Signaling via NF-kappaB in the nervous system

Nuclear factor kappa B (NF-kappaB) is an inducible transcription factor present in neurons and glia. Recent genetic models identified a role for NF-kappaB in neuroprotection against various neurotoxins. Furthermore, genetic evidence for a role in learning and memory is now emerging. This review highlights our current understanding of neuronal NF-kappaB in response to synaptic transmission and summarizes potential physiological functions of NF-kappaB in the nervous system. This article contains a listing of NF-kappaB activators and inhibitors in the nervous system, furthermore specific target genes are discussed. Synaptic NF-kappaB activated by glutamate and Ca2+ will be presented in the context of retrograde signaling. A controversial role of NF-kappaB in neurodegenerative diseases will be discussed. A model is proposed explaining this paradox as deregulated physiological NF-kappaB activity, where novel results are integrated, showing that p65 could be turned from an activator to a repressor of anti-apoptotic genes.

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

BACKGROUND: Brain 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-alpha) 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. RESULTS: Here 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-alpha/beta-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-kappaB) 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-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-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. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Endothelial function and insulin sensitivity during acute non-esterified fatty acid elevation: effects of fat composition and gender

Background and Aims: We have reported that adverse effects on flow-mediated dilation of an acute elevation of non-esterified fatty acids rich in saturated fat (SFA) are reversed following addition of long-chain (LC) n-3 polyunsaturated fatty acids (PUFA), and hypothesised that these effects may be mediated through alterations in insulin signalling pathways. In a subgroup, we explored the effects of raised NEFA enriched with SFA, with or without LC n-3 PUFA, on whole body insulin sensitivity (SI) and responsiveness of the endothelium to insulin infusion. Methods and Results: Thirty adults (mean age 27.8 y, BMI 23.2 kg/m2) consumed oral fat loads on separate occasions with continuous heparin infusion to elevate NEFA between 60-390 min. For the final 150 min, a hyperinsulinaemic-euglycaemic clamp was performed, whilst FMD and circulating markers of endothelial function were measured at baseline, pre-clamp (240 min) and post-clamp (390 min). NEFA elevation during the SFA-rich drinks was associated with impaired FMD (P=0.027) whilst SFA+LC n-3 PUFA improved FMD at 240 min (P=0.003). In males, insulin infusion attenuated the increase in FMD with SFA+LC n-3 PUFA (P=0.049), with SI 10% greater with SFA+LC n-3 PUFA than SFA (P=0.041). Conclusion: This study provides evidence that NEFA composition during acute elevation influences both FMD and SI, with some indication of a difference by gender. However our findings are not consistent with the hypothesis that the effects of fatty acids on endothelial function and SI operate through a common pathway. Trial registered at clinicaltrials.gov, NCT01351324.

Endocannabinoid signaling in autism

Autism spectrum disorder (ASD) is a complex behavioral condition with onset during early childhood and a lifelong course in the vast majority of cases. To date, no behavioral, genetic, brain imaging, or electrophysiological test can specifically validate a clinical diagnosis of ASD. However, these medical procedures are often implemented in order to screen for syndromic forms of the disorder (i.e., autism comorbid with known medical conditions). In the last 25 years a good deal of information has been accumulated on the main components of the “endocannabinoid (eCB) system”, a rather complex ensemble of lipid signals (“endocannabinoids”), their target receptors, purported transporters, and metabolic enzymes. It has been clearly documented that eCB signaling plays a key role in many human health and disease conditions of the central nervous system, thus opening the avenue to the therapeutic exploitation of eCB-oriented drugs for the treatment of psychiatric, neurodegenerative, and neuroinflammatory disorders. Here we present a modern view of the eCB system, and alterations of its main components in human patients and animal models relevant to ASD. This review will thus provide a critical perspective necessary to explore the potential exploitation of distinct elements of eCB system as targets of innovative therapeutics against ASD.

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbβ3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbβ3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbβ3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.

Growth factor receptor-bound protein 2 contributes to (hem)immunoreceptor tyrosine-based activation motif-mediated signaling in platelets

Rationale: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective: We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results: Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem)immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions: These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome.

Critical role for an acidic amino acid region in platelet signaling by the HemITAM (hemi-immunoreceptor tyrosine-based activation motif) containing receptor CLEC-2 (C-type lectin receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.

The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Comparison of CLEC-2 and GPVI signaling in platelets: the role of adaptor proteins

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLCy2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLCy2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x6−12Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

Ibrutinib inhibits platelet integrin αIIbβ3 outside-in signaling and thrombus stability but not adhesion to collagen

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbβ3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbβ3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here we show in mice that four months of pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling down-regulates Porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.