Publications by authors named "Rita Pepponi"

24 Publications

  • Page 1 of 1

Insight into the Role of the STriatal-Enriched Protein Tyrosine Phosphatase (STEP) in A Receptor-Mediated Effects in the Central Nervous System.

Front Pharmacol 2021 19;12:647742. Epub 2021 Apr 19.

National Centre for Drug Research and Evaluation, Istituto Superiore di Sanitá, Rome, Italy.

The STriatal-Enriched protein tyrosine phosphatase STEP is a brain-specific tyrosine phosphatase that plays a pivotal role in the mechanisms of learning and memory, and it has been demonstrated to be involved in several neuropsychiatric diseases. Recently, we found a functional interaction between STEP and adenosine A receptor (AR), a subtype of the adenosine receptor family widely expressed in the central nervous system, where it regulates motor behavior and cognition, and plays a role in cell survival and neurodegeneration. Specifically, we demonstrated the involvement of STEP in AR-mediated cocaine effects in the striatum and, more recently, we found that in the rat striatum and hippocampus, as well as in a neuroblastoma cell line, the overexpression of the AR, or its stimulation, results in an increase in STEP activity. In the present article we will discuss the functional implication of this interaction, trying to examine the possible mechanisms involved in this relation between STEP and ARs.
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http://dx.doi.org/10.3389/fphar.2021.647742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8090931PMC
April 2021

P2X7 Receptor Agonist 2'(3')-O-(4-Benzoylbenzoyl)ATP Differently Modulates Cell Viability and Corticostriatal Synaptic Transmission in Experimental Models of Huntington's Disease.

Front Pharmacol 2020 19;11:633861. Epub 2021 Feb 19.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Huntington's disease (HD) is a life-threatening neurodegenerative disorder. Altered levels and functions of the purinergic ionotropic P2X7 receptors (P2X7Rs) have been found in animal and cellular models of HD, suggesting their possible role in the pathogenesis of the disease; accordingly, the therapeutic potential of P2X7R antagonists in HD has been proposed. Here we further investigated the effects of P2X7R ligands in and HD experimental models. In ST14A/Q120 rat striatal cells, we found a reduction of P2X7R expression; however, the P2X7R agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) induced cellular death, and this effect was fully reversed by the antagonist periodate-oxidized adenosine 5'-triphosphate (OxATP). Moreover, in corticostriatal slices from symptomatic R6/2 mice, BzATP reduced the synaptic transmission to a larger extent than in wild-type (WT) mice. Such an effect was accompanied by a concomitant increase of the paired-pulse ratio, suggesting a presynaptic inhibitory action. This was confirmed to be the case, since while the effects of BzATP were unaffected by the P2X7R antagonist OxATP, they were blocked by the adenosine A receptor (AR) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), suggesting possible BzATP hydrolysis to 2'(3')-O-(4-benzoylbenzoyl)adenosine (Bz-adenosine) and consequent activation of ARs as a mechanism. Taken together, these data point out that 1) P2X7R expression and activity are confirmed to be altered in the presence of HD mutation; 2) in some experimental settings, such an abnormal functioning can be ascribed to presynaptic ARs activation.
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http://dx.doi.org/10.3389/fphar.2020.633861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933594PMC
February 2021

The activity of the Striatal-enriched protein tyrosine phosphatase in neuronal cells is modulated by adenosine A receptor.

J Neurochem 2020 02 10;152(3):284-298. Epub 2019 Oct 10.

National Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

We recently demonstrated that a tonic activation of adenosine A receptors (A Rs) is required for cocaine-induced synaptic depression and increase in the activity of STriatal-Enriched protein tyrosine Phosphatase (STEP). In this study, we elaborated on the relationship between A R and STEP using genetic, pharmacological, and cellular tools. We found that the activities of protein tyrosine phosphatases (PTPs), and in particular of STEP, are significantly increased in the striatum and hippocampus of a transgenic rat strain over-expressing the neuronal A R (NSEA ) with respect to wild-type (WT) rats. Moreover the selective A R agonist 4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride up-regulates PTPs and STEP activities in WT but not in NSEA rats, while the selective A R antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol restores the tyrosine phosphatase activities in NSEA , having no effects in WT rats. In addition, while cocaine induced the activation of PTP and STEP in WT rats, it failed to increase phosphatase activity in NSEA rats. A Rs modulate STEP activity also in the SH-SY5Y neuroblastoma cell line, where a calcium-dependent calcineurin/PP1 pathway was found to play a major role. In summary, the present study identified a novel interaction between A R and STEP that could have important clinical implications, since STEP has emerged as key regulator of signaling pathways involved in neurodegenerative and neuropsychiatric diseases and A Rs are considered a promising target for the development of therapeutic strategies for different diseases of the central nervous system. Read the Editorial Highlight for this article on page 270.
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http://dx.doi.org/10.1111/jnc.14866DOI Listing
February 2020

Adenosine A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes.

Sci Rep 2019 07 5;9(1):9782. Epub 2019 Jul 5.

Research Coordination and Support Service, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A receptors (AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.
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http://dx.doi.org/10.1038/s41598-019-46268-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611770PMC
July 2019

Adenosine A receptor as potential therapeutic target in neuropsychiatric disorders.

Pharmacol Res 2019 09 2;147:104338. Epub 2019 Jul 2.

National Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, Roma, Italy.

Adenosine A receptor (AR) is a G-protein coupled receptor that regulates several important functions in the central nervous system. Large amount of preclinical data suggests that the AR could represent a target for the development of new therapeutic strategies for different neuropsychiatric conditions. In this review we will recapitulate and discuss the most relevant studies on the role of ARs in neurodegenerative, neurodevelopmental and psychiatric diseases, which led to suggest a therapeutic use of AR agonists in certain diseases (Niemann-Pick disease, autism-spectrum disorders, schizophrenia) and AR antagonists in others (Alzheimer's disease, Parkinson's disease, attention-deficit hyperactivity disorder, fragile X syndrome, depression, anxiety). Moreover, we will try to analyze which are the main obstacles to the conduction of clinical trials with AR ligands for the treatment of neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.phrs.2019.104338DOI Listing
September 2019

Neuroprotective potential of adenosine A receptor partial agonists in experimental models of cerebral ischemia.

J Neurochem 2019 04 11;149(2):211-230. Epub 2019 Feb 11.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A receptors (A Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in in vitro and ex vivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation in vitro in human neuroblastoma (SH-SY5Y) cells both A R partial agonists increased cell viability. Considering the high level of expression of A Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7 min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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http://dx.doi.org/10.1111/jnc.14660DOI Listing
April 2019

Neuronal adenosine A receptor overexpression is neuroprotective towards 3-nitropropionic acid-induced striatal toxicity: a rat model of Huntington's disease.

Purinergic Signal 2018 09 16;14(3):235-243. Epub 2018 May 16.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

The A adenosine receptor (AR) is widely distributed on different cellular types in the brain, where it exerts a broad spectrum of pathophysiological functions, and for which a role in different neurodegenerative diseases has been hypothesized or demonstrated. To investigate the role of neuronal ARs in neurodegeneration, we evaluated in vitro and in vivo the effect of the neurotoxin 3-nitropropionic acid (3-NP) in a transgenic rat strain overexpressing ARs under the control of the neural-specific enolase promoter (NSEA rats). We recorded extracellular field potentials (FP) in corticostriatal slice and found that the synaptotoxic effect of 3-NP was significantly reduced in NSEA rats compared with wild-type animals (WT). In addition, after exposing corticostriatal slices to 3-NP 10 mM for 2 h, we found that striatal cell viability was significantly higher in NSEA rats compared to control rats. These in vitro results were confirmed by in vivo experiments: daily treatment of female rats with 3-NP 10 mg/kg for 8 days induced a selective bilateral lesion in the striatum, which was significantly reduced in NSEA compared to WT rats. These results demonstrate that the overexpression of the AR selectively at the neuronal level reduced 3-NP-induced neurodegeneration, and suggest an important function of the neuronal AR in the modulation of neurodegeneration.
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http://dx.doi.org/10.1007/s11302-018-9609-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107463PMC
September 2018

Expression, pharmacology and functional activity of adenosine A1 receptors in genetic models of Huntington's disease.

