Iris Lavon

Identification of the receptor component of the IκBα-ubiquitin ligase

NF-κB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-κB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.

Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression.

Intracerebroventricular or intravenous (IV) injection of neural precursor cells (NPCs) attenuates experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Although stem cell therapy was introduced initially for cell replacement, we examine here whether NPCs possess immunomodulatory effects.

High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-κB activation

Based on the essential involvement of NF-κB in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF-κB in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF-κB is normally retained in the cytoplasm by its inhibitor IκB, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant IκBα transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF-κB activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF-κB activation in certain systemic infections, possibly by coordinating innate immunity in the liver.

Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis.

Stem cell transplantation was introduced as a mean of cell replacement therapy, but the mechanism by which it confers clinical improvement in experimental models of neurological diseases is not clear. Here, we transplanted neural precursor cells (NPCs) into the ventricles of mice at day 6 after induction of chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Transplanted cells migrated into white matter tracts and attenuated the clinical course of disease. NPC transplantation down-regulated the inflammatory brain process at the acute phase of disease, as indicated by a reduction in the number of perivascular infiltrates and of brain CD3+ T cells, an increase in the number and proportion of regulatory T cells and a reduction in the expression of ICAM-1 and LFA-1 in the brain. Demyelination and acute axonal injury in this model are considered to result mainly from the acute inflammatory process and correlate well with the chronic neurological residua. In consequence to inhibition of brain inflammation, precursor cell transplantation attenuated the primary demyelinating process and reduced the acute axonal injury. As a result, the size of demyelinated areas and extent of chronic axonal pathology were reduced in the transplanted brains. We suggest that the beneficial effect of transplanted NPCs in chronic EAE is mediated, in part, by decreasing brain inflammation and reducing tissue injury.

Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors

We investigated the anti-inflammatory effects of acetylcholinesterase inhibitors (AChEI) at the cellular and molecular levels. AChEI suppressed lymphocyte proliferation and pro-inflammatory cytokine production, as well as extracellular esterase activity. Anti-inflammatory activity was mediated by the alpha7 nicotinic acetylcholine receptor (neuronal); the muscarinic receptor had the opposite effect. Treatment of the central nervous system (CNS) inflammatory disease, experimental autoimmune encephalomyelitis (EAE), with EN101, an anti-sense oligodeoxynucleotide, targeted to AChE mRNA, reduced the clinical severity of the disease and CNS inflammation intensity. The results of our experiments suggest that AChEI increase the concentration of extracellular acetylcholine (ACh), rendering it available for interaction with a nicotinic receptor expressed on lymphocytes. Our findings point to a novel role for AChEI which may be relevant in CNS inflammatory diseases such as EAE and multiple sclerosis. They also emphasize the importance of cholinergic balance in neurological disorders, such as Alzheimers disease and myasthenia gravis, in which these drugs are used.

Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades

We evaluated whether cell-free circulating DNA can be used as a noninvasive approach for detection of genetic/epigenetic alterations in brain tumors during the course of the disease. Paired tumor-serum samples from 70 patients with either high-grade astrocytomas (n = 41) or oligodendrogliomas of various grades were analyzed. The median interval between surgery and serum sampling was 1 month (range 0.5-168 months). DNA was extracted from whole blood, serum, and paraffin-embedded tumor sections. Loss of heterozygosity (LOH) in chromosomes 1p, 19q, and 10q was assessed by polymerase chain reaction (PCR)-based microsatellite analysis. The methylation status of O(6)-methyl guanine methyltransferase (MGMT) and phosphatase and tensin homolog promoters was studied by methylation-specific PCR. LOH and/or methylation that could identify DNA as tumor-specific was found in 80.5% of astrocytic tumors and in all oligodendrogliomas. The rate of serum detection of these biomarkers was 51% and 55%, respectively, with specificity around 100%. The rate of serum detection did not differ between low- and high-grade oligodendrogliomas. Statistically significant tumor-serum concordance was found for MGMT methylation in both astrocytic tumors (83%; P < .001) and oligodendroglial tumors (72%; P < .003) and for LOH of 10q (79%; P < .002) and 1p (62%; P < .03) in oligodendrogliomas. We conclude that serum DNA in glial tumors is informative for both LOH and aberrant gene promoter methylation analysis during the course of the disease. The sensitivity is moderate and specificity is high for both low- and high-grade tumors. Future studies should identify a panel of biomarkers that bear the highest potential for clinical application.

Gliomas display a microRNA expression profile reminiscent of neural precursor cells

Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.

Novel Mechanism whereby Nuclear Factor κB Mediates DNA Damage Repair through Regulation of O6-Methylguanine-DNA-Methyltransferase

O(6)-Methylguanine-DNA-methyltransferase (MGMT) and nuclear factor kappaB (NF-kappaB) are two key effectors associated with the development of resistance to alkylating agent-based chemotherapy. This prompted us to hypothesize that NF-kappaB might be involved in MGMT regulation. Consistent with this hypothesis, we have discovered two putative NF-kappaB binding sites within the MGMT promoter region and showed a specific and direct interaction of NF-kappaB at each of these sites. Forced expression of the NF-kappaB subunit p65 in HEK293 cells induced an increase in MGMT expression whereas addition of the NF-kappaB super repressor DeltaNIkappaB completely abrogated the induction. We also found a significant correlation between the extent of NF-kappaB activation and MGMT expression in the glioma cell lines and the human glial tumors tested and showed that it was independent of MGMT promoter methylation. Our results are of potential clinical significance because we show that cell lines with ectopic p65 or high constitutive NF-kappaB activity are less sensitive to nitrosourea treatment and that suppression of MGMT activity with O(6)-benzylguanine completely abolishes the chemoresistance acquired by NF-kappaB. The findings of our study strongly suggest that NF-kappaB plays a major role in MGMT regulation and that MGMT is most probably the major player in NF-kappaB-mediated chemoresistance to alkylating agents.

Suppression of neuroinflammation and immunomodulation by the acetylcholinesterase inhibitor rivastigmine

In this study we determined the influence of cholinergic up-regulation by rivastigmine, an acetylcholinesterase inhibitor, on central nervous system inflammation. Neuroinflammation was induced in experimental autoimmune encephalomyelitis (EAE). Rivastigmine markedly ameliorated clinical symptoms of EAE and the spatial memory deficits induced by EAE. It also reduced demyelination, microglia activation and axonal damage. Rivastigmine decreased the reactivity of encephalitogenic T-cells and the production of pro-inflammatory cytokines (TNF-alpha, IFN-gamma and IL-17) without affecting IL-10 production. These effects were abolished by alpha7 nicotinic acetylcholine receptor antagonists. Antigen presentation was also affected by this treatment. Thus, rivastigmine treatment had immunomodulatory activity in EAE.