Marina Pizzi

Neuroprotection by Aspirin and Sodium Salicylate Through Blockade of NF-κB Activation

Aspirin (acetylsalicylic acid) is a commonly prescribed drug with a wide pharmacological spectrum. At concentrations compatible with amounts in plasma during chronic anti-inflammatory therapy, acetylsalicylic acid and its metabolite sodium salicylate were found to be protective against neurotoxicity elicited by the excitatory amino acid glutamate in rat primary neuronal cultures and hippocampal slices. The site of action of the drugs appeared to be downstream of glutamate receptors and to involve specific inhibition of glutamate-mediated induction of nuclear factor kappa B. These results may contribute to the emerging theme of anti-inflammatory drugs and neurodegeneration.

Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection

The role of group-I metabotropic glutamate receptors (mGlu1 and 5) in neurodegeneration is still controversial. While antagonists of these receptors are consistently neuroprotective, agonists have been found to either amplify or attenuate excitotoxic neuronal death. At least three variables affect responses to agonists: (i) the presence of the NR2C subunit in the NMDA receptor complex; (ii) the existence of an activity-dependent functional switch of group-I mGlu receptors, similar to that described for the regulation of glutamate release; and (iii) the presence of astrocytes expressing mGlu5 receptors. Thus, a number of factors, including the heteromeric composition of NMDA receptors, the exposure time to drugs or to ambient glutamate, and the function of astrocytes clearing extracellular glutamate and producing neurotoxic or neuroprotective factors, must be taken into account when examining the role of group-I mGlu receptors in neurodegeneration/neuroprotection.

A Bioinformatics Analysis of Memory Consolidation Reveals Involvement of the Transcription Factor c-Rel

Consolidation of long-term memory (LTM) is a complex process requiring synthesis of new mRNAs and proteins. Many studies have characterized the requirement for de novo mRNA and protein synthesis; however, few studies have comprehensively identified genes regulated during LTM consolidation. We show that consolidation of long-term contextual memory in the hippocampus triggers altered expression of numerous genes encompassing many aspects of neuronal function. Like contextual memory formation, this altered gene expression required NMDA receptor activation and was specific for situations in which the animal formed an association between a physical context and a sensory stimulus. Using a bioinformatics approach, we found that regulatory elements for several transcription factors are over-represented in the upstream region of genes regulated during consolidation of LTM. Using a knock-out mouse, we found that c-rel, one of the transcription factors identified in our bioinformatics study, is necessary for hippocampus-dependent long-term memory formation.

Attenuation of Excitatory Amino Acid Toxicity by Metabotropic Glutamate Receptor Agonists and Aniracetam in Primary Cultures of Cerebellar Granule Cells

Abstract: Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration‐dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans‐1‐aminocyclopentane‐1, 3‐dicarboxylate or by quisqualate counteracts both glutamate‐ and kainate‐induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR‐evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration‐dependently counteracted glutsmate‐, kainate‐, or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans‐1‐aminocyclopentane‐1, 3‐dicarboxylate. In addition, aniracetam potentiated the mGluR‐coupled stimulation of phospholipase C, as revealed by the measurement of 3H‐inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

Bim and Noxa Are Candidates to Mediate the Deleterious Effect of the NF-κB Subunit RelA in Cerebral Ischemia

The transcription factor nuclear factor κB (NF-κB) is well known for its antiapoptotic action. However, in some disorders, such as cerebral ischemia, a proapoptotic function of NF-κB has been demonstrated. To analyze which subunit of NF-κB is functional in cerebral ischemia, we induced focal cerebral ischemia in mice with a germline deletion of the p52 or c-Rel gene or with a conditional deletion of RelA in the brain. Only RelA deficiency reduced infarct size. Interestingly, expression of the proapoptotic BH3 (Bcl-2 homology domain 3)-only genes Bim and Noxa in cerebral ischemia depended on RelA and the upstream kinase IKK (IκB kinase). RelA stimulated Bim and Noxa gene transcription in primary cortical neurons and bound to the promoter of both genes. Thus, the deleterious function in cerebral ischemia is specific for the NF-κB subunit RelA and may be mediated through Bim and Noxa.