Neurobiol Dis 2014 Nov 15;71:193-204. Epub 2014 Aug 15.

Istituto Superiore di Sanità, Department of Therapeutic Research and Medicines Evaluation, Viale Regina Elena 299, 00161 Rome, Italy.

Adenosine A1 receptor (A1R) stimulation exerts beneficial effects in response to various insults to the brain and, although it was found neuroprotective in a lesional model of Huntington's disease (HD), the features of this receptor in genetic models of HD have never been explored. In the present study we characterized the expression, affinity and functional effects of A1Rs in R6/2 mice (the most widely used transgenic model of HD) and in a cellular model of HD. Binding studies revealed that the density of A1Rs was significantly reduced in the cortex and the striatum of R6/2 mice compared to age-matched wild-type (WT), while receptor affinity was unchanged. The selective A1R agonist cyclopentyladenosine (CPA, 300nM) was significantly more effective in reducing synaptic transmission in corticostriatal slices from symptomatic R6/2 than in age-matched WT mice. Such an effect was due to a stronger inhibition of glutamate release from the pre-synaptic terminal. The different functional activities of A1Rs in HD mice were associated also to a different intracellular signaling pathway involved in the synaptic effect of CPA. In fact, while the PKA pathway was involved in both genotypes, p38 MAPK inhibitor SB203580 partially prevented synaptic effects of CPA in R6/2, but not in WT, mice; moreover, CPA differently modulated the phosphorylation status of p38 in the two genotypes. In vitro studies confirmed a different behavior of A1Rs in HD: CPA (100 nM for 5h) modulated cell viability in STHdh(Q111/Q111) (mhttHD cells), without affecting the viability of STHdh(Q7/Q7) (wthtt cells). This effect was prevented by the application of SB203580. Our results demonstrate that in the presence of the HD mutation A1Rs undergo profound changes in terms of expression, pharmacology and functional activity. These changes have to be taken in due account when considering A1Rs as a potential therapeutic target for this disease.
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http://dx.doi.org/10.1016/j.nbd.2014.08.013DOI Listing
November 2014

The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann-Pick type C patients.

J Neurosci 2013 Sep;33(39):15388-93

Department of Cell Biology and Neuroscience, and Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161 Rome, Italy.

Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.
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http://dx.doi.org/10.1523/JNEUROSCI.0558-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618463PMC
September 2013

Interferon-Beta combined with interleukin-2 restores human natural cytotoxicity impaired in vitro by ionizing radiations.

J Interferon Cytokine Res 2013 Jun 19;33(6):308-18. Epub 2013 Feb 19.

Department of Neuroscience, Chair of Pharmacology, School of Medicine, University of Rome Tor Vergata, Rome, Italy.

It is well known that ionizing radiations induce a marked downregulation of antigen-dependent and natural immunity for a prolonged period of time. This is due, at least in part, to radiation-induced apoptosis of different lymphocyte subpopulations, including natural killer (NK) cells. Aim of this study was to investigate the capability of Beta Interferon (β-IFN) and Interleukin-2 (IL2), alone or in combination, to restore the functional activity of the natural immune system. Mononuclear cells (MNCs) obtained from intact or in vitro irradiated human peripheral blood were treated in vitro with β-IFN immediately before or at the end of the 4-day treatment with IL2. Time-course analysis was performed on the NK activity, the total number and the apoptotic fraction of CD16+ and CD56+ cells, the 2 main NK effector cell subpopulations. The results indicate that radiation-induced impairment of natural cytotoxicity of MNC could be successfully antagonized by the β-IFN+IL2 combination, mainly when exposure to β-IFN preceded IL2 treatment. This radioprotective effect is paralleled by lower levels of radiation-induced apoptosis and increased expression of the antiapoptotic Bcl-2 protein. Since natural immunity can play a significant role in antitumor host's resistance, these results could provide the rational basis for a cytokine-based pharmacological strategy able to restore immune responsiveness and to afford possible therapeutic benefits in cancer patients undergoing radiotherapy.
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http://dx.doi.org/10.1089/jir.2012.0025DOI Listing
June 2013

BDNF prevents NMDA-induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved.

