Angelisa Frasca

A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1β

Interleukin-1beta (IL-1beta) is overproduced in human and rodent epileptogenic tissue and it exacerbates seizures upon brain application in rodents. Moreover, pharmacological prevention of IL-1beta endogenous synthesis, or IL-1 receptor blockade, mediates powerful anticonvulsive actions indicating a significant role of this cytokine in ictogenesis. The molecular mechanisms of the proconvulsive actions of IL-1beta are not known. We show here that EEG seizures induced by intrahippocampal injection of kainic acid in C57BL6 adult mice were increased by 2-fold on average by pre-exposure to IL-1beta and this effect was blocked by 3-O-methylsphingomyelin (3-O-MS), a selective inhibitor of the ceramide-producing enzyme sphingomyelinase. C2-ceramide, a cell permeable analog of ceramide, mimicked IL-1beta action suggesting that ceramide may be the second messenger of the proconvulsive effect of IL-1beta. The seizure exacerbating effects of either IL-1beta or C2-ceramide were dependent on activation of the Src family of tyrosine kinases since they were prevented by CGP76030, an inhibitor of this enzyme family. The proconvulsive IL-1beta effect was associated with increased Tyr(418) phosphorylation of Src-family of kinases indicative of its activation, and Tyr(1472) phosphorylation of one of its substrate, the NR2B subunit of the N-methyl-d-aspartate receptor, which were prevented by 3-O-MS and CGP76030. Finally, the proconvulsive effect of IL-1beta was blocked by ifenprodil, a selective NR2B receptor antagonist. These results indicate that the proconvulsive actions of IL-1beta depend on the activation of a sphingomyelinase- and Src-family of kinases-dependent pathway in the hippocampus which leads to the phosphorylation of the NR2B subunit, thus highlighting a novel, non-transcriptional mechanism underlying seizure exacerbation in inflammatory conditions.

AMPA receptor subunit 1 (GluR-A) knockout mice model the glutamate hypothesis of depression

Recent evidence indicates that glutamate homeostasis and neurotransmission are altered in major depressive disorder, but the nature of the disruption and the mechanisms by which it contributes to the syndrome are unclear. Glutamate can act via AMPA, NMDA, or metabotropic receptors. Using targeted mutagenesis, we demonstrate here that mice with deletion of the main AMPA receptor subunit GluR‐A represent a depression model with good face and construct validity, showing behavioral and neurochemical features of depression also postulated for human patients. GluR‐A−/− mice display increased learned helplessness, decreased serotonin and norepinephrine levels, and disturbed glutamate ho‐meostasis with increased glutamate levels and increased NMDA receptor expression. These results correspond well with current concepts regarding the role of AMPA and NMDA receptors in depression, postulating that compounds that augment AMPA receptor signaling or decrease NMDA receptor functions have antidepressant effects. GluR‐A−/− mice represent a model to investigate the pathophysiology underlying the depressive phenotype and to identify changes in neural plasticity and resilience evoked by the genetic alterations in glutamatergic function. Furthermore, GluR‐A−/− mice may be a valuable tool to study biological mechanisms of AMPA receptor modulators and the efficacy of NMDA antagonists in reducing behavioral or biochemical changes that correlate with increased helplessness.—Chourbaji, S., Vogt, M. A., Fumagalli, F., Sohr, R., Frasca, A., Brandwein, C, Hörtnagl, H., Riva, M. A., Sprengel, R., Gass, P. AMPA receptor subunit 1 (GluR‐A) knockout mice model the glutamate hypothesis of depression. FASEB J. 22, 3129–3134 (2008)

Chronic fluoxetine administration inhibits extracellular signal-regulated kinase 1/2 phosphorylation in rat brain.

Accumulating evidence indicates that antidepressants alter intracellular signalling mechanisms resulting in long‐term synaptic alterations which probably account for the delay in clinical action of these drugs. Therefore, we investigated the effects of chronic fluoxetine administration on extracellular signal‐regulated kinase (ERK) 1 and 2, a group of MAPKs that mediate signal transduction from the cell surface downstream to the nucleus. Our data demonstrate that 3‐week fluoxetine treatment resulted in long‐lasting reduction of phospho‐ERK 1 and 2. Such an effect depends on the length of the treatment given that no changes were observed after a single drug injection or after 2 weeks of treatment and it is region specific, being observed in hippocampus and frontal cortex but not in striatum. Finally, phospho‐ERK 1 and 2 were differently modulated within nucleus and cytosol in hippocampus but similarly reduced in the same compartments of the frontal cortex, highlighting the specific subcellular compartmentalization of fluoxetine. Conversely, imipramine did not reduce the hippocampal phosphorylation of both ERK subtypes whereas it selectively increased ERK 1 phosphorylation in the cytosolic compartment of frontal cortex suggesting a drug‐specific effect on this intracellular target. These results point to modulation of phosphorylation, rather than altered expression, as the main target in the action of fluoxetine on this pathway. The reduction of ERK 1/2 function herein reported may be associated with the therapeutic effects of fluoxetine in the treatment of depression.

