Christian Kaltschmidt

NF-kB: a crucial transcription factor for glial and neuronal cell function

Abstract Transcription factors provide the link between early membrane-proximal signalling events and changes in gene expression. NF-kB is one of the best-characterized transcription factors. It is expressed ubiquitously and regulates the expression of many genes, most of which encode proteins that play an important and often determining role in the processes of immunity and inflammation. Apart from its role in these events, evidence has begun to accumulate that NF-kB is involved in brain function, particularly following injury and in neurodegenerative conditions such as Alzheimers disease. NF-kB might also be important for viral replication in the CNS. An involvement of NF-kB in neuronal development is suggested from studies that demonstrate its activation in neurones in certain regions of the brain during neurogenesis. Brain-specific activators of NF-kB include glutamate (via both AMPA/KA and NMDA receptors) and neurotrophins, pointing to an involvement in synaptic plasticity. NF-kB can therefore be considered as one of the most important transcription factors characterized in brain to date and it might be as crucial for neuronal and glial cell function as it is for immune cells.

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Selective Activation of NF-κB by Nerve Growth Factor Through the Neurotrophin Receptor p75

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NTR activated the transcription factor nuclear factor kappa B (NF-κB) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-κB was not activated by BDNF or NT-3.

Constitutive NF-kappa B activity in neurons.

NF-kappa B is inducible transcription factor present in many cell types in a latent cytoplasmic form. So far, only immune cells including mature B cells, thymocytes, and adherent macrophages have been reported to contain constitutively active forms of NF-kappa B in the nucleus. A recent study showed that the human immunodeficiency virus type 1 (HIV-1) promoter is highly active in several brain regions of transgenic mice (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clements, Science 258:1804-1807, 1992). Since the activity of this viral enhancer is governed mainly by two binding sites for NF-kappa B, we were prompted to investigate the state of NF-kappa B activity in neurons. Primary neuronal cultures derived from rat hippocampus and cerebral cortex showed a high constitutive expression of an HIV-1 long terminal repeat-driven luciferase reporter gene, which was primarily dependent on intact NF-kappa B binding sites and was abolished upon coexpression of the NF-kappa B-specific inhibitor I kappa B-alpha. Indirect immunofluorescence and confocal laser microscopy showed that the activity of NF-kappa B correlated with the presence of the NF-kappa B subunits p50 and RelA (p65) in nuclei of cultured neurons. NF-kappa B was also constitutively active in neurons in vivo. As investigated by electrophoretic mobility shift assays, constitutive NF-kappa B DNA-binding activity was highly enriched in fractions containing neuronal nuclei prepared from rat cerebral cortex. Nuclear NF-kappa B-specific immunostaining was also seen in cryosections from mouse cerebral cortex and hippocampus. Only a subset of neurons was stained. Activated NF-kappa B in the brain is likely to participate in normal brain function and to reflect a distinct state of neuronal activity or differentiation. Furthermore, it may explain the high level of activity of the HIV-1 enhancer in neurons, an observation potentially relevant for the etiology of the AIDS dementia complex caused by HIV infection of the central nervous system.

NF-kappaB in the nervous system.

The transcription factor NF-kappaB has diverse functions in the nervous system, depending on the cellular context. NF-kappaB is constitutively activated in glutamatergic neurons. Knockout of p65 or inhibition of neuronal NF-kappaB by super-repressor IkappaB resulted in the loss of neuroprotection and defects in learning and memory. Similarly, p50-/- mice have a lower learning ability and are sensitive to neurotoxins. Activated NF-kappaB can be transported retrogradely from activated synapses to the nucleus to translate short-term processes to long-term changes such as axon growth, which is important for long-term memory. In glia, NF-kappaB is inducible and regulates inflammatory processes that exacerbate diseases such as autoimmune encephalomyelitis, ischemia, and Alzheimers disease. In summary, inhibition of NF-kappaB in glia might ameliorate disease, whereas activation in neurons might enhance memory. This review focuses on results produced by the analysis of genetic models.

Brain synapses contain inducible forms of the transcription factor NF-κB

Abstract We investigated the rat brain for the presence and activation state of the inducible transcription factor NF-κB. Two forms of NF-κB containing the transactivating p65 subunit were found in all brain regions investigated. The majority of NF-κB was in an inducible cytoplasmic form by virtue of its association with the inhibitory subunit IκB. Significant amounts of inducible NF-κB forms were present in synaptosomes, as suggested by electrophoretic mobility shift assay and Western blot analysis of subcellular brain fractions. A synaptic localization of NF-κB was further evident from immunostaining of inner and outer plexiform layers of the retina with an antibody directed against the p50 subunit of NF-κB. In cerebral cortex and striatium, NF-κB-specific antibodies showed a punctate immunostaining partially overlapping with that for the synaptic marker protein synaptophysin. NF-κB is thus the first transcription factor found in synapses of neurons. With its unusual subneuronal localization, the inducible transcription factor has the potential to function as retrograde messenger mediating stimulus-response coupling and long-term changes in gene expression following presynaptic stimulation.

Induction of a metastatogenic tumor cell type by neurotransmitters and its pharmacological inhibition by established drugs

The active migration of tumor cells, a crucial requirement for metastasis development and cancer progression, is regulated by signal substances including neurotransmitters. We investigated the migration of tumor cells within a three‐dimensional collagen matrix using time‐lapse videomicroscopy and computer‐assisted analysis of the migration path. Tumor cell migration is induced by norepinephrine, dopamine and substance P. We show that this induced migration, using MDA‐MB‐468 breast and PC‐3 prostate carcinoma cells, can be inhibited by using specific, clinically established receptor antagonists to the β2‐adrenoceptor, the D2 receptor, or the neurokinin‐1 receptor, respectively. All of the investigated neurotransmitters significantly activated the cyclic adenosine‐monophosphate response element binding protein (CREB). Furthermore, microarray analysis revealed changes of gene expression toward a highly motile tumor cell type, including an upregulation of the α2 integrin, which is an essential adhesion receptor for collagen in migration. The gene for the tumor suppressor gelsolin was downregulated. These 2 critical alterations were confirmed on the protein level by flow‐cytometry and immunoblotting, respectively. Neurotransmitters thus induce a metastatogenic tumor cell type by directly regulating gene expression and increased migratory activity, which can be prevented by established neurotransmitter antagonists.

Tumor necrosis factor α triggers proliferation of adult neural stem cells via IKK/NF-κB signaling

BackgroundBrain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-α) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation.ResultsHere we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-α/β-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-κB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-κB super-repressor IκB-AA1. Pharmacological blockade of IκB ubiquitin ligase activity led to comparable decreases in NF-κB activity and proliferation. In addition, IKK-β gene product knock-down via siRNA led to diminished NF-κB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFβ-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine.ConclusionTNF-mediated activation of IKK-β resulted in activation of NF-κB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-κB pathway resulted in strongly increased proliferation of NSCs.

NF-kappaB regulates spatial memory formation and synaptic plasticity through protein kinase A/CREB signaling.

ABSTRACT Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory. In a forebrain neuronal conditional NF-κB-deficient mouse model, we demonstrate here that the transcription factor NF-κB regulates spatial memory formation, synaptic transmission, and plasticity. Gene profiling experiments and analysis of regulatory regions identified the α catalytic subunit of protein kinase A (PKA), an essential memory regulator, as a new NF-κB target gene. Consequently, NF-κB inhibition led to a decrease in forskolin-induced CREB phosphorylation. Collectively, these results disclose a novel hierarchical transcriptional network involving NF-κB, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of NF-κB in learning and memory.

MCP-1 induces migration of adult neural stem cells

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.