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Endogenous noradrenaline (NA) has multiple bioactive functions and, in the central nervous system (CNS), has been implicated in modulating neuroinflammation via β-adrenergic receptors (β-ARs). Microglia, resident macrophages in the CNS, have a central role in the brain immune system and have been reported to be activated by NA. However, intracellular signaling mechanisms of the AR-mediated proinflammatory responses of microglia are not fully understood. Using a rapid and stable in vitro reporter assay system to evaluate IL-1β production in microglial BV2 cells, we found that NA and the β-AR agonist isoproterenol upregulated the IL-1β reporter activity. This effect was suppressed by β-AR antagonists. We further examined the involvement of EPAC (exchange protein directly activated by cAMP) and TPL2 (tumor progression locus 2, MAP3K8) and found that inhibitors for EPAC and TPL2 reduced AR agonist-induced IL-1β reporter activity. These inhibitors also suppressed NA-induced endogenous Il1b mRNA expression and IL-1β protein production. Our results suggest that EPAC and TPL2 are involved in β-AR-mediated IL-1β production in microglial cells, and extend our understanding of its intracellular signaling mechanism.

Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.

BACKGROUND: Patients suffering from the irritable bowel syndrome report exaggerated and sustained abdominal pain. We have shown that activation of protease activated receptor 2 (PAR2) on nociceptive (pain sensing) dorsal root ganglia (DRG) neurons evoke long term hyperexcitability but the mechanisms of this sustained pain signaling are unclear. Recent studies show that trypsin activates canonical PAR2 signaling causing endocytosis and this leads to sustained PAR2- endosomal signaling. Proteases such as elastase and cathepsin S, however, act at non-canonical sites and do not cause receptor endocytosis. We have shown that protease activity is increased in IBS tissues but it is unclear whether this leads to sustained neuronal excitation. AIMS: To examine whether proteases in tissues from IBS patients lead to sustained nociceptive signaling and, if so, what intracellular mechanisms are involved. METHODS: DRG neurons (T9-T13) from C57BL/6 mice were pre-incubated (10 min) with Trypsin (50nM) or supernatants (30 min) from colonic biopsies obtained from diarrhea predominant IBS patients or controls then washed with F12 media. We measured neuronal excitability by perforated patch-clamp, recording changes in rheobase (minimum current to fire action potential) and action potential discharged at twice rheobase immediately after washing with F12 media (time 0) or ~ 30 min later (time 30). PAR2 (10 μM I-343) and ERK1/2 (50 μM PD8059) inhibitors were applied 30 min before IBS supernatant or trypsin. Two way ANOVA and post hoc Tuckey’s tests were used to analyze the data. RESULTS: IBS supernatants increased DRG neuronal excitability acutely (time 0) as well as sustained hyperexcitability (time 30) (Table 1). This was blocked by the PAR2 antagonist I-343. This sustained excitability was also blocked by antagonist of ERK1/2. Sustained excitability evoked by trypsin was also inhibited by the ERK1/2 antagonist. CONCLUSIONS: Proteases in IBS tissues, acting through PAR2, evoke long term hyperexcitability of nociceptive signaling in DRG neurons that is ERK1/2 dependent. In addition, the sustained excitability evoked by the serine protease trypsin, which we previously have shown is mediated by PAR2-endosomal signaling, was also ERK1/2 dependent. Thus, serine proteases in IBS tissues may be signaling through similar pathways and these could provide a novel therapeutic target to treat pain in patients with IBS. FUNDING AGENCIES: CAGCONACYT

Janus kinases (JAKs) initiate the intracellular signaling cascade triggered by exposure of cells to cytokines and interferons. In order to achieve this, JAKs are bound to the intracellular domain of specific cytokine receptors immediately adjacent to the cell membrane. In this issue of Structure, Ferrao et al. (2016) provide structural details of such an interaction and in doing so, identify for the first time the motif used by type II cytokine receptors to recruit JAK1.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

GLI transcription factors have important roles in intracellular signaling cascade, acting as the main mediators of the HH-GLI signaling pathway. This is one of the major developmental pathways, regulated both canonically and non-canonically. Deregulation of the pathway during development leads to a number of developmental malformations, depending on the deregulated pathway component. The HH-GLI pathway is mostly inactive in the adult organism but retains its function in stem cells. Aberrant activation in adult cells leads to carcinogenesis through overactivation of several tightly regulated cellular processes such as proliferation, angiogenesis, EMT. Targeting GLI transcription factors has recently become a major focus of potential therapeutic protocols.

