[Show abstract][Hide abstract] ABSTRACT: Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 μM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.
Full-text · Article · Nov 2015 · Neuropharmacology
[Show abstract][Hide abstract] ABSTRACT: 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 ciclopentyladenosine (CPA, 300 nM) 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 activity of A1Rs in HD mice was 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 behaviour of A1Rs in HD: CPA (100 nM for 5hour) 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 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.
No preview · Article · Aug 2014 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Huntington’s disease is a devastating hereditary neurodegenerative disorder caused by CAG mutation within the IT15 gene encoding Huntingtin protein. Even though mutant and normal Huntingtin are ubiquitously expressed, the degenerative processes primarily occur within the striatum and particularly hit the GABAergic enkephalin neuronal subpopulation of medium spiny neurons particularly enriched with adenosine A2ARs, suggesting that the latter might play a role in HD. In agreement, variants in the ADORA2A gene influence the age at onset in HD and A2AR dynamics is largely altered by mutated Huntingtin. Adenosine receptors are involved in a number of processes critical for neuronal function and homeostasis, such as modulation of synaptic activity and excitotoxicity, the control of neurotrophin levels and functions as well as the regulation of protein degradation mechanisms. In the present review, we critically reviewed the current knowledge involving adenosine receptors in HD and discussed whether they represent a suitable therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status are significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Autism is a neurodevelopmental disorder characterized by social and communication impairments and repetitive behaviours. The inbred BTBR T+tf/J (BTBR) strain, a putative mouse model of autism, exhibits lower social interactions, higher repetitive self-grooming levels and unusual pattern of vocalizations as compared to C57BL/6J strain. First aim of the present study was to evaluate at adolescence (postnatal days 30-35) male BTBR and C57BL/6J performances in two different tasks involving either investigation of social cues (same strain partners) or non social ones (inanimate objects). In the social interaction test, BTBR mice showed a reduction of investigation of the social partner, due to a selective reduction of head sniffing, associated with a decrease in ultrasonic vocalizations. By contrast, no strain differences were detected in object investigations. Second aim of the study was to evaluate adult male BTBR and C57BL/6J performances in a fear conditioning task. Strain differences were evident during contextual retest: these strain differences primarily suggested a lack of behavioural flexibility in BTBR mice (i.e. realizing the occurrence of changes in the experimental paradigm). Subsequent electrophysiological analysis in hippocampal slices from adult BTBR and C57BL/6J mice revealed a significant reduction of Brain Derived Neurotrophic Factor (BDNF)-induced potentiation of synaptic transmission in BTBR mice. BDNF and tyrosine kinase B (TrkB) protein levels measured in the hippocampal region were also lower in BTBR as compared to C57BL/6J mice. These data confirm the presence of low levels of direct interaction with social stimuli in BTBR mice at adolescence, in the absence of any strain difference as for investigation of physical objects. At adulthood in BTBR mice clear signs of behavioural inflexibility were evident whereas both biochemical and electrophysiological data point to decreased BDNF signalling (likely due to a reduction in TrkB levels) in the hippocampus of this mouse strain.
Full-text · Article · Dec 2012 · Behavioural brain research
[Show abstract][Hide abstract] ABSTRACT: The striatum is a subcortical area involved in sensorimotor, cognitive and emotional processes. Adenosine A(2A) receptors (A(2A)Rs) are highly expressed in the striatum, and their ability to establish functional and molecular interactions with many other receptors attributes to a pivotal role in the modulation and integration of striatal neurotransmission. This review will focus on the interaction between A(2A)Rs and cannabinoid CB(1) receptors (CB(1)Rs), taking it as a paradigmatic example of synaptic integration. Indeed, A(2A)Rs can exert an opposite (permissive vs. inhibitory) influence on CB1-dependent synaptic effect. These apparently irreconcilable functions could depend on a different role of pre- vs. postsynaptic A(2A)Rs, on their interaction with other receptors (namely adenosine A(1), metabotropic glutamate 5 and dopamine D2 receptors), and on whether A(2A)Rs form or not heteromers with CB(1)Rs. Besides providing a good example of the intricate pattern of events taking place in striatal synapses, the A(2A)/CB(1)R interaction proves very informative to understand the physiology of the basal ganglia and the mechanisms of related diseases. This article is part of a Special Issue entitled: Brain Integration.