J Neurochem 2013 Apr 27;125(2):225-35. Epub 2013 Feb 27.

Section of Central Nervous System Pharmacology, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.
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http://dx.doi.org/10.1111/jnc.12177DOI Listing
April 2013

Potential therapeutic relevance of adenosine A2B and A2A receptors in the central nervous system.

CNS Neurol Disord Drug Targets 2012 Sep;11(6):664-74

Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy.

Adenosine A2B and, much more importantly, adenosine A2A receptors modulate many physiological and pathological processes in the brain. In this review, the most recent evidence concerning the role of such receptors and their potential therapeutic relevance is discussed. The low affinity of A2B receptors for adenosine implies that they might represent a good therapeutic target, since they are activated only under pathological conditions (when adenosine levels raise up to micromolar concentrations). The availability of selective ligands for A2B receptors would allow exploration of such an hypothesis. Since adenosine A2A receptors mediate both potentially neuroprotective and potentially neurotoxic effects, their role in neurodegenerative diseases is highly controversial. Nevertheless, A2A receptor antagonists have shown clear antiparkinsonian effects, and a great interest exists on the role of A2A receptors in Alzheimer's disease, brain ischaemia, spinal cord injury, drug addiction and other conditions. In order to establish whether such receptors represent a target for CNS diseases, at least two conditions are needed: the full comprehension of A2A-dependent mechanisms and the availability of ligands capable of discriminating among the different receptor populations.
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http://dx.doi.org/10.2174/187152712803581100DOI Listing
September 2012

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum.

J Neurochem 2009 Sep 17;110(6):1921-30. Epub 2009 Jul 17.

Section of Central Nervous System Pharmacology, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06282.xDOI Listing
September 2009

Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons.

Eur J Pharmacol 2009 Sep 18;617(1-3):28-32. Epub 2009 Jul 18.

Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A(2A) receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A(2A) receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pre-treatment with the adenosine A(2A) receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A(2A) receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity.
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http://dx.doi.org/10.1016/j.ejphar.2009.07.016DOI Listing
September 2009

A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.

PLoS Genet 2008 Nov 21;4(11):e1000266. Epub 2008 Nov 21.

Department of Environment and Primary Prevention, Experimental Carcinogenesis Division, Istituto Superiore di Sanità, Rome, Italy.

Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington's disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington's disease-like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington's disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington's disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.
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http://dx.doi.org/10.1371/journal.pgen.1000266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580033PMC
November 2008

Neuroprotective effects of thymosin beta4 in experimental models of excitotoxicity.

Ann N Y Acad Sci 2007 Sep;1112:219-24

Istituto Superiore di Sanità, Viale Regina Elena, 299 Rome, Italy.

The aim of this study was to evaluate the possible neuroprotective effects of thymosin beta(4) in different models of excitotoxicity. The application of thymosin beta(4) significantly attenuated glutamate-induced toxicity both in primary cultures of cortical neurons and in rat hippocampal slices. In in vivo experiments, the intracerebroventricular administration of thymosin beta(4) significantly reduced hippocampal neuronal loss induced by kainic acid. These results show that thymosin beta(4) induced a protective effect in models of excitotoxicity. The mechanisms underlying such an effect, as well as the real neuroprotective potential of thymosin beta(4), are worthy of further investigations.
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http://dx.doi.org/10.1196/annals.1415.033DOI Listing
September 2007

Combined effect of temozolomide and hyperthermia on human melanoma cell growth and O6-methylguanine-DNA methyltransferase activity.

Int J Oncol 2007 Feb;30(2):443-51

Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata-IRCCS, I-00167 Rome, Italy.

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37 degrees C or 41.5 degrees C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5 degrees C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.
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February 2007

A single cycle of treatment with temozolomide, alone or combined with O(6)-benzylguanine, induces strong chemoresistance in melanoma cell clones in vitro: role of O(6)-methylguanine-DNA methyltransferase and the mismatch repair system.

Int J Oncol 2006 Oct;29(4):785-97

Institute of Neurobiology and Molecular Medicine, Tor Vergata Research Area, National Research Council, I-00133 Rome, Italy.

Clinically achievable concentrations of temozolomide (TMZ) produce cytotoxic effects only in mismatch repair (MMR)-proficient cells endowed with low O6-methylguanine-DNA methyltransferase (MGMT) activity. Aim of the present study was to investigate the molecular mechanisms underlying acquired resistance of melanoma cells to TMZ and the effect of O6-benzylguanine (BG), a specific MGMT inhibitor, on the development of a TMZ-resistant phenotype. Three MMR-proficient melanoma cell clones with low or no MGMT activity were treated daily for 5 days with 50 micromol/l TMZ, alone or in combination with 5 micromol/l BG. Parental clones and sublines established after one or four cycles of treatment were analyzed for sensitivity to TMZ or TMZ+BG and for other parameters. The sublines established after one cycle of TMZ or TMZ+BG exhibited a marked increase in MGMT activity and resistance to TMZ alone. BG only partially reversed acquired resistance to the drug. In some cases, alterations in the MMR system accounted for MGMT-independent resistance to TMZ. Up-regulation of MGMT activity was associated with either demethylation of the MGMT promoter or hypermethylation of the body of the gene, and partially reversed by 5-aza-2'-deoxycytidine. The sublines established after four cycles of TMZ or TMZ+BG did not show a further increase in resistance to TMZ alone. However, two out of three sublines established after TMZ+BG treatment exhibited increased resistance to TMZ+BG. In conclusion, our data demonstrate that a single cycle of TMZ is sufficient to induce high levels of drug resistance in melanoma clones, principally, but not exclusively, via up-regulation of MGMT expression. Exposure to TMZ+BG favors the development of MGMT-independent mechanisms of TMZ resistance.
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October 2006

Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre- and postsynaptic sites in the rat striatum.

J Neurosci Res 2004 Jul;77(1):100-7

Department of Pharmacology, Istituto Superiore di Sanità, Rome, Italy.

Adenosine A(2A) receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A(2A) receptor antagonists in models of pre- and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A(2A) receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose-dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30-100 nM) significantly reduced 4-aminopyridine (4-AP)-induced paired-pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N-methyl-D-aspartate (NMDA; 12.5 and 50 microM). The A(2A) antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A(2A) agonist CGS 21680 (100 nM) showed a protective influence toward 50 microM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca(2+)](i) or the release of lactate dehydrogenase (LDH) induced by 100 and 300 microM NMDA, respectively. The ability of ZM 241385 to prevent QA-induced glutamate outflow and 4-AP-induced effects confirms that A(2A) receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA-induced effects suggest that A(2A) receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A(2A) antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.
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http://dx.doi.org/10.1002/jnr.20138DOI Listing
July 2004

Neuroprotective effects of the mGlu5R antagonist MPEP towards quinolinic acid-induced striatal toxicity: involvement of pre- and post-synaptic mechanisms and lack of direct NMDA blocking activity.

J Neurochem 2004 Jun;89(6):1479-89

Department of Pharmacology, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy.

The aim of this work was to investigate the potential neuroprotective effects of the metabotropic glutamate receptor 5 (mGlu5R) antagonist 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) towards quinolinic acid (QA)-induced striatal excitoxicity. Intrastriatal MPEP (5 nmol/0.5 micro L) significantly attenuated the body weight loss, the electroencephalographic alterations, the impairment in spatial memory and the striatal damage induced by bilateral striatal injection of QA (210 nmol/0.7 micro L). In a second set of experiments, we aimed to elucidate the mechanisms underlying the neuroprotective effects of MPEP. In microdialysis studies in naive rats MPEP (80-250 micro m through the dialysis probe) significantly reduced the increase in glutamate levels induced by 5 mm QA. In primary cultures of striatal neurons MPEP (50 micro m) reduced the toxicity induced by direct application of glutamate [measured as release of lactate dehydrogenase [LDH]). Finally, we found that 50 micro m MPEP was unable to directly block NMDA-induced effects (namely field potential reduction in corticostriatal slices, as well as LDH release and intracellular calcium increase in striatal neurons). We conclude that: (i) MPEP has neuroprotective effects towards QA-induced striatal excitotoxicity; (ii) both pre- and post-synaptic mechanisms are involved; (iii) the neuroprotective effects of MPEP do not appear to involve a direct blockade of NMDA receptors.
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http://dx.doi.org/10.1111/j.1471-4159.2004.02448.xDOI Listing
June 2004

Cholesterol perturbing agents inhibit NMDA-dependent calcium influx in rat hippocampal primary culture.