Steroidal compounds that are closely related structurally to ecdysone are grouped as ecdysteroids. Ecdysteroids identified in plants are named phytoecdysteroids, and those from animals are sometimes called zooecdysteroids. Among ecdysteroids, ecdysone and 20-hydroxyecdsone (20E) are integral to the growth and reproductive functions in arthropods. Ecdysteroids bind to ecdysone receptors (EcRs). EcRs belong to the nuclear receptor and are heterodimerized with another nuclear receptor ultraspiracle (USP) to transactivate the genes related to molting (genomic action). It is also known that ecdysteroids moderate the intracellular signaling cascade without any changes in gene expression (non-genomic action). Non-steroidal ecdysone agonists, such as diacylhydrazine (DAH)-type compounds, bind to the EcR/USP complex and activate the pathways for genomic action of 20E. Chemicals with different structures from DAHs are also reported as being ecdysone agonists.

The actions of estrogens have been associated with brain differentiation and sexual dimorphism in a wide range of vertebrates. Here we consider the actions of brain-derived ‘neuroestrogens’ in the forebrain and the accompanying differences and similarities observed between males and females in a variety of species. We summarize recent evidence showing that baseline and fluctuating levels of neuroestrogens within the auditory forebrain of male and female zebra finches are largely similar, and that neuroestrogens enhance auditory representations in both sexes. With a comparative perspective we review evidence that non-genomic mechanisms of neuroestrogen actions are sexually differentiated, and we propose a working model for nonclassical estrogen signaling via the MAPK intracellular signaling cascade in the songbird auditory forebrain that is informed by the way sex differences may be compensated. This view may lead to a more comprehensive understanding of how sex influences estradiol-dependent modulation of sensorimotor representations.

Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promises of hPSCs for applications in regenerative medicine, disease modeling, and developmental biology studies. While most studies of hPSCs have so far focused on identifying extrinsic soluble factors, intracellular signaling pathways, and transcriptional networks that are involved in regulating hPSC self-renewal and differentiation, a few promising studies have emerged in recent years to reveal some unique mechano-sensitive and -responsive properties of hPSCs and the effect of the physical aspects of the local cellular microenvironment on regulating hPSC behaviors. This Frontier Review is to highlight these recent studies of mechanobiology in hPSCs and to discuss the impact of advancing our understanding of mechanoregulation of hPSC behaviors on improving survival, self-renewal and differentiation of hPSCs using well-controlled synthetic micro/nanoscale cell culture tools.

As detailed in Chaps. 5, 6, and 7, the mainstay of anemia management includes ESA therapy, iron management, and blood transfusions. ESA therapy has evolved from a recombinant protein to a hyperglycosylated molecule, to a pegylated protein, to an erythropoietin-mimetic peptide. However, all of these developments have in common the fact that the molecule binds to the erythropoietin receptor and induces a common intracellular signaling cascade, which ultimately leads to the enhanced survival and proliferation of primitive erythroid cells.

We describe a novel clinical phenotype associating T- and B-cell lymphopenia, intermittent neutropenia, and atrial septal defects in 3 members of a consanguineous kindred. Their clinical histories included recurrent bacterial infections, viral infections, mucocutaneous candidiasis, cutaneous warts, and skin abscesses. Homozygosity mapping and candidate gene sequencing revealed a homozygous premature termination mutation in the gene STK4 (serine threonine kinase 4, formerly having the symbol MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apoptosis. STK4-deficient lymphocytes and neutrophils exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis. STK4 deficiency is a novel human primary immunodeficiency syndrome.

