Neuronal Migration and Axon Guidance

Question 1

Requirements of Development and/or Repair of Nervous System

Answer 1

o Early patterning
o Neurogenesis and neuronal fate specification
o Neuronal migration
o Neuronal contact formation
 Post-synaptic contacts on dendrites
 Pre-synaptic contacts dependent on the axonal growth cone  better studied

Question 2

Developmental Defects in Axon Guidance

Answer 2

• Developmental Defects in Axon Guidance – KIF21A – transport of essential cell components on axons
o KIF21A mutation – superior branch of oculomotor nerve is hypoplastic or absent

Question 3

Growth Cone Structure

Answer 3

• axons grow by extending growth cone – long process that grows outward to detect factors affecting directionality and growth
  o Filopodia – “fingers” – contain actin rich protrusions and dynamic microtubules
  o Lamellipodia – “webbing between fingers”
  o Long arm/axon – contains stable microtubules and cytoskeleton – associated proteins that carry cargo bidirectionally

Question 4

Growth Cone ELongation

Answer 4

o Dynamic process in which microtubules are added at the growing (distal) end
o F-actin Treadmilling – takes place in actin protrusions in filopedia
 Polymerization occurs at distal end and depolymerization occurs at the proximal end

Question 5

Growth Cone Turning

Answer 5

• Actin polymerization & microtubule stabilization occur in direction of turning
  o Cytochalasin – depolymerizes actin  axon turns away
  o Taxol – stabilizes microtubules  axon turns towards
  o Nocodazole – destabilizes microtubules  axon turns away

Question 6

Control of Axon Guidance

Answer 6

– 4 class of molecules that control axonal guidance – attraction or repulsion, substrate bound or soluble
o Expression and Function – is the molecule expressed in the right place, at the right time, and in a biologically relevant concentration
o Non-specific, generalized outgrowth cues – forms scaffold on which axons may grow
 Ex: fibronectin, laminin
o Soluble molecules – bind to receptors expressed on the filopodial surface
 Mediate attraction or repulsion
o Substrate bound (cell-attached) molecules – regulate axon guidance
 Cell-adhesion molecules (CAMs), cadherins, and integrins play a role in cell migration and axon guidance by affecting activity of intracellular protein kinases/phosphatases that ultimately regulate actin dynamics

Question 7

Neuronal Development Molecules

Answer 7

• neurons must get to midline and cross the midline without re-crossing
  o Netrin – soluble molecule – guides axons to the midline
   Expressed by floor plate and causes growth of axonal processes towards it
  o Robo/Slit – determine whether neurons cross the midline or not
  • Robo – receptor mediated repulsion
  • Slit – ligand that binds to Robo and stimulates repulsion
   Commissural/crossing axons
  • Comm - stimulates crossing of midline and keeps Robo levels low
  • Robo levels are increased once it crosses midline thus preventing re-cross
  o Semaphorins (Sema-3A) – repellant molecule which depolymerizes actin

Question 8

Initiation of Intracellular Events - Important Effectors

Answer 8

o Ion channels – activity dependent guidance
o G protein coupled receptors (GPCR) and release of intracellular Ca+ and 2 other effectors
o Calcium – changes influence the activity of critical protein kinases/phosphatases
 Kinase/phosphatase balance is critical to the activity of actin effectors
o Rho GTPase activity – effector of actin dynamics in axonal and dendritic spine growth
 Calcium enters the cell to activate changes in Rho to affect actin polymerization
o Cyclic nucleotide activity – a complex interplay between calcium and cyclic nucleotide-dependent effects on growth cone guidance
 Distinct calcium signaling can be modulated by cAMP  can influence an axon’s bidirectional turning response

Question 9

G protein Coupled Receptors

Answer 9

function in cell shape and migration as well as axon outgrowth
o	Most abundance receptor cell type in the brain; contains 7 transmembrane domains
o	Activation – binding of soluble agonist to its external domain causing dissociation of βγ subunit
	Linked intracellularly to an alpha-subunit  cascade of events

Question 10

Actin Dynamics Regulator

Answer 10

• regulated by ADF/cofilin – proteins that are cotransported with actin to the tips of growth cones
  o Phosphorylation of ADF/cofilin regulate its activity

Question 11

Mature Dendritic Spines

Answer 11

– represent the post synaptic process for the majority of glutamatergic synapses
o Changes in spine morphology are critical to learning and memory
 Influenced by many of the same molecules that are critical to axon guidance
o Calcium
 Rapid, high increases in calcium result in kinase activation and larger spines
 Slow changes stimulate phosphatase activity and spine shrinkage
 Localized changes in calcium affect adhesion molecule expression and localization  more synapses (activity) in particular neuron the more expansion of that neuron

Question 12

Axon Guidance in Mature PNS vs CNS

Answer 12

o Regeneration in the PNS is much better than in the CNS
 Regeneration is inhibited by repulsive adhesion molecules – harder to get regeneration in mature CNS due to higher expression of repulsive and axon inhibitory molecules
 Intrinsic properties of PNS vs. CNS neurons
 Environmental factors in the PNS vs. CNS
 Difference in glial ECM proteins that are upregulated –astrocytes make inhibitory CSPGs and don’t make growth promoting basal lamina proteins
 Differences in clearance of myelin debris/inhibitory myelin components

Question 13

Summary

Answer 13

o Molecules important to axon guidance include 4 classes of extracellular molecules (repulsive/attractive adhesion molecules as well as repulsive/attractive soluble molecules)
o Intracellular changes in calcium, Rho GTPase, and cyclic nucleotides - role in axon guidance in
o Molecules important to axon guidance during development are likely important to processes in which cell morphology is critical including cell migration, post-injury repair processes, and synaptic plasticity
o ADF/cofilin regulates actin dynamics; they are proteins that are cotransported w/ actin to tips of growth cones; phosphorylation of ADF/cofilin regulates activity
o Changes in the phosphorylation of ADF/cofilin are seen with neuronal activity, integrin engagement, Rho signaling, netrin, nerve growth factor