B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin-B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor-bearing) and dorsal (ephrin-B-bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5-10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin-B1 ectodomains cause slow (30-60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor-ligand binding, endocytosis occurs in the reverse direction (EphB2-Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B-type Eph/ephrin-mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction. (C) 2003 Wiley Periodicals, Inc

Axonal mRNA translation in neurological disorders.

It is increasingly recognized that local protein synthesis (LPS) contributes to fundamental aspects of axon biology, in both developing and mature neurons. Mutations in RNA-binding proteins (RBPs), as central players in LPS, and other proteins affecting RNA localization and translation are associated with a range of neurological disorders, suggesting disruption of LPS may be of pathological significance. In this review, we substantiate this hypothesis by examining the link between LPS and key axonal processes, and the implicated pathophysiological consequences of dysregulated LPS. First, we describe how the length and autonomy of axons result in an exceptional reliance on LPS. We next discuss the roles of LPS in maintaining axonal structural and functional polarity and axonal trafficking. We then consider how LPS facilitates the establishment of neuronal connectivity through regulation of axonal branching and pruning, how it mediates axonal survival into adulthood and its involvement in neuronal stress responses.ERC, Champalimaud Foundation Vision Awar

Hermes Regulates Axon Sorting in the Optic Tract by Post-Trancriptional Regulation of Neuropilin 1.

UNLABELLED: The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT: An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting.This work was supported by Wellcome Trust Programme Grants (085314) (CEH), European Research Council Advanced Grant (322817) (CEH), a Wellcome Trust Investigator Award (WAH), EMBO Long Term Fellowship (JMC), BBSRC studentship (HH) and Cambridge Gates Trust Scholarship (HH)

NF-protocadherin and TAF1 regulate retinal axon initiation and elongation in vivo

NF-protocadherin (NFPC)-mediated cell–cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs

Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome.

Axonal protein synthesis and degradation are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope labeling of amino acids in cell culture with single-pot solid-phase-enhanced sample preparation, we characterized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our analysis detects 350 basally translated axonal proteins on average, including several linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic landscape by extrinsic cues and uncover a logic underlying attraction versus repulsion

Therapeutic Hypothermia Protocol in a Community Emergency Department

Objectives: Therapeutic hypothermia (TH) has been shown to improve survival and neurological outcome in patients resuscitated after out of hospital cardiac arrest (OHCA) from ventricular fibrillation/ventricular tachycardia (VF/VT). We evaluated the effects of using a TH protocol in a large community hospital emergency department (ED) for all patients with neurological impairment after resuscitated OHCA regardless of presenting rhythm. We hypothesized improved mortality and neurological outcomes without increased complication rates.Methods: Our TH protocol entails cooling to 33 C for 24 hours with an endovascular catheter. We studied patients treated with this protocol from November 2006 to November 2008. All non-pregnant, unresponsive adult patients resuscitated from any initial rhythm were included. Exclusion criteria were initial hypotension or temperature less than 30 C, trauma, primary intracranial event, and coagulopathy. Control patients treated during the 12 months before the institution of our TH protocol met the same inclusion and exclusion criteria. We recorded survival to hospital discharge, neurological status at discharge, and rates of bleeding, sepsis, pneumonia, renal failure, and dysrhythmias in the first 72 hours of treatment.Results: Mortality rates were 71.1% (95% CI, 56-86%) for 38 patients treated with TH and 72.3% (95% CI 59-86%) for 47 controls. In the TH group, 8% of patients (95% CI, 0-17%) had a good neurological outcome on discharge, compared to 0 (95% CI 0-8%) in the control group. In 17 patients with VF/VT treated with TH, mortality was 47% (95% CI 21-74%) and 18% (95% CI 0-38%) had good neurological outcome; in 9 control patients with VF/VT, mortality was 67% (95% CI 28-100%), and 0% (95% CI 0-30%) had good neurological outcome. The groups were well-matched with respect to sex and age. Complication rates were similar or favored the TH group.Conclusions: Instituting a TH protocol for OHCA patients with any presenting rhythm appears safe in a community hospital ED. A trend towards improved neurological outcome in TH patients was seen, but did not reach significance. Patients with VF appeared to derive more benefit from TH than patients with other rhythms. [West J Emerg Med. 2010; 11(4):367-372.

Tctp in Neuronal Circuitry Assembly.

Although tctp expression in many areas of the human brain was reported more than 15 years ago, little was known about how it functions in neurons. The early notion that Tctp is primarily expressed in mitotic cells, together with reports suggesting a relative low abundance in the brain, has perhaps potentiated this almost complete disregard for the study of Tctp in the context of neuron biology. However, recent evidence has challenged this view, as a number of independent genome-wide profiling studies identified tctp mRNA among the most enriched in the axonal compartment across diverse neuronal populations, including embryonic retinal ganglion cells. Considering the emerging parallels between axon guidance and cancer cell invasion, the axonal expression of cancer-associated tctp was suggestive of it holding an unexplored role in the wiring of neuronal circuits. Our study revealed that Tctp is necessary for the accurate and timely development of axon projections during the formation of vertebrate retinal circuits via its association with the survival machinery of the axon. Globally, the findings indicate that compromised pro-survival signaling in Tctp-deficient axons results in mitochondrial dysfunction and a subsequent decrease in axonal mitochondrial density. These effects likely translate into a metabolic state inadequate to support the normal guidance and extension processes of a developing axon

Tumor protein Tctp regulates axon development in the embryonic visual system.

The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly.This work was supported by Fundação para a Ciência e Tecnologia (C.G.R.; fellowship SFRH/BD/33891/2009), Sir Edward Youde Memorial Fund, Croucher Foundation, Cambridge Commonwealth–European & International Trust (H.W.), Gates Cambridge Scholarship (J.Q.L.), and a Wellcome Trust Programme Grant (C.E.H.; grant 085314/Z/08/Z).This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.13106

Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus

Background: Blastomere injection of mRNA or antisense oligonucleotides has proven effective in analyzing early gene function in Xenopus. However, functional analysis of genes involved in neuronal differentiation and axon pathfinding by this method is often hampered by earlier function of these genes during development. Therefore, fine spatio-temporal control of over-expression or knock-down approaches is required to specifically address the role of a given gene in these processes