[Show abstract][Hide abstract] ABSTRACT: Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.
No preview · Article · Dec 2011 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: We hypothesized that chronic supplementation with branched chain amino acids (BCAAs) affects neurobehavioral development in vulnerable gene backgrounds.
A murine model of amyotrophic lateral sclerosis (ALS), G93A mice bearing the mutated human superoxide dismutase 1 (SOD1) gene, and control mice received from 4 to 16 wk of age dietary supplementation with BCAAs at doses comparable to human usage. Motor coordination, exploratory behaviors, pain threshold, synaptic activity and response to glutamatergic stimulation in primary motor cortex slices were evaluated between the 8th and 16th week. The glial glutamate transporter 1 (GLT-1) and metabotropic glutamate 5 receptor (mGlu5R) were analyzed by immunoblotting in cortex, hippocampus and striatum. BCAAs induced hyperactivity, decreased pain threshold in wild-type mice and exacerbated the motor deficits of G93A mice while counteracting their abnormal pain response. Electrophysiology on G93A brain slices showed impaired synaptic function, reduced toxicity of GLT-1 blocking and increased glutamate toxicity prevented by BCAAs. Immunoblotting indicated down-regulation of GLT-1 and mGlu5R in G93A, both effects counteracted by BCAAs.
These results, though not fully confirming a role of BCAAs in ALS-like etiology in the genetic model, clearly indicate that BCAAs' complex effects on central nervous system depend on gene background and raise alert over their spread use.
No preview · Article · Apr 2011 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2011) 116, 273–280.
An interaction between adenosine A2A receptors (A2ARs) and cannabinoid CB1 receptors (CB1Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB1R and A2AR in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A2AR activation by CGS21680 inhibited CB1R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A2AR activation prevented, while A2AR inhibition facilitated, the CB1R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB1R function by A2ARs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB1R and A2AR may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.
Full-text · Article · Nov 2010 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: In the last few years, accumulating evidence has shown the existence of an important cross-talk between adenosine A(2A) receptors (A(2A)Rs) and brain-derived neurotrophic factor (BDNF). Not only are A(2A)Rs involved in the mechanism of transactivation of BDNF receptor TrkB, they also modulate the effect of BDNF on synaptic transmission, playing a facilitatory and permissive role. The cAMP-PKA pathway, the main transduction system operated by A(2A)Rs, is involved in such effects. Furthermore, a basal tonus of A(2A)Rs is required to allow the regulation of BDNF physiological levels in the brain, as demonstrated by the reduced protein levels measured in A(2A)Rs KO mice. The crucial role of adenosine A(2A)Rs in the maintenance of synaptic functions and BDNF levels will be reviewed here and discussed in the light of possible implications for Huntington's disease therapy, in which a joint impairment of BDNF and A(2A)Rs seems to play a pathogenetic role.
Full-text · Article · Sep 2010 · The Scientific World Journal
[Show abstract][Hide abstract] ABSTRACT: The effect of chronic treatment with the selective adenosine A(2A) receptor agonist CGS 21680 on N-Methyl-d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntington's disease (HD). Starting from 8weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5mg/kg i.p.) for 3weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A(2A) receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.
[Show abstract][Hide abstract] ABSTRACT: Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A(2A)R blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A(2A)R blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A(2A)Rs in HD.
