Lecture 4 - Neuronal Migration and Axonal Pathfinding

Question 1

Where and how does migration in histogenesis of neuron and glia occur?

Answer 1

Neural crest cells migrate throughout the body and differentiate into many different cell types

In adult mammals, neural stem cells in the subventricular zone (SVZ) proliferate to generate new neuroblasts. Neuroblasts migrate rostrally along the rostral migratory stream (RMS) to the olfactory bulb (OB)

Question 2

Describe the process of neurogenesis and gliogenesis

Answer 2

a) Self-renewing neuroepithelial cells lining the ventricles may generate some neurons. Neuroepithelial cells are transformed into radial glial cells as neurogenesis begins.

b) Radial glia produce intermediate progenitor cells and oligodendrocyte precursor cells (OPCs), which in turn produce neurons and oligodendrocytes, respectively. Radial glia can also become astrocytes, astrocytic progenitors and ependymal cells.

c) In adults, type B cells in the cortical subventricular zone produce transit-amplifying cells (known as type C cells), which in turn produce OPCs as well as neurons

Question 3

How does corticogenesis occur in the mammalian brain?

Answer 3

Laminar architecture of the mammalian neocortex
Six-layers - most superficial layer is layer I while the deepest is VI
Layers of the cortex are created in an inside-out order

Question 4

Corticogenesis in the mammalian brain

What are the two major neuronal populations?

Answer 4

Cortical projection neurons
- Glutamatergic, excitatory neurons
- From cortical proliferative areas
- Radial migration to the cortical plate (CP) through the intermediate zone (IZ)

Cortical interneurons
- GABAergic
- From subcortical zone known as medial ganglionic eminence (MGE)
- Tangential and radial migration

Question 5

How does somal translocation of cortical neurons occur?

Answer 5

Somal translocation is mediated by microtubules in the neuron

• The leading process extends as driven by
  microtubule extension
• The centrosome moves forward into the leading process, while the trailing end of the cell contracts (arrows)
• The nucleus moves forward toward the centrosome (nucleokinesis) and the trailing process retracts

Question 6

How does radial migration of cortical neurons occur? (5 steps)

Answer 6

Radial migration is mediated by the radial glia during the process of locomotion

Main steps in the radial migration of
newborn cortical neurons:
1. Generation of newborn neurons
2. Multipolar tangential migration
3. Multipolar-unipolar transition
4. Attachment and locomotion
5. Detachment
The cell adhesion molecule N-cadherin
has a key role in these processes.

Question 7

What is the role of Reelin?

Answer 7

Reelin is a secreted extracellular matrix glycoprotein produced by Cajal-Retzius cells in the marginal zone

Signal promoting migration along and/or detachment from radial glial cell

Migrating neurons express Reelin receptors ApoER2 (Apolipoprotein E receptor 2) and VLDLR (Very low density lipoprotein receptor). The adaptor protein Disabled-1 (Dab1) is a key intermediate of Reelin signaling

Autosomal recessive mutations in the RELN gene in mouse underlie the Reeler mutant and in humans has been linked to the rare developmental disorder Lissencephaly 2 with brain malformation.

Question 8

How does neuronal differentiation occur in vitro? (5 steps)

Answer 8

1. Neuritogenesis
2. Neurite outgrowth
3. Pathfinding
4. Targeting
5. Synaptogenesis

During neuronal differentiation, neuritogenesis occurs at thin, actin-rich peripheral protrusions surrounding the soma. Subsequently, neurite outgrowth, pathfinding, and targeting are driven by the growth cone. After reaching the correct target, the growth cone stops growing and forms synapse with the post-synaptic cell.

The structure of the growth cone can be divided into two cytoplasmic domains: the central domain (C-domain) contains microtubules, and the peripheral domain (P-domain) contains F-actin. The P-domain consists of filopodia (bundles of longer F-actin) and lamellipodia (randomly oriented networks of shorter F-actin between the filopodia).

Question 9

The assembly and disassembly of F-actin are orchestrated by a variety of actin-binding proteins (or actin-modulating proteins) that function downstream of Rho-family GTPases (including Rho, Rac, and Cdc42). What are some of these proteins?

