Francesca Biagioni

The role of norepinephrine in epilepsy: from the bench to the bedside.

This article provides a brief review of the role of norepinephrine (NE) in epilepsy, starting from early studies reproducing the kindling model in NE-lesioned rats, through the use of specific ligands for adrenergic receptors in experimental models of epilepsy, up to recent advances obtained by using transgenic and knock-out mice for specific genes expressed in the NE system. Data obtained from multiple experimental models converge to demonstrate the antiepileptic role of endogenous NE. This effect predominantly consists in counteracting the development of an epileptic circuit (such as in the kindling model) rather than increasing the epileptic threshold. This suggests that NE activity is critical in modifying epilepsy-induced neuronal changes especially on the limbic system. These data encompass from experimental models to clinical applications as recently evidenced by the need of an intact NE innervation for the antiepileptic mechanisms of vagal nerve stimulation (VNS) in patients suffering from refractory epilepsy. Finally, recent data demonstrate that NE loss increases neuronal damage following focally induced limbic status epilepticus, confirming a protective effect of brain NE, which has already been shown in other neurological disorders.

Endogenous Activation of mGlu5 Metabotropic Glutamate Receptors Contributes to the Development of Nigro-Striatal Damage Induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine in Mice

We combined the use of knock-out mice and subtype-selective antagonists [2-methyl-6-(phenylethynyl)pyridine (MPEP) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB1893)] to examine whether endogenous activation of mGlu5 metabotropic glutamate receptors contributes to the pathophysiology of nigro-striatal damage in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. High doses of MPTP (four injections of 20 mg/kg, i.p., every 2 hr) induced a high mortality rate and a nearly total degeneration of the nigro-striatal pathway in wild-type mice. mGlu5 knock-out mice were less sensitive to MPTP toxicity, as shown by a higher survival and a milder nigro-striatal damage. Protection against MPTP (80 mg/kg) toxicity was also observed after MPEP injections (four injections of 5 mg/kg, i.p., 30 min before each MPTP injection). MPEP treatment did not further increase neuroprotection against 80 mg/kg of MPTP in mGlu5 knock-out mice, indicating that the drug acted by inhibiting mGlu5 receptors. In wild-type mice, MPEP was also neuroprotective when challenged against lower doses of MPTP (either 30 mg/kg, single injection, or four of 10 mg/kg injections). The action of MPEP was mimicked by SIB1893 but not by the mGlu1 receptor antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester. MPEP did not change the kinetics of 1-methyl-4-phenylpyridinium ion formation in the striatum of mice injected with MPTP. We conclude that mGlu5 receptors act as amplifiers of MPTP toxicity and that mGlu5 receptor antagonists may limit the extent of nigro-striatal damage in experimental models of parkinsonism.

miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer

Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.

Induction of Dickkopf-1, a Negative Modulator of the Wnt Pathway, Is Required for the Development of Ischemic Neuronal Death

Expression of Dickkopf-1 (Dkk-1), a secreted protein that negatively modulates the Wnt pathway, was induced in the hippocampus of gerbils and rats subjected to transient global cerebral ischemia as well as in cultured cortical neurons challenged with an excitotoxic pulse. In ischemic animals, the temporal and regional pattern of Dkk-1 expression correlated with the profile of neuronal death, as assessed by Nissl staining and Dkk-1 immunostaining in adjacent hippocampal sections. Treatment of ischemic animals with either Dkk-1 antisense oligonucleotides or lithium ions (which rescue the Wnt pathway acting downstream of the Dkk-1 blockade) protected vulnerable hippocampal neurons against ischemic damage. The same treatments protected cultured cortical neurons against NMDA toxicity. We conclude that induction of Dkk-1 with the ensuing inhibition of the canonical Wnt signaling pathway is required for the development of ischemic and excitotoxic neuronal death.