Full-text · Article · Oct 2009 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Aug 2009 · European journal of pharmacology
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2009 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown an impairment of the endocannabinoid system in experimental models of Huntington's disease. In transgenic R6/2 mice, created by inserting exon 1 of the human IT15 mutant gene into the mouse, and exhibiting 150 CAG repeats as well as signs of HD, a progressive decline of CB(1) receptor expression and an abnormal sensitivity to CB(1) receptor stimulation have been reported. Here, by using isotope-dilution liquid chromatography-mass spectrometry, we investigated whether the levels of three endogenous neuroprotective substances, the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and palmitoylethanolamide (PEA), are altered in different brain areas of transgenic R6/2 versus wild-type (WT) mice at two different disease phases, i.e. in pre-symptomatic (4.5 weeks) or overtly symptomatic (10 weeks) R6/2 mice versus age-matched WT mice (n=4/group). Except for a approximately 25% decrease in 2-AG levels in the cortex, no significant changes in endocannabinoid and PEA levels were observed in pre-symptomatic R6/2 versus WT mice. By contrast, in symptomatic R6/2 mice the levels of all three compounds were significantly (approximately 30-60%) decreased in the striatum, whereas little changes were observed in the hippocampus, and a approximately 28% decrease of 2-AG levels, accompanied by a approximately 50% increase of AEA levels, was found in the cortex. These findings show that endocannabinoid levels change in a disease phase- and region-specific way in the brain of R6/2 mice and indicate that an impaired endocannabinoid system is a hallmark of symptomatic HD, thus suggesting that drugs inhibiting endocannabinoid degradation might be used to treat this disease.
No preview · Article · Feb 2008 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Both BDNF and its tyrosine kinase receptors (TrkB) are highly expressed in the hippocampus, where an interaction with adenosine A(2A) receptors (A(2A)Rs) has been recently reported. In the present paper, we evaluated the role of A(2A)Rs in mediating functional effects of BDNF in hippocampus using A(2A)R knock-out (KO) mice. In hippocampal slices from WT mice, application of BDNF (10 ng/mL) increased the slope of excitatory post-synaptic field potentials (fEPSPs), an index of synaptic facilitation. This increase of fEPSP slope was abolished by the selective A(2A) antagonist ZM 241385. Similarly, genetic deletion of the A(2A)Rs abolished BDNF-induced increase of the fEPSP slope in slices from A(2A)R KO mice The reduced functional ability of BDNF in A(2A)R KO mice was correlated with the reduction in hippocampal BDNF levels. In agreement, the pharmacological blockade of A(2)Rs by systemic ZM 241385 significantly reduced BDNF levels in the hippocampus of normal mice. These results indicate that the tonic activation of A(2A)Rs is required for BDNF-induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus.
Full-text · Article · Feb 2008 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: The effect of chronic treatment with the selective adenosine A2A receptor antagonist SCH 58261 on the behavioral and electrophysiological alterations typical of R6/2 mice (a transgenic mouse model of Huntington's disease, HD), has been studied. Starting from 5 weeks of age, R6/2 and wild type (WT) mice were treated daily with SCH 58261 (0.01 mg/kg i.p.) for 7 days. In the following weeks, the ability of mice to perform in the rotarod, plus maze and open field tests were evaluated. In addition, with electrophysiological experiments in corticostriatal slices we tested whether the well-known increased NMDA vulnerability of R6/2 mice was prevented by SCH 58261 treatment. We found that chronic treatment with SCH 58262: i) fully prevented the alterations in emotional/anxious responses displayed by R6/2 mice; ii) did not prevent the impairment in motor coordination; iii) abolished the increase in NMDA-induced toxicity observed in the striatum of HD mice. On balance, targeting A2A receptors seems to have some beneficial effects in HD even though, given the complexity of A2A receptor pharmacology and HD pathogenesis, further studies are necessary to clarify whether A2A receptor antagonists have therapeutic potential in HD.
No preview · Article · Dec 2007 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A(2A) receptors (A(2A)Rs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A(2A)Rs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A(2A)R knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A(2A)R KO vs. WT mice. Having found an even marked reduction in the striatum of A(2A)R KO mice, and as both BDNF and A(2A)Rs have been implicated in the pathogenesis of Huntington's disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A(2A)Rs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A(2A)R antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A(2A)Rs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.
Full-text · Article · Oct 2007 · Purinergic Signalling