Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Here, we transfected activated and inhibitory mutants of Trk effectors into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling us to observe the contributions of individual signaling mediators. While Ras was both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induced distinct morphologies. Activated Raf-1 caused axon lengthening comparable to NGF, while active Akt increased axon caliber and branching. Our results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons.

CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-aminoacid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18 104-140. A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18 104-140 and a more active truncated fragment, CAP18 104-135 , were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18 104-140 may have therapeutic utility for conditions associated with elevated concentrations of LPS.

We have established functions of the stimulus dependent MAPKs, ERK1/2 and ERK5 in DRG, motor neuron, and Schwann cell development. Surprisingly, many aspects of early DRG and motor neuron development were found to be ERK1/2 independent and Erk5 deletion had no obvious effect on embryonic PNS. In contrast, Erk1/2 deletion in developing neural crest resulted in peripheral nerves that were devoid of Schwann cell progenitors, and deletion of Erk1/2 in Schwann cell precursors caused disrupted differentiation and marked hypomyelination of axons. The Schwann cell phenotypes are similar to those reported in neuregulin-1 and ErbB mutant mice and neuregulin effects could not be elicited in glial precursors lacking Erk1/2. ERK/MAPK regulation of myelination was specific to Schwann cells, as deletion in oligodendrocyte precursors did not impair myelin formation, but reduced precursor proliferation. Our data suggest a tight linkage between developmental functions of ERK/MAPK signaling and biological actions of specific RTK-activating factors.

We have defined functions of MEK in regulating gliogenesis in developing cerebral cortex using loss and gain of function mouse genetics. Radial progenitors deficient in both Mek1 and Mek2 fail to transition to the gliogenic mode in late embryogenesis, and astrocyte and oligodendroglial precursors fail to appear. In exploring mechanisms, we found that the key cytokine regulated gliogenic pathway is attenuated. Further, the Ets transcription family member Etv5/Erm is strongly regulated by MEK and Erm overexpression can rescue the gliogenic potential of Mek deleted progenitors. Remarkably, Mek1/2 deleted mice surviving postnatally exhibit cortices almost devoid of astrocytes and oligodendroglia and exhibit neurodegeneration. Conversely, expression of constitutively active MEK1 leads to a major increase in numbers of astrocytes in the adult brain. We conclude that MEK is essential for acquisition of gliogenic competence by radial progenitors, and that levels of MEK activity regulate gliogenesis in the developing cortex.

Disrupted ERK1/2 (MAPK3/MAPK1) MAPK signaling has been associated with several developmental syndromes in humans; however, mutations in ERK1 or ERK2 have not been described. We demonstrate haplo-insufficient ERK2 expression in patients with a novel Ϸ1 Mb micro-deletion in distal 22q11.2, a region that includes ERK2. These patients exhibit conotruncal and craniofacial anomalies that arise from perturbation of neural crest development and exhibit defects comparable to the DiGeorge syndrome spectrum. Remarkably, these defects are replicated in mice by conditional inactivation of ERK2 in the developing neural crest. Inactivation of upstream elements of the ERK cascade (B-Raf and C-Raf, MEK1and MEK2) or a downstream effector, the transcription factor serum response factor resulted in analogous developmental defects. Our findings demonstrate that mammalian neural crest development is critically dependent on a RAF/MEK/ERK/ serum response factor signaling pathway and suggest that the craniofacial and cardiac outflow tract defects observed in patients with a distal 22q11.2 micro-deletion are explained by deficiencies in neural crest autonomous ERK2 signaling.22q11 microdeletion ͉ human syndromes ͉ MAP kinase

SUMMARY Human genetic studies have revealed an association between GTP cyclohydrolase 1 polymorphisms, which decrease tetrahydrobiopterin (BH4) levels, and reduced pain in patients. We now show that excessive BH4 is produced in mice by both axotomized sensory neurons and macrophages infiltrating damaged nerves and inflamed tissue. Constitutive BH4 overproduction in sensory neurons increases pain sensitivity, whereas blocking BH4 production only in these cells reduces nerve injury-induced hypersensitivity without affecting nociceptive pain. To minimize risk of side effects, we targeted sepiapterin reductase (SPR), whose blockade allows minimal BH4 production through the BH4 salvage pathways. Using a structure-based design, we developed a potent SPR inhibitor and show that it reduces pain hypersensitivity effectively with a concomitant decrease in BH4 levels in target tissues, acting both on sensory neurons and macrophages, with no development of tolerance or adverse effects. Finally, we demonstrate that sepiapterin accumulation is a sensitive biomarker for SPR inhibition in vivo.

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF Nav1.8 mice). We found that constitutive BRAF pathway activation in BRAF Nav1.8 mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF Nav1.8 mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis-or dry skin-elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation. IntroductionThe ability of the brain to discriminate pain from itch in order to make binary decisions -eliciting either withdrawal or scratching behavior -is critically dependent on the functional connectivity of the somatosensory system. Itch information, along with pain, is conveyed by primary afferents of the dorsal root ganglion (DRG) to the spinal cord and of the trigeminal ganglion neurons to the trigeminal subnucleus caudalis of the brainstem, respectively, which in turn supplies input to the somatosensory cortex through spinothalamic tract or trigeminothalamic tract neurons (1-4). At the molecular level, emerging evidence suggests that activation of GPCRs in sensory neurons is likely responsible for relaying distinct types of acute stimulus-evoked itch (5-7). In addition, several transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRPA1, have been implicated in mediating histaminergic and nonhistaminergic itch, respectively (8-10). In the spinal cord, gastrin-releasing peptide (GRP) receptor (GRPR) and neurons expressing GRPR are key mediators dedicated to the coding of itch sensation (11-13). In contrast to acute itch, chronic itch may arise from an altered or diseased state of the

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders.