin 1 beta (50 U/ml), tumor necrosis factor alpha (1,000 U/ml), and interferon gamma (1,000 U/ml), an increase of cell death vs. control islets was demonstrated by TUNEL and cell death detection ELISA method. Islet death was associated with apoptosis and mitochondrial swelling as evidenced by electron microscopy. This effect was correlated with a marked decrease of Bcl‐2 mRNA expression (without any major change of Bax mRNA) and a marked increase of inducible nitric oxide synthase mRNA. Since peripheral benzodiazepine receptors constitute the aspecific mitochondrial permeability transition pore, and that it has been suggested to be involved in cytokine‐induced cell death, we evaluated the effects of the cytotoxic cytokines on PBR density and mRNA expression. We demonstrated that cytokine treatment of human islets induced an increase of maximum density of 3H1‐(2‐chlorophenyl‐N‐methyl‐1‐methylpropyl)‐3‐ isoquinolinecarboxamide binding sites, (5,110 ± 193 vs. 3,421 ± 336 fmol/mg proteins, P < 0.05) with no significant change in the affinity constant value (9.45 ± 0.869 vs. 8.7 ± 1.159 nM). Moreover, an increase of the expression of peripheral benzodiazepine receptor mRNA was observed, suggesting an increased transcription from the coding gene. These results suggest a possible role of peripheral benzodiazepine receptors in the organism response to tissue damage associated with inflammatory mediator production. J. Cell. Biochem. 84: 636–644, 2002.