Answer 9

Profilin
CapZ
WASp/Scar
Arp 2/3 complex
ADF/cofilin
Gelsolin

Question 10

Assembly and disassembly of F-actin

What is the function of Profilin?

Answer 10

G-actin-binding protein: promotes actin assembly on the barbed-end

Question 11

Assembly and disassembly of F-actin

What is the function of CapZ?

Answer 11

A barbed-end capping protein; stops F-actin elongation

Question 12

Assembly and disassembly of F-actin

What is the function of WASp/Scar?

Answer 12

Activators of Arp2/3 complex

Question 13

Assembly and disassembly of F-actin

What is the function of Arp2/3 complex?

Answer 13

Initiates new F-actin as a branch on the side of an existing filament

Question 14

Assembly and disassembly of F-actin

What is the function of ADF/cofilin?

Answer 14

Stimulates F-actin depolymerisation and debranching at the pointed-end

Question 15

Assembly and disassembly of F-actin

What is the function of Gelsolin?

Answer 15

Binds to the side of F-actin and cuts it into two pieces

Question 16

How does regulation of actin dynamics during neuritogenesis by LIM kinase 1 and cofilin occur? — studying neuritogenesis in vitro

Answer 16

An increase in intracellular cAMP concentration by activation of adenylate cyclase (AC) simultaneously triggers two independent cascades: (1) increased LIM kinase 1 expression, and (2) novel expression of VDCCs. Expression of these proteins may depend on CREB-mediated gene transcription. Cofilin is an actin severing protein - it binds G-actin and depolymerizes actin filaments. The increase in LIM kinase 1 expression induces cofilin phosphorylation, which abolishes its ability to bind and depolymerize actin. This in turn promotes polymerization of actin monomers, resulting in induction of neuritogenesis. Conversely, novel expression of VDCCs negatively regulates LIM kinase 1 expression. VDCCs allows the cells to increase intracellular Ca2+ concentration in response to membrane depolarization. Activation of calcineurin by Ca2+ influx through VDCCs strongly suppresses LIM kinase 1 expression. The decreased amount of LIM kinase 1 induces marked cofilin dephosphorylation, resulting in actin depolymerization and subsequent inhibition of neuritogenesis.

Question 17

Model organisms used to study axon guidance (Drosophila Melanogaster)

What was found about axons crossing the midline?

Answer 17

In the Drosophila CNS, some axons cross the midline (like the red axons in A), but others do not (B). Those that do cross, called commissural axons, cross only once. Comm functions as a switch to control this decision (C). Comm is ON in commissural neurons as they cross, but OFF in ipsilateral neurons and post-crossing commissural neurons. Comm regulates the sensitivity of axons to the midline repellent Slit (yellow in C). It does this by controlling the intracellular trafficking of Robo (blue), the Slit receptor

Question 18

What are the four major families of axonal instructive guidance cues and receptors?

Answer 18

Netrin - Un-5/DCC
Slit - Robo
Ephrin - Eph receptors
Semaphorin - Semaphorin receptors

Question 19

What do signaling pathways downstream of guidance cues converge on?

Answer 19

They converge on Rho GTPases which regulate cytoskeletal organisation

Question 20

How do semaphorins cause growth cone collapse?

Answer 20

At least 2 pathways are elicited by Sema3A. One includes Rac-GEF activation the small GTPase Rac1 that leads to changes in actin, while a second leads to the loss of integrins-mediated substrate adhesion

Collapsin Response Mediator Protein (CRMP) can bind tubulin directly and this provides a connection between axon guidance and microtubule organization in a region of the growth cone that lies behind the actin-rich region.

Question 21

What is the effect of combinatorial regulation of axon guidance receptor signaling?

Answer 21

(a) Combinatorial assembly of semaphorin receptors increases the diversity of ligands to which a family of guidance receptors can respond.

(b) Combinatorial codes of Robo receptors confer different, graded responses to a single Slit guidance cue.

(c) Addition of UNC-5 subunits to a DCC receptor complex can convert the guidance response from attractive to repulsive.

(d) Interactions between receptors allow combinatorial integration of a variety of different guidance pathways.