Activation of Multiple Metabotropic Glutamate Receptor Subtypes Prevents NMDA-induced Excitotoxicity in Rat Hippocampal Slices

Metabotropic glutamate receptors (mGluRs) belong to a relative large receptor family consisting of multiple members with important roles in a number of brain functions. We report here that activation of mGluRs prevents the neurotoxic effect induced by N‐methyl‐D‐aspartate (NMDA) in slices from the rat hippocampus. Neuroprotection was elicited when slices were simultaneously exposed to both the selective mGluR agonist (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (tACPD) and NMDA. Persisting stimulation of mGluRs after the toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD was also evoked by its active isomer, (1 S,3R)‐ACPD, and was prevented by the selective mGluR antagonist (+)‐α‐methyl‐4‐carboxyphenyl‐glycine (500 μM), confirming that mGluR activation is involved in the mechanism of action of tACPD. The effect of 100 μM tACPD was reproduced by 100 μM quisqualate, an agonist for mGluR1 and mGluR5, and by 1 μM of (2S,1s,2s)‐2‐carboxycyclopropyl‐glycine, a preferential agonist of mGluR2 and mGluR3 subtypes. No neuroprotection was induced by L‐2‐amino‐4‐phospnonobutyrate, a selective agonist for mGluR4, mGluR6, mGluR7 and mGluR8, at 500 μM. Since the NMDA‐mediated cell death in hippocampal slices is considered relevant to ischaemia‐induced brain injury, these results indicate that mGluRs may be important safety devices used by neurons to decrease their sensitivity to excitotoxic stimuli and increase their chance of survival.

Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3β

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3β (GSK3β), an event inactivating this kinase. Leptin-mediated GSK3β phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Leprdb/db mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3β phosphorylation and mimicked by the GSK3β inhibitor SB216763. At concentrations preventing GSK3β phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3β activity.

NF‐κB p50/RelA and c‐Rel‐containing dimers: opposite regulators of neuron vulnerability to ischaemia

Diverse nuclear factor‐κB subunits mediate opposite effects of extracellular signals on neuron survival. While RelA is activated by neurotoxic agents, c‐Rel drives neuroprotective effects. In brain ischaemia RelA and p50 factors rapidly activate, but how they associate with c‐Rel to form active dimers and contribute to the changes in diverse dimer activation for neuron susceptibility is unknown. We show that in both cortical neurons exposed to oxygen glucose deprivation (OGD) and mice subjected to brain ischaemia, activation of p50/RelA was associated with inhibition of c‐Rel/RelA dimer and no change p50/c‐Rel. Targeting c‐Rel and RelA expression revealed that c‐Rel dimers reduced while p50/RelA enhanced neuronal susceptibility to anoxia. Activation of p50/RelA complex is known to induce the pro‐apoptotic Bim and Noxa genes. We now show that c‐Rel‐containing dimers, p50/c‐Rel and RelA/c‐Rel, but not p50/RelA, promoted Bcl‐xL transcription. Accordingly, the OGD exposure induced Bim, but reduced Bcl‐xL promoter activity and decreased the content of endogenous Bcl‐xL protein. These findings demonstrate that within the same neuronal cell, the balance between activation of p50/RelA and c‐Rel‐containing complexes fine tunes the threshold of neuron vulnerability to the ischaemic insult. Selective targeting of different dimers will unravel new approaches to limit ischaemia‐associated apoptosis.

CHF5074, a novel γ-secretase modulator, attenuates brain β-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease

Background and purpose:  We evaluated the effects of 1‐(3′,4′‐dichloro‐2‐fluoro[1,1′‐biphenyl]‐4‐yl)‐cyclopropanecarboxylic acid (CHF5074), a new γ‐secretase modulator, on brain β‐amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP).

Prevention of neuron and oligodendrocyte degeneration by interleukin-6 (IL-6) and IL-6 receptor/IL-6 fusion protein in organotypic hippocampal slices

We investigated the effects of IL-6 and a chimeric derivative of IL-6 and soluble IL-6 receptor (IL6RIL6 chimera) on excitotoxic injury in rat organotypic hippocampal slices. Brief application of N-methyl-d-aspartate (NMDA) induced astrocyte reactivity, neuron cell death, and oligodendrocyte degeneration, the latter caused by secondary activation of AMPA/kainate receptors. Both these cytokines rescued neurons and oligodendrocytes, albeit the chimeric compound was much more potent and efficient than IL-6. No change was produced on reactive astrocytosis. The cytokines preserved myelin basic protein (MBP) production in slices exposed to excitotoxic insult, and when applied singularly for a week, they also enhanced both MBP and proteolipid protein expression. These effects occurred through activating the signal transducer gp130 and were associated with stimulation of transcription factors STAT1 and STAT3. Our results suggest that IL-6 and IL6RIL6 may prove to be valuable in treating neurodegenerative and demyelinating diseases.