Pharmacological Activation of mGlu4 Metabotropic Glutamate Receptors Reduces Nigrostriatal Degeneration in Mice Treated with 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

We examined whether selective activation of mGlu4 metabotropic glutamate receptors attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal damage in mice. C57BL mice were treated with a single dose of MPTP (30 mg/kg, i.p.) preceded, 30 min earlier, by a systemic injection of the mGlu4 receptor enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). PHCCC was injected either subcutaneously in cremophor EL or intraperitoneally in saline containing 50% DMSO. PHCCC treatment (3 or 10 mg/kg) significantly reduced MPTP toxicity, as assessed by measurements of the striatal levels of dopamine and its metabolites and by tyrosine hydroxylase, dopamine transporter, and glial fibrillary acidic protein immunostaining in the corpus striatum and substantia nigra. In another set of experiments, a higher cumulative dose of MPTP (80 mg/kg divided into four injections with 2 h of interval) was injected to mGlu4−/− mice and their Sv129/CD1 wild-type controls. A higher dose was used in these experiments because Sv129/CD1 mice are less sensitive to MPTP toxicity. Systemic administration of PHCCC was protective in wild-type mice but failed to affect nigrostriatal damage in mGlu4−/− mice. Finally, unilateral infusion of PHCCC in the external globus pallidus protected the ipsilateral nigrostriatal pathway against MPTP toxicity. These data support the view that mGlu4 receptors are potential targets for the experimental treatment of parkinsonism.

Induction of the Wnt Antagonist, Dickkopf-1, Contributes to the Development of Neuronal Death in Models of Brain Focal Ischemia

Inhibition of the canonical Wnt pathway has been implicated in the pathophysiology of neuronal death. Here, we report that the secreted Wnt antagonist, Dickkopf-1 (Dkk-1) is rapidly induced in neurons after induction of focal brain ischemia. In rats undergoing transient focal ischemia in response to brain infusion of endothelin-1, Dkk-1 was induced in neurons of the ischemic core and the penumbra region. Induction of Dkk-1 was associated with a reduced expression of β-catenin (a downstream signaling molecule of the canonical Wnt pathway), and was not observed in neurons expressing the protective protein, heat shock protein-70. Treatment with lithium ions, which, inter alia, rescue the canonical Wnt pathway, was highly protective against ischemic damage. Dkk-1 was also induced in cortical neurons of mice undergoing permanent middle cerebral artery (MCA) occlusion. This model allowed us to compare wild-type mice with doubleridge mice, which are characterized by a reduced expression of Dkk-1. Doubleridge mice showed an attenuated reduction of β-catenin and a reduced infarct volume in response to MCA occlusion, providing a direct demonstration that Dkk-1 contributes to the pathophysiology of ischemic neuronal damage. These data rise the interesting possibility that Dkk-1 antagonists or drugs that rescue the Wnt pathway might be neuroprotective in stroke.

Induction of the wnt inhibitor, dickkopf-1, is associated with neurodegeneration related to temporal lobe epilepsy.

Summary:  Inhibition of the Wnt pathway by the secreted glycoprotein, Dickkopf‐1 (Dkk‐1) has been related to processes of excitotoxic and ischemic neuronal death. We now report that Dkk‐1 is induced in neurons of the rat olfactory cortex and hippocampus degenerating in response to seizures produced by systemic injection of kainate (12 mg/kg, i.p.). There was a tight correlation between Dkk‐1 expression and neuronal death in both regions, as shown by the different expression profiles in animals classified as “high” and “low” responders to kainate. For example, no induction of Dkk‐1 was detected in the hippocampus of low responder rats, in which seizures did not cause neuronal loss. Induction of Dkk‐1 always anticipated neuronal death and was associated with a reduction in nuclear levels of β‐catenin, which reflects an ongoing inhibition of the canonical Wnt pathway. Intracerebroventricular injections of Dkk‐1 antisense oligonucleotides (12 nmol/2 μL) substantially reduced kainate‐induced neuronal damage, as did a pretreatment with lithium ions (1 mEq/kg, i.p.), which rescue the Wnt pathway by acting downstream of the Dkk‐1 blockade. Taken collectively, these data suggest that an early inhibition of the Wnt pathway by Dkk‐1 contributes to neuronal damage associated with temporal lobe epilepsy. We also examined Dkk‐1 expression in the hippocampus of epileptic patients and their controls. A strong Dkk‐1 immunolabeling was found in six bioptic samples and in one autoptic sample from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Dkk‐1 expression was undetectable or very low in autoptic samples from nonepileptic patients or in bioptic samples from patients with complex partial seizures without neuronal loss and/or reactive gliosis in the hippocampus. Our data raise the attractive possibility that drugs able to rescue the canonical Wnt pathway, such as Dkk‐1 antagonists or inhibitors of glycogen synthase kinase‐3β, reduce the development of hippocampal sclerosis in patients with temporal lobe epilepsy.

