Patrizia Debetto

The P2X7 purinergic receptor: from physiology to neurological disorders

Purine nucleotides are well established as extracellular signaling molecules. P2X receptors are ATP‐gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X7, have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in progressive dilation of the channel pore and the development of permeability to molecules as large as 900 Da. The P2X7 receptor was originally described in cells of hematopoietic origin, including macrophages, microglia, and certain lymphocytes, and mediates the influx of Ca2+ and Na+ ions, as well as the release of proinflammatory cytokines. P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin‐1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7 receptors provides an inflammatory stimulus, and P2X7 receptor‐deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7 receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. The P2X7 receptor may thus represent a critical communication link between the nervous and immune systems, while providing a target for therapeutic exploitation. This review discusses the current biology and cellular signaling pathways of P2X7 receptor function, as well as insights into the role for this receptor in neurological/psychiatric diseases, outstanding questions, and the therapeutic potential of P2X7 receptor antagonism.—Skaper, S. D., Debetto, P., Giusti, P. The P2X7 purinergic receptor: from physiology to neurological disorders. FASEB J. 24, 337–345 (2010). www.fasebj.org

{alpha}-Synuclein and Parkinson's disease

Alpha‐synuclein (α‐syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in α‐syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinsons disease (PD). The physiological functions of α‐syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of α‐syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, α‐syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. α‐Syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which α‐syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin‐1, chaperone 14‐3‐3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that α‐syn‐based models represent a paradigm, which is closest to the human pathology.— Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D., Giusti, P. α‐Synuclein and Parkinsons disease.

A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues

In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one fluorometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 mumole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 mumoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSG determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.

Mechanisms of chromium toxicity in mammalian cell cultures

Our observations about the cytotoxic and cytogenetic effects of hexavalent and trivalent chromium compounds in mammalian cells cultured in vitro are reviewed. Additional data concerning the induction of chromosomal aberrations and sister chromatid exchanges, the inhibition of nucleic acid and protein synthesis, the interference with nucleotide metabolism, and the modification of membrane-linked enzyme activity are reported. A possible mechanism of chromium action is proposed.

Evaluation of the prescription and utilization patterns of statins in an Italian local health unit during the period 1994–2003

ObjectivesThe prescription pattern of statins in the Local Health Unit (LHU) of Treviso (northern Italy) over a 10-year period was evaluated, with the aim of evaluating the persistence with and adherence to therapy.MethodsData on 21,393 subjects who received at least one prescription for statins during the period between January 1, 1994 and December 31, 2003 were retrieved from the LHU database in order to track the pharmacological history of individual patients. The data included age, sex, drug formulation, strength, number of drug packages prescribed, and prescription date. The adopted indicators for drug utilization included the Defined Daily Dose (DDD), the Received Daily Dose (RDD), and a surrogated Prescribed Daily Dose (sPDD), extrapolated from available prescription data. An Adherence to Therapy Index (ATI) was calculated from the ratio between the amount of drug actually prescribed and the amount of sPDD. Based on the ATI, patients were grouped into non-adherent, poor-adherent, and good-adherent groups. The distribution of adherence level among patient-age classes and statin-prescribed patients in primary or secondary prevention was evaluated.ResultsAll drug-utilization indicators showed an increase in statin use over the study period in terms of both the number of prescribed patients and the sPDD. Persistence with and adherence to therapy remained low, with a 50% discontinuation rate in the first year, and persistent patients did not follow the therapy regularly. Patients in secondary prevention were the most adherent to their drug regimen, although only 41% of these had a good compliance.ConclusionsOur findings suggest an increase in statin use which is, however, accompanied by poor patient persistence with and adherence to statin therapy.

Synergism between fluoxetine and the mGlu2/3 receptor agonist, LY379268, in an in vitro model for antidepressant drug-induced neurogenesis

We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.

