Altered oxidative stress profile in the cortex of mice fed an enriched branched-chain amino acids diet: possible link with amyotrophic lateral sclerosis?

Branched-chain amino acids (BCAAs), valine, isoleucine, and leucine, are widely used among athletes as dietary integrators. Although the occurrence of untoward effects of BCCA supplementation, with particular regard to neurological disturbances, cannot be excluded, no specific studies have been performed so far. The aim of this work was to evaluate the effects of a diet enriched in BCAAs on the expression of oxidative stress pathway genes in the brain of C57Bl/6J mice. Animals were fed a standard or a BCAA diet for 95 days starting from postnatal day 21 until sacrifice. BCAA treatment, at doses comparable to human usage, significantly down-regulated the expression of some antioxidant genes, while up-regulating the expression of some oxygen transporters. In conclusion, it appears that BCAAs administered by diet could alter some specific oxidative stress pathways in the brain. Caution should thus be exercised in the widespread use of BCAAs as dietary integrators in sports practice.

Three years of experience: the Italian registry and safety data update.

At the end of 2006, a pharmacovigilance program on natalizumab was settled by the Italian Pharmaceutical Agency, and on January 2007, multiple sclerosis patients poorly responding to the immunomodulating therapies or with an aggressive clinical form of disease from onset initiated to be registered and to receive the medication. On February 2010, almost 3,000 cases have been treated with natalizumab. The drop-out rate is 10%. Almost 800 cases received cycles of natalizumab for more than 18 months. One case of PML was reported and other adverse events are similar to those described in phase III studies. The majority of cases remained stable, while in 25% of cases, an improvement of disability was documented.

The pharmacovigilance program on natalizumab in Italy: 2 years of experience.

At the end of 2006 a country-based surveillance program on natalizumab therapy in multiple sclerosis was settled in Italy by a collaborative effort of the Italian Drug Agency (AIFA) and a group of experts and neurologists appointed by the National Society of Neurology (SIN). After 2 years, 1,818 patients are registered in the database. The majority of cases (88.6%) failed the therapy with beta interferon or glatiramer acetate and had relapses or accumulated disability during immunomodulating treatment, while 11.4% of patients enrolled in the surveillance study were not previously treated with immunomodulating therapies and had a rapidly evolving clinical course. Almost 10% of the patients treated with natalizumab interrupted, for various different reasons, the therapy. Treatment was well tolerated and side effects were similar to those reported in the registrative studies. The majority of treated cases are stable or ameliorated.

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.

Regenerative medicine in Huntington's disease: Strengths and weaknesses of preclinical studies.

Huntington's disease (HD) is an inherited neurodegenerative disorder, characterized by impairment in motor, cognitive and psychiatric domains. Currently, there is no specific therapy to act on the onset or progression of HD. The marked neuronal death observed in HD is a main argument in favour of stem cells (SCs) transplantation as a promising therapeutic perspective to replace the population of lost neurons and restore the functionality of the damaged circuitry. The availability of rodent models of HD encourages the investigation of the restorative potential of SCs transplantation longitudinally. However, the results of preclinical studies on SCs therapy in HD are so far largely inconsistent; this hampers the individuation of the more appropriate model and precludes the comparative analysis of transplant efficacy on behavioural end points. Thus, this review will describe the state of the art of in vivo research on SCs therapy in HD, analysing in a translational perspective the strengths and weaknesses of animal studies investigating the therapeutic potential of cell transplantation on HD progression.

Adenosine-dopamine receptor-receptor interactions as an integrative mechanism in the basal ganglia.

Increasing evidence suggests that antagonistic interactions between specific subtypes of adenosine and dopamine receptors in the basal ganglia are involved in the motor depressant effects of adenosine receptor agonists and the motor stimulant effects of adenosine receptor antagonists, such as caffeine. The GABAergic striatopallidal neurons are regulated by interacting adenosine A2A and dopamine D2 receptors. On the other hand, the GABAergic striatonigral and striatoentopeduncular neurons seem to be regulated by interacting adenosine A1 and dopamine D1 receptors. Furthermore, behavioural studies have revealed interactions between adenosine A2A and dopamine D1 receptors that occur at the network level. These adenosine-dopamine receptor-receptor interactions might offer new therapeutic leads for basal ganglia disorders.

