Maurizio Memo

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.

Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path?

Cell-cycle-related proteins, such as cyclins or cyclin-dependent kinases, are re-expressed in neurons committed to death in response to a variety of insults, including excitotoxins, hypoxia and ischemia, loss of trophic support, or beta-amyloid peptide. In some of these conditions events that are typical of the mid-G1 phase, such as cyclin-dependent kinase 4/6 activation, are required for the induction of neuronal death. In other cases, the cycle must proceed further and recruit steps that are typical of the G1/S transition for death to occur. Finally, there are conditions in which cell-cycle proteins might be re-expressed, but do not contribute to neuronal death. We hypothesize that cell-cycle signaling becomes a mandatory component of neuronal demise when other mechanisms are not enough for neurons to reach the threshold for death. Under this scheme, the death threshold is set by the extent of DNA damage. Whenever the extent of DNA damage is below this threshold, a cell-cycle signaling becomes crucial for the induction of neuronal death through p53-dependent or -independent pathways.

Nuclear factor-κB/rel proteins : A point of convergence of signalling pathways relevant in neuronal function and dysfunction

Nuclear factor-kappaB (NF-kappaB)/Rel designates a family of transcription factors participating in the activation of a wide range of genes crucially involved in immune and inflammatory function. NF-kappaB/Rel proteins have been demonstrated recently in primary neurons and in several brain areas. Functional significance of these proteins is still not understood completely, but since certain subsets of neurons appear to contain constitutively active DNA-binding activity, it seems likely that they may participate in normal brain function. A growing body of evidence is accumulating for a specific activation of NF-kappaB/Rel proteins in the CNS, and in particular in neuronal cells, during neurodegenerative processes associated to etiologically unrelated conditions. Whether NF-kappaB activation is part of the neurodegenerative process or of protective mechanisms is a matter of debate. This issue will be reviewed here with particular attention to the available reports on the activity of NF-kappaB/Rel proteins in both experimental paradigms of neurodegeneration and post-mortem brain tissue of patients affected by various neurological diseases. We hypothesize that NF-kappaB/Rel proteins may represent the point of convergence of several signalling pathways relevant for initiating or accelerating the process of neuronal dysfunction and degeneration in many neurological diseases, including Parkinsons disease, Alzheimers disease, CNS viral infections, and possibly others. If NF-kappaB/Rel proteins represent an integrating point of several pathways potentially contributing to neuronal degeneration, molecules that finely modulate their activity could represent a novel pharmacological approach to several neurological diseases.

Redox proteomics identification of 4-hydroxynonenal-modified brain proteins in Alzheimer’s disease: Role of lipid peroxidation in Alzheimer’s disease pathogenesis

Numerous studies have shown that neuronal lipids are highly susceptible to oxidative stress including in those brain areas directly involved in the neurodegenerative process of Alzheimers disease (AD). Lipid peroxidation directly damages membranes and also generates a number of secondary biologically active products (toxic aldehydes)that are capable of easily attacking lipids, proteins, and DNA. Accumulating evidence has demonstrated regionally increased brain lipid peroxidation in patients with AD; however, extensive studies on specific targets of lipid peroxidation‐induced damage are still missing. The present study represents a further step in understanding the relationship between oxidative modification of protein and neuronal death associated with AD. We used a proteomics approach to determine specific targets of lipid peroxidation in AD brain, both in hippocampus and inferior parietal lobule, by coupling immunochemical detection of 4‐hydroxynonenal‐bound proteins with 2‐D polyacrylamide gel electrophoresis and MS analysis. We identified 4‐hydroxynonenal‐bound proteins in the hippocampus and inferior parietal lobule brain regions of subjects with AD. The identified proteins play different biological functions including energy metabolism, antioxidant system, and structural proteins, thus impairing multiple molecular pathways. Our results provide further evidence for the role of lipid peroxidation in the pathogenesis of AD.

Impaired Adult Neurogenesis Associated with Short-Term Memory Defects in NF-κB p50-Deficient Mice

Neurogenesis proceeds throughout adulthood in the brain of most mammalian species, but the molecular mechanisms underlying the regulation of stem/progenitor cell proliferation, survival, maturation, and differentiation have not been completely unraveled. We have studied hippocampal neurogenesis in NF-κB p50-deficient mice. Here we demonstrate that in absence of p50, the net rate of neural precursor proliferation does not change, but some of the steps leading to the final neuron differentiation status are hampered, resulting in ∼50% reduction in the number of newly born neurons in the adult mutant hippocampus. Additionally, in p50−/− mice, we observed a selective defect in short-term spatial memory performance without impairment of hippocampal-dependent spatial long-term memory and learning. Our results highlight the role of NF-κB p50 in hippocampal neurogenesis and in short-term spatial memory.