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Dynamic Regulation of Glutamatergic Postsynaptic Activity in Rat Prefrontal Cortex by Repeated Administration of Antipsychotic Drugs

Antipsychotics are the mainstay for the treatment of schizophrenia. Although these drugs act at several neurotransmitter receptors, they are expected to elicit different neuroadaptive changes at structures relevant for schizophrenia. Because glutamatergic dysfunction plays a role in the pathophysiology of schizophrenia, we focused our analysis on glutamatergic neurotransmission after repeated treatment with antipsychotic drugs. Rats were exposed to a 2-week pharmacological treatment with the first generation antipsychotic haloperidol and the second generation antipsychotic olanzapine. By using Western blot and immunoprecipitation techniques, we investigated the expression, trafficking, and interaction of essential components of glutamatergic synapse in rat prefrontal cortex. Prolonged treatment with haloperidol, but not olanzapine, dynamically affects glutamatergic synapse by selectively reducing the synaptic level of the obligatory N-methyl-d-aspartate (NMDA) subunit NR1, the regulatory NMDA subunit NR2A, and its scaffolding protein postsynaptic density 95 as well as the trafficking of subunit 1 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor to the membrane. In addition, haloperidol alters total as well as phosphorylated levels of calcium calmodulin kinase type II at synaptic sites and its interaction with the regulatory NMDA subunit NR2B. Our data suggest that the glutamatergic synapse is a vulnerable target for prolonged haloperidol treatment. The global attenuation of glutamatergic function in prefrontal cortex might explain, at least in part, the cognitive deterioration observed in patients treated with haloperidol.

Prenatal stress alters glutamatergic system responsiveness in adult rat prefrontal cortex

Exposure to stress during gestation alters brain development resulting in permanent alterations that may increase susceptibility to subsequent cognitive or neuropsychiatric disorders. In this manuscript we examined the effects of prenatal stress on critical determinants of the glutamatergic synapse under basal conditions as well as in response to acute stress. The main finding of this work is that gestational stress altered the responsiveness of the glutamatergic system following a challenge at adulthood. In fact, while in control animals acute swim stress enhanced the phosphorylation levels of the NMDA receptor subunits NR‐1(Ser896) and NR‐2B(Ser1303) as well as the phosphorylation levels of α calcium/calmodulin‐dependent protein kinase II (Thr286), a crucial sensor of calcium fluctuations, prenatal stress prevented or attenuated such activation. This dynamic modulation is restricted to prefrontal cortex since no changes were observed in the hippocampus, in line with the different maturational profile of these brain regions. Changes were also observed in the phosphorylation of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate subunit GluR‐1(Ser831) which, however, relied on the acute stress exposure and were independent of gestational stress. These effects point to a unique interference of chronic prenatal stress with the responsiveness of specific determinants of the glutamatergic synapse at adulthood in a region specific manner. The inability to mount an homeostatic glutamatergic response to subsequent stress at adulthood may impair the normal responses of the cell to challenging situations.

Long-lasting pro-ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology.

Purpose:  Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT).

In vivo imaging of glia activation using 1H-magnetic resonance spectroscopy to detect putative biomarkers of tissue epileptogenicity

Purpose:  Long‐lasting activation of glia occurs in brain during epileptogenesis, which develops after various central nervous system (CNS) injuries. Glia is the cell source of the biosynthesis and release of molecules that play a role in seizure recurrence and may contribute to epileptogenesis, thus representing a putative biomarker of epilepsy development and severity. In this study, we set up an in vivo longitudinal study using 1H‐magnetic resonance spectroscopy (MRS) to measure metabolite content in the rat hippocampus that could reflect the extent and the duration of glia activation. Our aim was to explore if glia activation during epileptogenesis, or in the chronic epileptic phase, can be used as a biomarker of tissue epileptogenicity (i.e., a measure of epilepsy severity).

Neuropeptide Y overexpression using recombinant adenoassociated viral vectors

SummaryGene therapy may represent a promising alternative treatment of epileptic patients who are resistant to conventional anti-epileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant adeno-associated viral (AAV) vectors have been most widely used. Preclinical studies using a selection of “therapeutic” genes injected into the rodent brain to correct the compromised balance between inhibitory and excitatory transmission in epilepsy, showed significant reduction of seizures and inhibition of epileptogenesis. In particular, transduction of neuropeptide genes, such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. Recent findings showed a long-lasting NPY over-expression in the rat hippocampus by local application of recombinant AAV vectors associated with reduced generalization of seizures, delayed kindling epileptogenesis, and strong reduction of chronic spontaneous seizures. These results establish a proof-of-principle evidence of the efficacy of gene therapy as anticonvulsant treatment. Additional investigations are required to address safety concerns and possible side effects in more detail.

GABA synthesis in Schwann cells is induced by the neuroactive steroid allopregnanolone

J. Neurochem. (2009) 112, 980–990.