Stimulation of adenosine A2A receptors reduces intracellular cholesterol accumulation and rescues mitochondrial abnormalities in human neural cell models of Niemann-Pick C1.

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.

Reduced social interaction, behavioural flexibility and BDNF signalling in the BTBR T+ tf/J strain, a mouse model of autism.

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.

Adenosine A(2A) receptors are required for normal BDNF levels and BDNF-induced potentiation of synaptic transmission in the mouse hippocampus.

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.

Behavioral and electrophysiological effects of the adenosine A2A receptor antagonist SCH 58261 in R6/2 Huntington's disease mice.

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.

Adenosine A(2A) receptors modulate BDNF both in normal conditions and in experimental models of Huntington's disease.

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.

The cannabinoid receptor agonist WIN 55,212-2 attenuates the effects induced by quinolinic acid in the rat striatum.

The ability of CB(1) receptors to regulate the release of glutamate in the striatum, together with the finding that, in experimental models of Huntington disease (HD), both endocannabinoid levels and CB(1) receptor densities are reduced, has prompted the investigation on the neuroprotective role of the cannabinoids in HD. Quinolinic acid (QA) is an excitotoxin that, when injected in the rat striatum reproduces many features of HD and that acts by stimulating glutamate outflow. The aim of the present study was to test the ability of the cannabinoid receptor agonist WIN 55,212-2 to prevent the effects induced by QA in the rat striatum. In microdialysis experiments, probe perfusion with WIN 55,212-2 significantly and dose-dependently prevented the increase in extracellular glutamate induced by QA. In electrophysiological recordings in corticostriatal slices, the application of WIN 55,212-2 prevented QA-induced reduction of the field potential amplitude. Both effects of WIN 55,212-2 were prevented by the CB(1) receptor antagonist AM 251. In in vivo experiments, intrastriatal WIN 55,212-2 significantly attenuated the striatal damage induced by QA, although no significant effects were observed on a behavioural ground. These data demonstrate that the stimulation of CB(1) receptors might lead to neuroprotective effects against excitotoxic striatal toxicity.

Is the functional interaction between adenosine A(2A) receptors and metabotropic glutamate 5 receptors a general mechanism in the brain? Differences and similarities between the striatum and the hippocampus.

The aim of the present paper was to examine, in a comparative way, the occurrence and the mechanisms of the interactions between adenosine A(2A) receptors (A(2A)Rs) and metabotropic glutamate 5 receptors (mGlu5Rs) in the hippocampus and the striatum. In rat hippocampal and corticostriatal slices, combined ineffective doses of the mGlu5R agonist 2-chloro-5-hydroxyphenylglycine (CHPG) and the A(2A)R agonist CGS 21680 synergistically reduced the slope of excitatory postsynaptic field potentials (fEPSPs) recorded in CA1 and the amplitude of field potentials (FPs) recorded in the dorsomedial striatum. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway appeared to be involved in the effects of CGS 21680 in corticostriatal but not in hippocampal slices. In both areas, a postsynaptic locus of interaction appeared more likely. N-methyl-D: -aspartate (NMDA) reduced the fEPSP slope and FP amplitude in hippocampal and corticostriatal slices, respectively. Such an effect was significantly potentiated by CHPG in both areas. Interestingly, the A(2A)R antagonist ZM 241385 significantly reduced the NMDA-potentiating effect of CHPG. In primary cultures of rat hippocampal and striatal neurons (ED 17, DIV 14), CHPG significantly potentiated NMDA-induced lactate dehydrogenase (LDH) release. Again, such an effect was prevented by ZM 241385. Our results show that A(2A) and mGlu5 receptors functionally interact both in the hippocampus and in the striatum, even though different mechanisms seem to be involved in the two areas. The ability of A(2A)Rs to control mGlu5R-dependent effects may thus be a general feature of A(2A)Rs in different brain regions (irrespective of their density) and may represent an additional target for the development of therapeutic strategies against neurological disorders.

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects.

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.

Permissive role of adenosine A2A receptors on metabotropic glutamate receptor 5 (mGluR5)-mediated effects in the striatum.

The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) have been reported to functionally interact in the striatum. The aim of the present work was to verify the hypothesis that the state of activation of A2A Rs could influence mGlu5R-mediated effects in the striatum. In electrophysiological experiments (extracellular recording in rat corticostriatal slices), the ability of the selective mGlu5R agonist CHPG to potentiate the reduction of the field potential amplitude induced by NMDA was prevented not only by the selective mGlu5R antagonist MPEP, but also by the selective A2AR antagonist ZM 241385. Analogously, the application of CHPG potentiated NMDA-induced toxicity (measured by LDH release) in cultured striatal neurons, an effect that was abolished by both MPEP and ZM 241385. Finally, the A2AR agonist CGS 21680 potentiated CHGP effects, an action that was reproduced and abolished, respectively, by forskolin (an activator of the cAMP/protein kinase A, PKA, pathway) and KT 5720 (a PKA inhibitor). The results indicate that A2ARs exert a permissive role on mGlu5R-induced effects in the striatum. Such an interaction may represent an additional target for the development of therapeutic strategies towards striatal disorders.

