Mariagrazia Grilli

IMF-κB and Rel: Participants in a Multiform Transcriptional Regulatory System

Publisher Summary This chapter highlights NF-кB/Rel protein complex. Associations of the binding and regulatory subunits of NF-кB operate at the center of a variety of different signal pathways. NF-кB can process and integrate instructions that come from the extracellular environment and detect intracellular events originating in either the cytoplasm or the nucleus. NF-кB then transmits this information with astonishing rapidity to the transcriptional machinery by directly binding to a range of different DNA sequences in gene control regions. NF-кB comprises members of a family of dimer-forming proteins with homology to the rel oncogene. NF-кB has the striking appearance of being constitutively present only in B cells of the “appropriate” stage for Ig к light chain gene expression. The functional role of NF-кB is demonstrated by mutational analysis of the к intronic enhancer. The property that gives NF-кB a widespread significance in cellular regulation is its role as a mediator of inducible gene transcription. The key features of NF-кB transcriptional control are that it is fast and versatile and is used in many different gene systems. It also has the important ability to carry signals from the cytoplasm into the nucleus and trans -activate specific genes by binding directly to their promoters.

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.

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.

Interleukin‐10 modulates neuronal threshold of vulnerability to ischaemic damage

Interleukin‐10 (IL‐10) is a powerful suppressor of cellular immune responses, with a postulated role in brain inflammation. First, we have evaluated the role of this cytokine in ischaemic brain damage using IL‐10 knockout (IL‐10–/–) mice. The middle cerebral artery (MCA) was occluded in either IL‐10–/– or wild‐type animals of corresponding strain (C57Bl/6) and age. Infarct volume was assessed 24 h later in serial brain sections. Brain infarct produced by MCA occlusion was 30% larger in the IL‐10–/– than in wild‐type mice (21.8 ± 1.2 vs. 16.9 ± 1.0 mm3, respectively; P < 0.01; Students t‐test). To further characterize these findings, studies were extended to in vitro models. Primary neuronal cortical cultures derived from IL‐10–/– animals were more susceptible to both excitotoxicity and combined oxygen–glucose deprivation compared with cell cultures from wild‐type mice. Moreover, when added to the culture medium, recombinant murine IL‐10 (0.1–100 ng/mL) exerted a concentration‐dependent prevention of neuronal damage induced by excitotoxicity in both cortical and cerebellar granule cell cultures taken from either strain. The accordance of in vivo and in vitro data allows us to suggest a potential neuroprotective role of IL‐10 against cerebral ischaemia when administered exogenously or made available from endogenous sources.

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.

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.

Age-progressing cognitive impairments and neuropathology in transgenic CRND8 mice

Patients with Alzheimers disease suffer from progressive cognitive impairments and show distinct post-mortem neuropathology, including beta-amyloid plaques. Transgenic (Tg) CRND8 mice carry a mutated human amyloid precursor protein gene and show age-related increases in beta-amyloid production and plaque deposition. It was previously reported that during the early stages of plaque deposition, Tg CRND8 mice demonstrated Morris maze impairments. However, it is unknown if Tg mice would be impaired at an earlier age prior to plaque deposition or more impaired at a later age with more extensive plaque deposition. In the current study, we describe Tg CRND8 age-progressing beta-amyloid neuropathology and cognitive abilities in greater detail. At all ages, Tg mice showed normal short-term memory in the Y-maze. Pre-plaque Tg and age-matched Non-Tg mice did not differ in learning the spatial Morris water maze. However, both early and late plaque Tg mice showed impairments during acquisition. In addition, although early plaque Tg mice performed well in the probe trial, late plaque Tg mice demonstrated impaired probe trial performance. Therefore compared to their Non-Tg littermates, Tg CRND8 mice demonstrate cognitive impairments that progressed with age and seemed to coincide with the onset of beta-amyloid plaque deposition.

Neuropathic pain-induced depressive-like behavior and hippocampal neurogenesis and plasticity are dependent on TNFR1 signaling.

Patients suffering from neuropathic pain have a higher incidence of mood disorders such as depression. Increased expression of tumor necrosis factor (TNF) has been reported in neuropathic pain and depressive-like conditions and most of the pro-inflammatory effects of TNF are mediated by the TNF receptor 1 (TNFR1). Here we sought to investigate: (1) the occurrence of depressive-like behavior in chronic neuropathic pain and the associated forms of hippocampal plasticity, and (2) the involvement of TNFR1-mediated TNF signaling as a possible regulator of such events. Neuropathic pain was induced by chronic constriction injury of the sciatic nerve in wild-type and TNFR1(-/-) mice. Anhedonia, weight loss and physical state were measured as symptoms of depression. Hippocampal neurogenesis, neuroplasticity, myelin remodeling and TNF/TNFRs expression were analyzed by immunohistochemical analysis and western blot assay. We found that neuropathic pain resulted in the development of depressive symptoms in a time dependent manner and was associated with profound hippocampal alterations such as impaired neurogenesis, reduced expression of neuroplasticity markers and myelin proteins. The onset of depressive-like behavior also coincided with increased hippocampal levels of TNF, and decreased expression of TNF receptor 2 (TNFR2), which were all fully restored after mice spontaneously recovered from pain. Notably, TNFR1(-/-) mice did not develop depressive-like symptoms after injury, nor were there changes in hippocampal neurogenesis and plasticity. Our data show that neuropathic pain induces a cluster of depressive-like symptoms and profound hippocampal plasticity that are dependent on TNF signaling through TNFR1.