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.

Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids

We found that primary cultures of rat cerebellar granule cells, although definitely postmitotic and terminally differentiated, express the tumour‐suppressor phosphoprotein p53. In particular, granule cells both expressed significant levels of p53 mRNA and positively reacted to an anti‐p53 antibody, from the first day of culturing. During neurone differentiation, p53 mRNA content did not significantly change, at least up to 12 days in vitro, while p53 immunoreactivity increased gradually. p53 expression appeared to be further modulable being upregulated after stimulation of glutamate ionotropic receptors by glutamate or kainate. Although qualitatively similar, p53 induction by glutamate and kainate differed in terms of intensity and time‐course. The glutamate increase of p53 immunoreactivity appeared within 30 min after the treatment and lasted for at least 2 h. Kainate‐induced increase of p53 immunoreactivity was delayed, becoming apparent within 2 h and lasting for at least 8 h. Both kainate‐ and glutamate‐induced increases of p53 immunoreactivity were prevented by the non‐competitive NMDA receptor antagonist MK 801. As shown by the electrophoretic mobility shift analysis, both glutamate and kainate induced increases of p53 DNA binding activity. Blockade of p53 induction by a specific p53 antisense oligonucleotide resulted in a partial reduction of excitotoxicity with a complete inhibition of the excitatory amino acids induced apoptosis.

Possible role of NF-κB and p53 in the glutamate-induced pro-apoptotic neuronal pathway

Apoptosis is now recognized as an important component in many progressive and acute neurodegenerative diseases. Extracellular signals and intracellular mechanisms triggering and regulating apoptosis in neuronal cells are still a matter of investigation. Here we review data from our and other laboratories with the aim to elucidate the nature of some proteins which are known to be involved in cell cycle regulation as well as in promoting degeneration and apoptosis of neurons. The following molecules will be taken into consideration: NF-κB, p53, p21 and MSH2. These proteins are activated by neurotoxic experimental conditions which involve the stimulation of selective receptors for the excitatory aminoacid glutamate. Thus, we hypothesize their contribution to an intracellular pathway responsible for the glutamate-induced neuronal death. Identification of such mechanisms could be relevant for understanding the apoptosis associated with various neurodegenerative diseases as well as for developing novel strategies of pharmacological intervention.

IDENTIFICATION AND CHARACTERIZATION OF A KAPPA B/REL BINDING SITE IN THE REGULATORY REGION OF THE AMYLOID PRECURSOR PROTEIN GENE

Several observations support the hypothesis that pathogenetic mechanisms of β amyloid formation in Alzheimers disease may involve alterations in amyloid precursor protein (APP) gene expression. In this regard, molecular dissection of the APP gene transcriptional regulation is of primary importance. We report evidence that members of the family of transcription factors NFκB/Rel can specifically recognize two identical sequences located in the 5′-regulatory region of APP. These sequences, which we refer to as APPκB sites, interact preferentially with p50-containing members of the family. In particular, p50 homodimers and p50/p65 and p50/c-Rel heterodimers act as transcriptional activators at the APPκB site. Finally, the nuclear complex specifically binding to the APPκB sites proves to be an integral part of neurons and lymphocytes.

Metabotropic glutamate receptor mrna expression in rat spinal cord

IT has recently become clear that, analogous to the situation observed in the brain, glutamatergic neurotransmission in the spinal cord relies upon the activation of ionotropic and metabotropic glutamate receptors (mGluRs). Although electrophysiological studies have emphasized the relevance of mGluR activation in the modulation of spinal neuron responses to glutamate, a detailed study of the molecular forms of receptors belonging to the mGluR family in the spinal cord is still lacking. Using a reverse transcriptase-polymerase chain reaction technique with primers specifically recognizing each cloned mGluR subtype, we found that rat adult spinal cord specifically expresses high levels of mRNA encoding mGluR1a but not mGluR1b. The expression of mRNAs for mGluRs 5a and 5b was also found at levels roughly comparable to that observed in mGluR5-positive brain areas. mGluR2, mGluR3, mGluR4 and mGluR7 mRNAs appeared to be expressed at lower levels, while mGluR6 and mGluR8 mRNAs were not detectable.