Protective role of group-II metabotropic glutamate receptors against nigro-striatal degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice

To examine how mGlu2/3 metabotropic glutamate receptors affect nigro-striatal degeneration, we used the agonist, LY379268, and the antagonist, LY341495, in mice challenged with the nigro-striatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In control mice, high doses of MPTP (20 mg/kg, i.p., injected four times with 2 h of interval) induced a nearly total degeneration of the nigro-striatal pathway, as shown by measurements of striatal dopamine (DA) levels and by immunohistochemical analysis of tyrosine hydroxylase, high affinity dopamine transporter, and glial fibrillary acidic protein in the corpus striatum and substantia nigra. Lower cumulative doses of MPTP (30 mg/kg, i.p., injected only once) produced a partial lesion of the nigro-striatal pathway (about 50% reduction of striatal DA content). Systemic injection of LY379268 (1 mg/kg, i.p., 30 min prior to each injection of MPTP) partially reduced the extent of nigro-striatal degeneration induced by high doses of MPTP. Similar results were obtained by continuously delivering LY379268 (1 mg/kg/d for 7 d) by means of a subcutaneous osmotic minipump. The protective effect of LY379268 was antagonized by LY341495 (also delivered by the osmotic minipump). In mice challenged with the lower cumulative dose of MPTP, injection of LY379268 did not produce a significant neuroprotective effect. In contrast, the lesion was amplified by the antagonist, LY341495. Neither LY379268 nor LY341495 influenced the central bioavailability and the local half-life of MPTP, as shown by measurements of the toxin and its active metabolite, MPP(+), in the striatum. We conclude that mGlu2/3 receptors play a protective role against MPTP toxicity, and that the efficacy of the agonist, LY379268, critically depends on the extent of the nigro-striatal lesion.

TGF-β1 protects against Aβ-neurotoxicity via the phosphatidylinositol-3-kinase pathway

beta-Amyloid (A beta) injection into the rat dorsal hippocampus had a small neurotoxic effect that was amplified by i.c.v. injection of SB431542, a selective inhibitor of transforming growth factor-beta (TGF-beta) receptor. This suggested that TGF-beta acts as a factor limiting A beta toxicity. We examined the neuroprotective activity of TGF-beta1 in pure cultures of rat cortical neurons challenged with A beta. Neuronal death triggered by A beta is known to proceed along an aberrant re-activation of the cell cycle, and involves late beta-catenin degradation and tau hyperphosphorylation. TGF-beta1 was equally protective when added either in combination with, or 6 h after A beta. Co-added TGF-beta1 prevented A beta-induced cell cycle reactivation, whereas lately added TGF-beta1 had no effect on the cell cycle, but rescued the late beta-catenin degradation and tau hyperphosphorylation. The phosphatidylinositol-3-kinase (PI-3-K) inhibitor, LY294402, abrogated all effects. Thus, TGF-beta1 blocks the whole cascade of events leading to A beta neurotoxicity by activating the PI-3-K pathway.

Occurrence of neuronal inclusions combined with increased nigral expression of α-synuclein within dopaminergic neurons following treatment with amphetamine derivatives in mice

In recent years several clinical and research findings have demonstrated the involvement of the presynaptic protein alpha-synuclein in a variety of neurodegenerative disorders which are known as synucleinopathies. Although the function of this protein in the physiology of the cell remains unknown, it is evident that both genetic alterations or a mere overexpression of the native molecule produces a degeneration of nigral dopamine-containing neurons leading to movement disorders, as demonstrated in inherited Parkinsons disease. In the present study, we investigated whether widely abused drugs such as methamphetamine and methylenedioxymethamphetamine (ecstasy), which are known to damage the nigrostriatal dopamine pathway of mice, increase the expression of alpha-synuclein within dopamine neurons of the substantia nigra pars compacta. The results of this study demonstrate that nigrostriatal dopamine denervation and occurrence of intracellular inclusions in nigral neurons produced by amphetamine derivatives are related to increased expression of alpha-synuclein within dopamine neurons of the substantia nigra. This lends substance to the hypothesis that increased amounts of native alpha-synuclein may be per se a detrimental factor for the dopamine neurons.