FEBS Lett 2004 May;566(1-3):25-9

Laboratory of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

The present study was carried out to investigate the potential involvement of cholesterol-rich membrane microdomains in the mobilization of calcium induced by NMDA-receptors (NMDA-R). We herein provide evidence that agents interfering with plasma membrane cholesterol (namely, filipin and methyl-beta-cyclodextrin (Cdex)) inhibit the NMDA-stimulated influx of calcium in hippocampal cells in culture. Filipin-treated cells maintained their morphology and were able to respond with a calcium influx to high K(+) challenge, whereas Cdex altered both cellular parameters. These results suggest that the NMDA-R can be located in cholesterol-rich membrane microdomains or alternatively that the mechanisms coupling their dynamics in the post-synaptic membrane are dependent on the integrity of the microdomains.
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http://dx.doi.org/10.1016/j.febslet.2004.03.113DOI Listing
May 2004

Role of mismatch repair in the induction of chromosomal aberrations and sister chromatid exchanges in cells treated with different chemotherapeutic agents.

Cancer Chemother Pharmacol 2003 Sep 19;52(3):185-92. Epub 2003 Jun 19.

Department of Public Health and Cell Biology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

Purpose: The mismatch repair (MMR) system plays a major role in mediating the cytotoxicity and clastogenicity of agents generating O(6)-methylguanine in DNA. Loss of MMR has also been associated with tumor cell resistance to the cytotoxic effects of 6-thioguanine and cisplatin and with hypersensitivity to N-(2-chloroethyl)- N'-cyclohexyl- N-nitrosourea (CCNU). The aim of the present investigation was to elucidate the role played by the MMR system in the generation of chromosomal damage in cells exposed to 6-thioguanine, cisplatin or CCNU.

Methods: The MMR-proficient cell lines TK6 and HCT116/3-6, and their MMR-deficient counterparts MT1 and HCT116, were treated with 6-thioguanine, cisplatin or CCNU, and analyzed for cell growth inhibition and chromosomal damage. As a control, similar experiments were performed with the methylating agent temozolomide.

Results: Cytotoxicity, chromosomal aberrations and sister chromatid exchanges induced by 6-thioguanine and temozolomide were significantly reduced in the MMR-deficient cell lines with respect to their MMR-proficient counterparts. In contrast, although conferring some protection against cytotoxicity, the loss of MMR did not affect cytogenetic damage induced by cisplatin. CCNU produced comparable levels of cytotoxicity, chromosomal aberrations and sister chromatid exchanges in both MMR-proficient and MMR-deficient cell lines.

Conclusions: The MMR system is involved in the generation of chromosomal damage in cells exposed to 6-thioguanine. The system does not play a relevant role in the generation of chromosomal damage in cells treated with CDDP and does not confer protection against the clastogenic effects of CCNU, at least in the cell lines investigated.
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http://dx.doi.org/10.1007/s00280-003-0660-6DOI Listing
September 2003

The effect of O6-alkylguanine-DNA alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide, 1,3-bis(2-chloroethyl)1-nitrosourea, and cisplatin.

J Pharmacol Exp Ther 2003 Feb;304(2):661-8

Istituto Dermopatico Dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O(6)-benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ- or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC(50) values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.
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http://dx.doi.org/10.1124/jpet.102.043950DOI Listing
February 2003

High-frequency microsatellite instability is associated with defective DNA mismatch repair in human melanoma.

J Invest Dermatol 2002 Jan;118(1):79-86

Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy.

Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.
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http://dx.doi.org/10.1046/j.0022-202x.2001.01611.xDOI Listing
January 2002