Melanopsin expressing retinal ganglion cells represent a third class of ocular photoreceptors and are involved in irradiance detection and non-image-forming responses to light including pupil constriction, circadian entrainment, and regulation of sleep. Over recent years, there has been a rapid increase in our understanding of the anatomical variety of pRGC subtypes, the regions of the brain which they innervate, and the behavioral responses of melanopsin-based light detection. However, by contrast, our understanding of the intracellular signaling cascade initiated following activation of melanopsin has, until recently, remained poorly characterized. This chapter focus on the melanopsin signaling pathway, detailing the cellular mechanisms of phototransduction that occur within pRGCs, highlighting recent advances, but also the gaps in our understanding of this important light detecting system.

Long-term depression (LTD) at parallel fiber-Purkinje cell (PF-PC) synapses is thought to be the cellular correlate of cerebellar associative learning. The molecular processes are, in brief, phosphorylation of AMPA-type glutamate receptors (AMPARs) and their subsequent removal from the surface of the PF-PC synapse. In order to elucidate the fundamental mechanisms for cerebellar LTD and further the understanding of its computational role, we have investigated its systems biology and proposed the following hypotheses, some of which have already been experimentally verified: (1) due to the mitogen-activated protein kinase (MAPK)-protein kinase C (PKC) positive feedback loop, phosphorylation of AMPARs is an all-or-none event; (2) the inositol 1,4,5-triphosphate receptor detects concurrent PF and climbing fiber inputs, forming the cellular basis for associative learning, and (3) the local concentration of nitric oxide in the PC dendrite reflects the relevance of a given context, enabling context-dependent selection of learning modules within the cerebellum. In this review, we first introduce theoretical studies on cerebellar LTD, mainly focusing on our own published work, followed by a discussion of the effects of stochasticity, localization, diffusion, and scaffolding. Neurons embody two features that are apparently contradictory, yet necessary for synaptic memory: stability and plasticity. We will also present models for explaining how neurons solve this dilemma. In the final section, we propose a conceptual model in which a cascade of excitable dynamics with different time scales, i.e., Ca(2+)-induced Ca(2+) release, the MAPK-PKC positive feedback loop, and protein kinase Mzeta (PKMzeta)-induced PKMzeta synthesis, provides a mechanism for stable memory that is still amenable to modifications.

Exposure of stress precipitates a coordinated series of responses which may lead to changes in brain functions. Most of the changes in brain function are mediated by stress-induced activation of neurotransmitter systems in the brain, which include neurotransmitter receptors and these receptors-mediated signal transduction pathways. The signal transduction pathways are ultimately responsible for stress-induced changes in neuronal functions. Some of these changes are beneficial, however, depending upon the nature and duration of stressful stimuli, the changes in neuronal functions may be harmful, which may lead to many disorders. In the present chapter, we describe comprehensively the effects of stress on neurotransmitter receptors and various molecules of signal transduction pathways in brain

Recent studies have demonstrated that apoptosis induction via the intracellular signaling cascade, consisting mainly of the p53 gene, plays an important role in cell death due to various cell injury factors. It has been reported that hsp72 protein is involved in thermotolerance induction, thereby at least partly regulating thermosensitivity. In this chapter, we review various aspects of hyperthermia, namely, thermosensitivity, sublethal thermal damage repair (SLTDR), cell phase response to heat, step-down and step-up heating, thermotolerance, apoptosis, heat shock protein (hsp), and p53 protein and its status at the cellular and molecular levels, and introduce articles concerning these published by our group.

Previous studies show that fluid shear stress on cell membranes causes metabolic changes in cells. One question that arises is what physical effects shear stress has on the fluidity of artificial lipid bilayers. As demonstrated by Haidekker et al, shear stress can change membrane fluidity of endothelial cells within 5 seconds, and membrane fluidity recovered completely after flow stopped. Due to the speed and reversibility of the change in membrane fluidity caused by shear stress, we hypothesized that the change in fluidity may be caused by the direct effect of shear stress on the packing of the lipid bilayer itself, rather than an effect mediated through an intracellular signaling cascade. This research studied the effect of shear stress on a protein-free membrane by inducing shear stress on a supported lipid bilayer in a micro-volume flow chamber. The effect of changing fluid flow in step increments on the membrane fluidity was measured using z-scan fluorescence correlation spectroscopy (FCS).