Rosa Luisa Potenza

Adenosine A2A 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 A2A receptors (A2ARs) has been recently reported. In the present paper, we evaluated the role of A2ARs in mediating functional effects of BDNF in hippocampus using A2AR 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 A2A antagonist ZM 241385. Similarly, genetic deletion of the A2ARs abolished BDNF‐induced increase of the fEPSP slope in slices from A2AR KO mice The reduced functional ability of BDNF in A2AR KO mice was correlated with the reduction in hippocampal BDNF levels. In agreement, the pharmacological blockade of A2Rs by systemic ZM 241385 significantly reduced BDNF levels in the hippocampus of normal mice. These results indicate that the tonic activation of A2ARs is required for BDNF‐induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus.

Nonmotor symptoms in Parkinson's disease: Investigating early-phase onset of behavioral dysfunction in the 6-hydroxydopamine-lesioned rat model

To investigate the psychiatric symptoms accompanying the early phases of Parkinsons disease (PD), we injected adult rats with 10.5 μg 6‐hydroxydopamine (6‐OHDA) bilaterally into the dorsal striatum. The resulting neurodegeneration led, 12 weeks after injection, to a mild (36%) reduction of striatal dopamine. We tested the behavioral response of sham and 6‐OHDA‐lesioned animals at different time points after injection to evaluate the onset and progression of behavioral abnormalities. The results showed that such a mild reduction of dopamine levels was associated with a decrease in anxiety‐like behavior, an increase in “depression”‐like behavior, and a marked change in social behavior. Learning and memory abilities were not affected. Overall, the PD rat model used here displays behavioral alterations having face validity with psychiatric symptoms of the pathology and thus appears to be a valuable tool for investigating the neural bases of the early phases of PD.

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 Huntingtons 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 A2A receptor blockade differentially influences excitotoxic mechanisms at pre‐ and postsynaptic sites in the rat striatum

Adenosine A2A 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 A2A receptor antagonists in models of pre‐ and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A2A 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 μM). The A2A antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A2A agonist CGS 21680 (100 nM) showed a protective influence toward 50 μM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca2+]i or the release of lactate dehydrogenase (LDH) induced by 100 and 300 μM NMDA, respectively. The ability of ZM 241385 to prevent QA‐induced glutamate outflow and 4‐AP‐induced effects confirms that A2A receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA‐induced effects suggest that A2A receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A2A antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.

Effects of the adenosine A2A receptor antagonist SCH 58621 on cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor availability in a rat model of striatal neurodegeneration

Abstract Inhibition of adenosine A2A receptors (A2ARs) is neuroprotective in several experimental models of striatal diseases. However, the mechanisms elicited by A2AR blockade are only partially known, and critical aspects about the potential beneficial effects of A2AR antagonism in models of neurodegeneration still await elucidation. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitotoxicity obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons and prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the 2 brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain-derived neurotrophic factor levels. Because COX-2 activity has been linked to excitotoxic processes and because brain-derived neurotrophic factor depletion has been observed in mouse models as well as in patients with Huntington disease, we suggest that the final outcome of A2AR blockade (namely neuroprotection vs neurodegeneration) is likely to depend on the balance among its various and region-specific effects.

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.

Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by interfering with the neuroinflammatory component of the disease.

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.

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 µL) 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 µL). 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 µm through the dialysis probe) significantly reduced the increase in glutamate levels induced by 5 mm QA. In primary cultures of striatal neurons MPEP (50 µm) reduced the toxicity induced by direct application of glutamate [measured as release of lactate dehydrogenase [LDH]). Finally, we found that 50 µm 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.

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease.

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntingtons 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.

Neuroprotective Effects of Thymosin β4 in Experimental Models of Excitotoxicity

Abstract:  The aim of this study was to evaluate the possible neuroprotective effects of thymosin β4 in different models of excitotoxicity. The application of thymosin β4 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 β4 significantly reduced hippocampal neuronal loss induced by kainic acid. These results show that thymosin β4 induced a protective effect in models of excitotoxicity. The mechanisms underlying such an effect, as well as the real neuroprotective potential of thymosin β4, are worthy of further investigations.