SYAPTIC PLASTICITY AND AXONAL GUIDANCE 1. The Neuron: basic Mechanisms of Action 2. Axon Guidance and Nerve Growth: Basic Principles 3. Short Range Guidance: 1. Eph-Ephrins / 2. Semaphorins 4. Long Range Cues: Semaphorins / Netrin-Slit / Nogo / Others 5. Learning and Memory - Guidance and Neuronal Adaptation in the Adult - Regulation of Pathways SLIT & NETRINS Netrins • Netrins are a small family of highly conserved guidance molecules (~70-80kDa). • One in worms (c.elegans) UNC6 • Two in Drosophila Netrin -A and -B • Two in chick, netrin-1 and -2. • In mouse and humans a third netrin identified netrin-3 (netrin-2-like). • In all species there are axons that project to the midline of the nervous system. • The midline attracts these axons and netrin plays a role in this. Netrins • Netrin-1 is produced by the floor plate • Netrin-2 is made in the ventral spinal cord except for the floor-plate • Both netrins become associated with the ECM and the receptor DCC • Model: commissural axons first encounter gradient of netrin-2, which brings them into the domain of netrin-1 Netrins Roof plate Commissural neuron 0 125 250 375 Commissural neurons extend ventrally Floor plate and then toward floor plate, if within 250µm from the floor plate Netrins • Netrins are bifunctional molecules, attracting some axons and repelling others. • C.elegans axons migrating away from the UNC-6 netrin source are misrouted in the unc-6 mutant. • The repulsive activity of netrin first shown in vertebrates for populations of motor axons that project away from the midline. • The receptors that mediate the attractive and repulsive effects of netrins are also highly conserved. • Growth cone attraction involves the transmembrane receptors of the DCC family. • Repulsion involves the transmembrane receptors of the UNC-5 family. Netrin=unc6 Ligand DCC=unc40 attract unc5 = repulsion Unc-6 minus Wild type Unc-40+ Unc-5+ motor sensory neurons neuons UNC-6 Unc-5 minus Unc-40 minus Slits • Slit proteins are large ~190kDa extracellular matrix proteins containing leucine-rich repeats and epidermal growth factor-like repeats. • Slit has a role in axon guidance at the midline. • Receptor (Robo) is expressed by axons that navigate the midline and prevents them crossing • Robo expressing axons run longitudinally and never cross the midline. • In Robo mutants axons freely cross the midline. • Commissural axons up-regulate Robo after they have crossed the midline • Slit is repellent ligand • 3 vertebrate slit proteins are expressed at the midline and have chemorepulsive activity for olfactory bulb axons, hippocampal axons and spinal motor axons in culture SLIT-NETRIN SIGNALING DUAL ROLE FOR ROBO IN GUIDANCE (a) Mammalian Robo1 transduces part of the signal for repulsion by directly interacting with srGAPs. The binding of Slit to Robo1 recruits srGAP to the receptor's cytoplasmic tail. This is associated with the activation of RhoA and the inhibition of Cdc42. This shift in the balance of GTPase activation may lead to growth cone collapse and repulsion. (b) Robo1 can also silence signalling by the netrin receptor DCC in a direct form of crosstalk between the two receptors. The binding of Slit to Robo1 induces an interaction between the cytoplasmic domains of Robo1 and DCC (*). This involves the CC1 domain in Robo1 and the P3 domain in DCC. This direct interaction prevents DCC from transmitting the signal for attraction upon netrin binding. At present, it is unknown if Robo1 transduces the srGAP-mediated signal for repulsion and silences attraction by DCC simultaneously. It is unknown how phosphorylation of the Robo CC1 domain by Abl (dashed arrow) affects signalling by the srGAP pathway or the ability of Robo to silence attraction by DCC. Switching sensitivity at the midline As they cross the floor plate, vertebrate commissural axons lose sensitivity to the midline attractant, netrin, and acquire sensitivity to Slit and semaphorin repellents. This switch may be mediated in part by silencing of netrin attraction by Slit. Switching sensitivity at the midline Drosophila commissural axons also become sensitive to Slit only after crossing. This appears to reflect Comm’s role in regulating the intracellular traficking of Robo. Shh (sonic hedge hoc) is an Axonal Chemoattractant Commissural Axon Turning Induced by Shh-Expressing Cells or Tissues, and Its Cyclopamine-Dependence (A) Schematic representation of the commissural axon turning assay. Explants of the entire E11 rat spinal cord were embedded in a three-dimensional collagen matrix and cocultured for 40 hr with various tissues or cells expressing Shh and/or Netrin-1. (B, C) and left images (D, E, H, I, L, M, P, Q, T, and U): without cyclopamine; other right images (F, G, J, K, N, O, R, S, V, and W): with 10 M cyclopamine. Commissural axon trajectories were detected by TAG-1 immunohistochemistry. D, dorsal; V, ventral; sc, spinal cord. (B–O) Shh causes commissural axon turning within spinal cord explants in a cyclopaminedependent manner. COS cells expressing Shh (D–E) or Netrin-1 (H–I) and notochord