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.

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.

Effects of SCH 58261, an adenosine A(2A) receptor antagonist, on quinpirole-induced turning in 6-hydroxydopamine-lesioned rats. Lack of tolerance after chronic caffeine intake.

In unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats, a rodent model of Parkinson's disease (PD), the adenosine A(2A) receptor antagonist SCH 58261 significantly increased (+180%, p <.01) the number of rotations induced by a low dose of quinpirole (a dopamine D(2) receptor agonist), while it did not significantly modify the effects of a comparably low dose of SKF 38393 (a dopamine D(1) receptor agonist). The dose-dependent potentiating effects of SCH 58261 on quinpirole-induced turning were similar in caffeine-treated rats (1 g/l in drinking water over 14 days) and control animals (tap water). The selective potentiating effects of SCH 58261 on D(2)-dependent turning confirm the existence of a potent and specific A(2A)/D(2) receptor-receptor interaction. The persistence of the potentiating effects of SCH 58261 after chronic caffeine intake suggests that no tolerance should develop towards the antiparkinsonian effects of adenosine A(2A) receptor antagonists with chronic treatment.

The selective mGlu(5) receptor agonist CHPG inhibits quinpirole-induced turning in 6-hydroxydopamine-lesioned rats and modulates the binding characteristics of dopamine D(2) receptors in the rat striatum: interactions with adenosine A(2a) receptors.

In 6-hydroxydopamine-lesioned rats, the selective mGlu(5) receptor agonist (RS)-2-Cholro-5-Hydroxyphenylglycine (CHPG, 1-6 microg/10 microl intracerebroventricularly) significantly inhibited contralateral turning induced by quinpirole and, to a lesser extent, that induced by SKF 38393. The inhibitory effects of CHPG on quinpirole-induced turning were significantly potentiated by an adenosine A(2A) receptor agonist (CGS 21680, 0.2 mg/kg IP) and attenuated by an A(2A) receptor antagonist (SCH 58261, 1 mg/kg IP). In rat striatal membranes, CHPG (100-1,000 nM) significantly reduced the affinity of the high-affinity state of D(2) receptors for the agonist, an effect potentiated by CGS 21680 (30 nM). These results show the occurrence of functional interactions among mGlu(5), adenosine A(2A), and dopamine D(2) receptors in the regulation of striatal functioning, and suggest that mGlu(5) receptors may be regarded as alternative/integrative targets for the development of therapeutic strategies in the treatment of Parkinson's disease.

Verapamil and flunarizine inhibit phencyclidine-induced effects: an EEG and behavioural study in rats.

The influence of verapamil and flunarizine on phencyclidine-induced effects has been studied in adult male rats. Both verapamil (25 and 100 micrograms/10 microliters, i.c.v.) and flunarizine (40 and 60 mg/kg, i.p.) significantly reduced behavioural (mean intensity of ataxia, mean duration of head weaving) and the EEG (increase in the mean voltage of background activity of the EEG) effects induced by phencyclidine 5 mg/kg (i.p.). It was reported previously that nimodipine and diltiazem significantly potentiate effects induced by phencyclidine. The contrasting results obtained with verapamil and flunarizine, suggest that these drugs may modulate effects induced by phencyclidine by acting at sites other than NMDA receptor-coupled "L"-type calcium channel.

Adenosine A2A and group I metabotropic glutamate receptors synergistically modulate the binding characteristics of dopamine D2 receptors in the rat striatum.

There is experimental evidence for the existence of interactions between metabotropic glutamate (mGlu), adenosine and dopamine receptors in the striatum. In membrane preparations from rat striatum the group I and II mGlu receptor agonist 1-aminocyclopentane-1S-3R-dicarboxylic acid (1S-3R-ACPD) was found to modulate the binding characteristics of D2 receptors in a similar manner as the A2A receptor agonist 2-[p-(2-carboxyethyl)phenthylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), with a significant decrease in the affinity of the high-affinity state of D2 receptors for dopamine. The effect of 1S-3R-ACPD was mimicked by (+/-)-trans-ACPD (t-ACPD; a racemic mixture of 1S-3R-ACPD and its inactive isomer 1R-3S-ACPD) and by the selective group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (DHPG) and it was counteracted by the selective group I mGlu receptor antagonist 1-aminoindan-1,5-dicarboxilic acid (AIDA), but not by the the group II and III mGlu receptor antagonist (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG) or the adenosine receptor antagonist 8-phenyltheophylline. Furthermore, a strong synergistic effect was observed when the striatal membranes were exposed to both CGS 21680 and 1S-3R-ACPD. In agreement with the biochemical results, in unilaterally 6-OH-dopamine lesioned rats 1S-3R-ACPD counteracted the turning behaviour induced by the D2 receptor agonist quinpirole, but not by the D1 receptor agonist SKF 38393, and it synergistically potentiated the antagonistic effect of CGS 21680 on quinpirole-induced turning behaviour.

Cromakalim (BRL 34915) counteracts the epileptiform activity elicited by diltiazem and verapamil in rats.

1. The effects of BRL 34915 (cromakalim), a potassium channel opener, have been tested on the epileptiform activity elicited by high dose/concentrations of some calcium antagonists in in vivo (diltiazem) and in vitro (diltiazem and verapamil) experiments in rats. 2. Diltiazem (150-300 mg kg-1, i.p.) induced behavioural and electroencephalographic (EEG) seizures that were completely prevented by cromakalim (10 nmol/10 microliters, i.c.v.). Whereas, pentobarbitone (5-10 mg kg-1, i.p.) only prevented the behavioural component of the seizures. 3. In hippocampal slices, verapamil (1.5-2.0 mM) produced, within 30-60 min of perfusion, a CA1 epileptiform bursting in 80% of the experiments. This epileptiform activity was prevented by the cromakalim concentration (50 microM) that did not affect the control CA1 synaptic transmission per se. Pentobarbitone also prevented verapamil-induced epileptiform bursting only at the concentration (100 microM) that also reduced control CA1 synaptic transmission. 4. Diltiazem (1.5 mM) produced a biphasic excitatory-depressant effect within 60 min of perfusion. A CA1 epileptiform bursting appeared in 100% of the experiments within 30 min of perfusion. These excitatory effects were followed by a depression phase, characterized by a reduction of the magnitude of CA1 excitatory postsynaptic potentials (e.p.s.ps) and population spike. 5. The diltiazem-induced epileptiform bursting was prevented by cromakalim at a concentration (50 microM) that did not affect the control CA1 synaptic transmission per se. Pentobarbitone also prevented the diltiazem-induced epileptiform bursting only at a concentration (100 microM) that also reduced the control CA1 synaptic transmission. Both cromakalim (50 microM) and pentobarbitone (100 microM) failed to affect the depressant effects of diltiazem on CA1 hippocampal area. On the contrary, high (3.3mM) calcium solutions prevented both the excitatory and the depressant effects of 1.5 mm diltiazem within 60 min.6. These data indicate an involvement of potassium currents in the epileptiform activity elicited by high doses of diltiazem and verapamil.

Gangliosides and Guillain-Barré syndrome.