Generation of a α-synuclein-based rat model of Parkinson's disease

Two missense mutations (A30P and A53T) in the gene for alpha-synuclein (alpha-syn) cause familial Parkinsons disease (PD) in a small cohort. There is increasing evidence to propose that abnormal metabolism and accumulation of alpha-syn in dopaminergic neurons play a role in the development of familial as well as sporadic PD. The complexity of the mechanisms underlying alpha-syn-induced neurotoxicity, however, has made difficult the development of animal models that faithfully reproduce human PD pathology. We now describe and characterize such a model, which is based on the stereotaxic injection into rat right substantia nigra pars compacta of the A30P mutated form of alpha-syn fused to a protein transduction domain (TAT). The TAT sequence allows diffusion of the fusion protein across the neuronal plasma membrane and results in a localized dopaminergic loss. Dopaminergic cell loss was evaluated both by tyrosine hydroxylase immunohistochemistry and by HPLC analysis of dopamine and its catabolite 3,4 dihydroxyphenylacetic acid. Infusion of TAT-alpha-synA30P induced a significant 26% loss in dopaminergic neurons. This dopaminergic loss was accompanied by a time-dependent impairment in motor function, evaluated utilizing the rotarod and footprint tests. In comparison to chemical neurotoxin-based (e.g. 6-hyroxydopamine, MPTP) animal models of PD, the alpha-syn-based PD animal model offers the advantage of mimicking the early stages and slow development of the human disease and should prove valuable in assessing specific aspects of PD pathogenesis in vivo and in developing new therapeutic strategies.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1 The effects of norbormide on the contractility of endothelium‐deprived rat, guinea‐pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2 In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5–50 μm) induced a concentration‐dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10–800 μm) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3 In resting rat and guinea‐pig aortae, guinea‐pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10–100 μm) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2+‐free medium. Norbormide (up to 100 μm) was ineffective in phenylephrine‐contracted guinea‐pig and rat aorta. 4 In A7r5 cells, a cell line from rat aorta, norbormide prevented high K+‐but not 5‐hydro‐xytryptamine‐induced intracellular calcium transients. 5 These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.

Toxic effect of chromium on cellular metabolism

The cytotoxic action of Cr(VI) in intact mammalian cells is strictly related to its carrier-mediated transport across the plasma membrane and its reduction to Cr(III) inside the cell. A marked decrease in the ratio of reduced/oxidized glutathione in rat thymocytes treated with dichromate indicates an involvement of glutathione in the reduction of Cr(VI) to Cr(III) in these cells. Intracellular chromium is shown to interfere with specific steps of cellular energy metabolism in that it produces severe unbalance of purine ribonucleotide pools in hamster fibroblast cultures and inhibition of mitochondrial oxygen consumption in rat thymocytes at micromolar concentrations. To what extent the effects caused by Cr(VI) are directly related to the reduction process or to the subsequent binding of Cr(III), and possibly of Cr(V), to biological molecules playing a critical role in cell physiology, remains to be elucidated.

alpha-Synuclein- and MPTP-generated rodent models of Parkinson's disease and the study of extracellular striatal dopamine dynamics: a microdialysis approach.

The classical animal models of Parkinsons disease (PD) rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine and, more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These neurotoxins elicit motor deficits in different animal species although MPTP fails to induce a significant dopaminergic neurodegeneration in rats. In the attempt to better reproduce the key features of PD, in particular the progressive nature of neurodegeneration, alternative PD models have been developed, based on the genetic and neuropathological links between -synuclein ( -syn) and PD. In vivo microdialysis was used to investigate extracellular striatal DA dynamics in MPTP- and -syn-generated rodent models of PD. Acute and sub-acute MPTP intoxication of mice both induce prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion and nigral neuron death, mainly through reactive oxygen species generation. Although MPTP induces DA reduction, neurochemical and motor recovery starts immediately after the end of treatment, suggesting that compensatory mechanisms are activated. Thus, the MPTP mouse model of PD may be unsuitable for closely reproducing the features of the human disease and predicting potential long-term therapeutic effects, in terms of both striatal extracellular DA and behavioral outcome. In contrast, the -syn-generated rat model of PD does not suffer from a massive release of striatal DA during induction of the nigral lesion, but rather is characterized by a prolonged reduction in baseline DA and nicotine-induced increases in dialysate DA levels. These results are suggestive of a stable nigrostriatal lesion with a lack of dopaminergic neurochemical recovery. The -syn rat model thus reproduces the initial stage and slow development of PD, with a time-dependent impairment in motor function. This article will describe the above experimental PD models and demonstrate the utility of microdialysis for their characterization.