Lecture 9

Question 1

Outline the difference in the roles of the axon and dendrites in terms of the information they are dealing with

Answer 1

Axons carry information away from the cell body whereas dendrites integrate incoming information

Question 2

What two ways can axons be polarised

Answer 2

Morphologically and molecularly

Question 3

Describe the different cytoskeletal structure seen in axons and dendrites

Answer 3

Axons possess highly polarised microtubules whereas dendrites do no. In axons the microtubules are all orientated in the same way with the + end directed towards the growth cone and the – end towards the soma. In contrast dendrites do show some coordination in microtubule orientation but this is not as uniform as in the axons

Question 4

What factors help to coordinate the microtubules in axons and dendrites into these different orientations

Answer 4

Microtubule associate proteins

Question 5

Describe molecularly how the microtubules are stabilised differently in axons and dendrites and the proteins mediating this

Answer 5

Microtubules are stabilised through cross-linking by microtubule associated proteins which are different in themselves and the way the microtubules are cross-linked. In axons the tau MAP is used to cross-link microtubules whereas in in dendrites MAP2 cross-links the microtubules

Question 6

As well as the cytoskeleton what other region of the cell shows differences depending on the neuronal process and how is this different

Answer 6

The plasma membrane is also compartmentalised through the restriction of molecules by an actin-dependant diffusion barrier

Question 7

How was the compartmentalisation of the plasma membrane in neurons first visualised

Answer 7

Immunostaining for different proteins in the neuron. For example the cell adhesion molecule L1 was found to be localised to the distal axon and growth cone whereas the glutamate receptor (GluR1) was found to be restricted to the soma and proximal axon hillock.

Question 8

Describe the experimentation that determined that localised proteins within the neurons were unable to move across membrane compartments

Answer 8

Optical tweezers were used to move a bead conjugated to antibodies against L1. The antibodies could bind to L1 but the manipulation was unable to move the molecule over the boundary into the cell body. It was later found that it was impossible to move beads between the axon and the cell body although it was possible to move them between the soma and dendrites.

Question 9

Why was a kinesin-GFP fusion protein used to determine how neuronal polarity is established

Answer 9

Kinesin is a + end-directed motor protein and hence is responsible for moving cellular machinery towards the growth cone. By fusing it to GFP you can visualise which of the early immature neurites becomes the axon by watching where the GFP-kinesin fusion protein becomes localised

Question 10

What is significant about the way in which the axon is selected from immature neurites

Answer 10

It appears to be random after a number of the neurites are tried out

Question 11

What is different about the post-translational modifications of the microtubules depending on the neurite

Answer 11

Growth cones and neurites contain dynamic microtubules which are tyrosinated. Stabilised microtubules are present in newly polarised axons and are acetylated

Question 12

How can we experimentally trigger microtubule stabilisation and axon selection

Answer 12

If you treat one of the neurites of the neuron with taxol this will stabilise the microtubules and results in that neurite being selected as the axon

Question 13

Which experiments showed that all neurites have the ability to become the axon

Answer 13

If you allow one of the neurites to be selected and allow it to begin to develop into an axon before severing it one of the other neurites will then be selected to become the axon

Question 14

What are the three theoretical models in determining axon selection

Answer 14

Diffusible inhibitor limiting component and mechanical tension

Question 15

A negative feedback loop controls axon selection T or F

Answer 15

F - there is an additional requirement for a positive feedback loop that acts as an activator

Question 16

Describe the role of HRas and PI3-K on axon selection

Answer 16

HRas and PI3-K provide the positive feedback loop that activates axon selection. Overexpression of either HRas or PI3-K leads to multiple axons being generated.

Question 17

What is seen in terms of the levels of HRas and PI3-K when selecting a neurite to become the axon

Answer 17

HRas is seen to accumulate in growth cones at the point in which the neurite is selected

Question 18

The PI3-K inhibitor (LY294002) prevents the induction of multiple axons T or F

Answer 18

T

Question 19

Explain how HRas and PI3-K interact to trigger axon selection

Answer 19

Activated HRas activates PI3-K by its phosphorylation. PI3-K then acts to catalyse the phosphorylation of PIP2 to PIP3. PIP3 then acts to phosphorylate Akt/PKB which then becomes localised to the growth cone where it acts to promote microtubule stabilisation. PIP3 also acts on Rac which in-turn acts to effect actin dynamics and promote axon elongation. Finally PIP3 also acts to inhibit GSK3β whose normal role is to inhibit microtubule stabilisation through its regulation of MAPs. This inhibition of the inhibition of microtubule stabilisation promotes stability.

Question 20

What is the effect on axon stability if GSK3β is pharmacologically inhibited

Answer 20

This will results in multiple axons

Question 21

What is the significance of Par-3 and SAD kinases in neuronal polarity

Answer 21

PI3-K also affects the Par-3 complex and SAD kinases. The Par-3 complex is localised to the growth cone of neurons and the Par1/2 complex consisting of LKB1 and SAD kinases promote axonogenesis

Question 22

What are the other roles for the portioning defective genes

Answer 22

Par gene mutants fail to development asymmetric cell divisions. These genes are required to establish polarity after sperm entry. Following sperm entry the cortical reaction is induced which imitates polarisation through the antagonistic interactions of the Par-3 and Par-1/2 complex. These interactions affect the downstream microtubule organisation which differentially localises components of the cell this is essential for asymmetric division

Question 23

What are the effects of knocking out the SAD kinases and LKB1

Answer 23

Knockout of both SAD kinases prevents the axon from forming properly. There is a subsequent failure of the tubulin acetylation and hence stabilisation of microtubules. Instead the dynamic microtubules are found to be tyrosinated. Knockout of the LKB1 kinases results in a total failure to make axons

Question 24

What is the specific role of LKB1 in axon specification

Answer 24

LKB1 is required for axon initiation in vivo. LKB1 is phosphorylated by PKA and p-LKB1 activates SAD kinases which promote microtubule associated protein (MAP) stability and axon initiation

Question 25

What can be said about the PI3-K and PKA pathways in axon stabilisation and specification

Answer 25

PKA and PI3K pathways are two redundant and parallel pathways acting to control axon specification. The PI3K pathway leads to an inhibition of an inhibition (stimulation) of axon specification whereas the PKA pathway directly stimulates axon specification

Question 26

Describe how the clinic has led to the identification of additional factors involved in axon polarity

Answer 26

Several genes underlying mental retardation such as lissencephaly have been found to affect cortical development and migration of neuronal progenitors. Other diseases including tubulinopathies indicate the role of the microtubules in axonal polarity. Finally TGF-β receptor mutations that cause milder mental retardation have been implicated as involved in axon polarity. Whilst TGF-β in vitro can initiate axons its knockdown prevents the ability of neurons to form axons both in vitro and vivo

Question 27

What inhibitory guidance cue family of molecules are responsible for the collapse of differing growth cones

Answer 27

Semaphorins

Question 28

Inhibitory guidance cue molecules can be membrane-bound or secretory T or F

Answer 28

T – Sema3A is secreted whilst semamphorins found on retinal axons are membrane-bound

Question 29

What is the effect of semaphorins on the growth cone

Answer 29

Semaphorins can cause growth cones to turn/reorganise by causing a collapsing effect primarily on F-actin

Question 30

What is the significance of sema3A in axon specification

Answer 30

Sema3A is expressed in a gradient from basal to apical and attracts dendrites basally and promotes dendrite formation at the expense of axons in vitro. Sema3A increases [cGMP] and suppresses [cAMP]. This acts to inhibit PKA phosphorylation of LKB1 and GSK3β. Hence axons form where Sema3A is not present