New embryological evidence for the formation of quadricuspid aortic valves in the Syrian hamster (Mesocricetus auratus).

A Syrian hamster embryo, aged 11 days and 4 h post-coitus, had a developing quadricuspid aortic valve. The septation of the cardiac outflow tract was confined to the distal part of the conotruncus. There was a conspicuous recess in the anlage that normally gives rise to the left aortic valve cushion. Globular endothelial cells arranged in several layers were present at the luminal side of the recess. The present findings support the hypothesis that in the Syrian hamster, quadricuspid aortic valves result from the division of one of the three mesenchymal anlagen that give rise to normal aortic valves. In addition, they indicate that the division of the anlage is due to the invagination of the endothelial layer that covers its luminal side. The invagination of the endothelium starts at a very early stage of the valvulogenesis, namely, when the conotruncal ridges begin to fuse at the distal portion of the embryonic cardiac outflow tract.

Anatomy and formation of congenital bicuspid and quadricuspid pulmonary valves in Syrian hamsters.

BACKGROUND: Congenital bicuspid and quadricuspid pulmonary valves have received little attention because of their limited clinical relevance. However, knowledge of the mechanisms by which these anomalous valves develop is essential to obtain a more accurate survey of the etiological factors implicated in the malformations of the cardiac outflow tract in mammals. The present study was designed to assess the anatomical features of bicuspid and quadricuspid pulmonary valves in Syrian hamsters as well as to elucidate the mechanisms involved in the formation of these defective valves. METHODS: The sample examined consisted of 206 adults and 28 embryos belonging to a laboratory-inbred family of Syrian hamsters with a high incidence of congenital anomalies of the pulmonary and aortic valves. The study was carried out using histological techniques for light microscopy, semithin sections, and scanning electron microscopy. RESULTS: The pulmonary valve was tricuspid in 140 of the 206 adult hamsters, and in 124 of these tricuspid valves the dorsal commissure was more or less extensively fused. Another 45 hamsters possessed a bicuspid pulmonary valve with the sinuses oriented ventrodorsally. In 43 of these bicuspid valves, a raphe was located in the dorsal pulmonary sinus. The pulmonary valve was quadricuspid in a further nine specimens. The remaining 12 hamsters had a tricuspid pulmonary valve with a raphe-like ridge located in the right pulmonary sinus. In seven of these valves, the dorsal commissure showed a more or less extensive fusion. The embryos examined, aged between 11 days, 3 hours and 12 days, 6 hours postcoitum, were at the beginning of the valvulogenesis. In five of the 28 embryos, the pulmonary valve consisted of three mesenchymal valve cushions, right, left, and dorsal. In a further 17 embryos, the right and left valve cushions were more or less fused toward the lumen of the pulmonary artery. In the remaining six embryos, the left and dorsal valve cushions were normal, whereas the right cushion was divided into two lobes. CONCLUSIONS: The present findings suggest that in the Syrian hamster: (1) bicuspid pulmonary valves result from the extensive fusion of the right and left pulmonary valve cushions at the beginning of the valvulogenesis, (2) the partial fusion of the right and left pulmonary valve cushions leads to the formation of tricuspid pulmonary valves with a more or less extensive fusion of the dorsal commissure, (3) quadricuspid pulmonary valves result from the partition of one of the three valve cushions at a very early stage of the valvulogenesis, and (4) the partial division of the right pulmonary valve cushion may lead to the development of tricupsid pulmonary valves with a raphe-like ridge located in the right pulmonary sinus. In addition, the present findings, together with previous observations in Syrian hamsters, indicate that in this species the mechanisms by which bicuspid and quadricuspid pulmonary valves develop are similar to those by which bicuspid and quadricuspid aortic valves form, respectively. However, the primary factor or factors that induce the malformations of the pulmonary valve operate independently from those inducing the malformations of the aortic valve.

Pirenzepine versus ranitidine in gastroprotection during antiblastic chemotherapy: a double-blind study.

Corticosteroids and cytostatic drugs possess a documented lesive action on upper digestive mucosa, making epigastric pain and/or pyrosis common complaints among patients on antitumor treatment. The selective antimuscarinic pirenzepine and the H2-receptor antagonist ranitidine were tested against the ulcerogenic action of antiblastic chemotherapy. Thirty-eight patients affected with malignant lymphoproliferative disorders were endoscopically examined and the endoscopic pictures were quantified by using an arbitrary score. According to a double-blind randomized sequence, 19 out of the 38 patients received pirenzepine 100 mg/die/p.o. and the other 19 received ranitidine 300 mg/die/p.o. along with antitumoral therapy for periods of 3-6 months. Seven patients died of hematologic complications before ending the treatment. In the 31 surviving patients (13 pirenzepine- and 18 ranitidine-treated) a second endoscopic examination was performed at the end of the treatment and the lesion score repeated. No significant difference was found between initial and final scores in both groups. The antisecretive action of the two drugs may account for their effectiveness, but other mechanisms such as cytoprotection cannot be ruled out.