tissue (L–M), but not control COS cells (B–C), elicit commissural axon turning. Cyclopamine inhibits the attractant activity of COS cells expressing Shh (F–G) and of the notochord (N–O), but not of COS cells expressing Netrin-1 (J–K). (P–W) Cyclopamine blocks the Netrin-1-independent attractant activity of the floor plate. Both wild-type (P–Q) and Netrin-1 mutant (T–U) floor plate tissue can elicit commissural axon turning, but cyclopamine blocks only the latter (V–W), not the former (R–S). Scale bars are 200 m. Charron et alCell, Vol. 113, 11–23, April 4, 2003, Shh Is an Axonal Chemoattractant Two models could account for the effects of Shh in axon guidance. In Model 1, Shh acts directly as a chemoattractant. In Model 2, Shh acts by repatterning the spinal cord, altering the expression of other guidance cues that then secondarily reorient axon growth. Shh Is an Axonal Chemoattractant Two sets of morphogens, Shh and BMPs, are first used to pattern neural progenitors in the spinal cord, and then appear to be reused as guidance cues for commissural axons. In the early neural tube, Shh and BMP protein concentration gradients act to specify neural cell fate in the ventral and dorsal spinal cord, respectively. Later, the axons of differentiated commissural neurons are repelled from the dorsal midline by BMPs and attracted to the ventral midline by the combined chemoattractant effects of Netrin-1 and Shh. Netrin-1 also provides an essential permissive activity, allowing invasion of the otherwise non-permissive ventral spinal cord. rp, roof plate; fp,floor plate; c, commissural neurons. Ectoderm becomes Neural Tissue Dorsal-ventral Position •The default of ectoderm is to become neural, but it also becomes skin. •Ectoderm has receptors BMP (bone morphogenic protein) which causes non- neural development. •When BMPs are blocked by Chordin, Follistatin and Noggin neural tissue results •Different concentrations of the SHH and BMPs will determine gene expression. •BMP are expressed dorsally and SHH is expressed ventrally. Neurons at the midline are experiencing equal levels of both. •This system only works with both signals. If one were lost you would have a homogeneous cell population. Graded Shh activity and ventral neural tube patterning A model for the influence of Shh on the specification of ventral neuronal fates. Left : presumed gradient of Shh activity in the ventral neural tube (blue dots), distributed in a ventral-high, dorsal- low profile within the ventral neural epithelium, and the position of five classes of neurons that are generated inn response to graded Shh signalling. V0–V3 = four different classes of ventral interneurons. MN indicates motor neurons, and FP the floor plate. To the right is shown the profile of neuronal generation in intermediate neural plate explants grown in different concentrations of the recombinant amino-terminal fragment of Shh, termed Shh-N. D, dorsal neural tube; V, ventral neural tube. The more dorsal the position of neuronal subtype generation in vivo, the lower the concentration of Shh required to induce the same neuronal subpopulation in vitro. Three Phases of Ventral Neural Patterning (A) Graded Shh signaling initiates dorsoventral restrictions in the domains of class I and class II protein expression within the ventral neural tube. Class I proteins are repressed by Shh signals and class II proteins require Shh signaling. Individual class I and class II proteins have different Shh concentration requirements for repression or activation. (B) Cross-repressive interactions between class I and class II proteins that abut a common progenitor domain boundary refine and maintain progenitor domains. (C) The profile of expression of class I and class II proteins within an individual progenitor domain controls neuronal fate. BMP Proteins BMP Proteins ADAPTATION A model of growth-cone adaptation in a gradient of guidance cues. a, The guidance cue triggers a cytoplasmic signal (S) and two parallel processes of desensitization (D) and resensitization or adaptation (A), which regulate the output (O) of the signalling cascade, mediated through elevation of Ca2+, leading to cytoskeletal rearrangements associated with attractive or repulsive responses. b, Asymmetry in the output determines the direction of growth. With time, the effect of resensitization or adaptation compensates for that of desensitization, leading to chemotaxis. c, Assuming the onset of desensitisation precedes that of resensitization or adaptation, the asymmetric desensitization (higher towards the gradient) before the onset of resensitization or adaptation results in repulsive turning after the initial attraction, hence the zig- zag trajectory of growth. Blocking resensitization or adaptation by protein synthesis inhibitors results in a persistent asymmetric output after the initial densensitization, leading to the end result of repulsion. NOGO : AXONAL REGENERATION Wallerian degeneration: degradation of the axonal cytoskeleton Strittmatter SM at Yale Univ. Repulsive factors and axon regeneration in the CNS.