Cases of Guillain-Barré syndrome (GBS) associated with parenteral use of gangliosides have been reported in several European countries. To evaluate the hypothesis of association between ganglioside exposure and occurrence of GBS, a case-control study was conducted. GBS cases discharged during 1989 from public and private hospitals in three Italian provinces were identified: 42 GBS cases and 420 controls matched on age and gender were enrolled. Data of onset of symptoms of GBS was taken from clinical records. Exposure status of subjects was ascertained through the regional computerized drug prescription monitoring system. The odds ratio of association between ganglioside use, in the 30 days prior to onset of symptoms, and GBS was 9.1 (95% confidence interval 2.8-29.4). Although there are formidable difficulties in distinguishing prodromal therapy of GBS from drug causation, the association with ganglioside therapy is strong and supportive of the hypothesis of a role of ganglioside preparations in the occurrence of GBS.

Diphenylhydantoin potentiates the EEG and behavioural effects induced by N-methyl-D-aspartate antagonists in rats.

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors are involved in the electrical and behavioural generalization of epileptiform activity within the brain. In rats, both competitive and non-competitive NMDA antagonists induce three dose-dependent stages of EEG patterns: 1) increase in cortical desynchronization periods; 2) increase in amplitude of cortical high frequency (20-30 Hz), low voltage (30-50 microV) background activity; 3) appearance of cortical slow (2-3 Hz) wave-sharp wave complexes. These EEG changes are accompanied by stimulatory-depressive behavioural effects such as stereotypy (circling, head weaving) and ataxia. In the present study, the influence of the prototypic anticonvulsant diphenylhydantoin (DPH) has been tested on the EEG and behavioural effects induced by the non-competitive NMDA antagonists phencyclidine (PCP) and dizocilpine (MK-801) and by the competitive NMDA antagonist cis-4-phosphonomethyl-2-piperidine-carboxylic acid (CGS 19755). Even though DPH (up to 100 mg/kg IP) did not markedly affect basal cortical EEG activity, at doses of 10-100 mg/kg IP it potentiated all the EEG effects induced by the NMDA antagonists. These data support involvement of NMDA neurotransmission in the pharmacological effects of DPH.

Different capability of N-methyl-D-aspartate antagonists to elicit EEG and behavioural phencyclidine-like effects in rats.

Phencyclidine (PCP), a drug inducing schizophrenia-like symptoms in humans, is reported to be a non-competitive antagonist at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors. In rats, PCP produces three dose-dependent stages of EEG patterns: 1) increase of cortical desynchronization duration; 2) increase of the amplitude of the high-frequency (20-30 Hz) low-voltage (30-50 microV) cortical background activity; 3) appearance of cortical slow (2-3 Hz) wave-sharp wave complexes. These EEG changes are accompanied by stimulatory-depressive effects such as stereotypy (circling, head weaving) and ataxia. In the present study, the EEG and behavioural effects induced by systemic administration of the NMDA antagonists dizocilpine (MK 801), dextromethorphan (DM), [(+)-alpha-(4-chlorophenyl)-4- [(phenyl)methyl-1-piperidine ethanol] (SL 82.0715), (+)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), cis-4-phosphonomethyl-2-piperidine-carboxylic acid (CGS 19755) have been compared to those of PCP in rats. The rank of potency for inducing PCP-like EEG stages 1-3 was as follows: MK 801 > PCP > CGS 19755 > CPP. These drugs also induced PCP-like behavioural effects. On the contrary, DM and SL 82.0715, administered up to the dose of 100 mg/kg IP, failed to induce PCP-like behavioural effects and elicited only the stage 1 of PCP-like EEG. These results strongly suggest the involvement of NMDA neurotransmission in the behavioral and EEG effects of PCP.

Behavioural and electoencephalographic interactions between haloperidol and PCP/sigma ligands in the rat.

Phencyclidine (PCP) and sigma ligands produce a typical excitatory behaviour in rats, characterized by circling and head- and body-weaving. Excitatory amino acid antagonists such as 2-amino 5-phosphonovaleric acid (AP5) or 3-(+/-)-2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) also produce a PCP-like excitatory behaviour in rats. In the present paper, the interactions between PCP/sigma drugs or excitatory amino acid receptor antagonists and haloperidol have been investigated in rats. In addition, the influence of two other butyrophenones having a different affinity for the sigma/haloperidol receptors, such as spiperone and 3-(4-(3(4-fluorobenzoyl)-propyl-piperazino-l-yl-isoquinolino (HR 375), has been tested on the behavioural and EEG effects of PCP/sigma drugs and excitatory amino acid antagonists. PCP (2.5-5 mg/kg IP), (+) or (-) SKF 10,047 (1-15 mg/kg IP), (+) or (-) cyclazocine (2-8 mg/kg IP) and AP5 (0.5 mumol ICV) dose-dependently and significantly (P less than 0.01) antagonized the haloperidol-induced catalepsy in the horizontal bar and podium tests in rats. On the other hand, either haloperidol (1 mg/kg IP) or spiperone (1 mg/kg IP) reduced the head-weaving induced by (+) SKF 10,047, PCP, or AP5. On the contrary, HR 375 (6 mg/kg IP) was ineffective in blocking the excitatory effects of these drugs. In addition, either haloperidol (1 mg/kg IP) or spiperone (1 mg/kg IP), but not HR 375 (6 mg/kg IP) reduced the amplitude increase of the fast (20-30 Hz) frequency/low (30-50 microV) voltage background cortical activity elicited by PCP or (+) SKF 10,047. The results demonstrate an interaction between dopamine and excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The stimulation of cholecystokinin receptors in the rostral nucleus accumbens significantly antagonizes the EEG and behavioural effects induced by phencyclidine in rats.

The influence of cholecystokinin (CCK), bilaterally injected into the rostral nucleus accumbens, on the EEG and behavioural effects induced by phencyclidine (PCP) has been studied in rats. CCK (10 ng) significantly inhibited PCP-induced EEG effects (increase of spectral power with respect to pre-drug tracing; increase of relative power distribution in the slowest frequency bands), and behavioural effects (circling and ataxia). The inhibitory effects of CCK were completely antagonized by 1 ng PD 135-158, a selective CCKB receptor antagonist, but not by lorglumide (1 microgram), a selective CCKA receptor antagonist. Since the effects induced by PCP in rodents have been proposed to be an experimental correlate of the psychotic symptoms it induces in humans, these results indicate that CCK may act as a neuroleptic. They also suggest that CCKB receptors located in the rostral nucleus accumbens may be involved in the neuroleptic-like activity of CCK.

The mGlu5 receptor agonist CHPG stimulates striatal glutamate release: possible involvement of A2A receptors.

The intrastriatal perfusion of the selective metabotropic glutamate (mGlu)5 receptor agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG, 1000 microM) significantly increased (approximately + 100%, p < 0.05) glutamate extracellular levels with respect to basal values. The potentiating effect of CHPG was prevented by the selective mGlu5 receptor antagonist 2-methyl-6(phenyl-ethynyl)-pyridine (MPEP, 250 microM)) and by the adenosine A2A receptor antagonist SCH 58261 (2 mg/kg, i.p.). The results show that mGlu5 receptors are involved in the regulation of striatal glutamate release and suggest an involvement of adenosine A2A receptors in mGlu5 receptor-mediated effects.

Age-related decline in the functional response of striatal group I mGlu receptors.

In order to verify whether striatal group I metabotropic glutamate (mGlu) receptors undergo functional alteration in ageing, the effects induced by the selective agonist 3,5-dihydroxyphenylglycine (DHPG) in the striatum of young (3 months) and aged (24-25 months old) rats were compared. The ability of DHPG to stimulate phosphoinositide (PI) hydrolysis (striatal slices), to influence striatal dopamine release (in vivo microdialysis) and to potentiate the effects of NMDA on extracellular field potential amplitude (extracellular recordings on striatal slices) was reduced in the striatum of old vs young rats. These results show an age-dependent reduction in the functional response of striatal group I mGlu receptors, which may be one of the factors underlying the reduced ability aged striatum to integrate information.