Lecture 22- Axon guidance V

Question 1

What are the main repulsive cues we discuss?

Answer 1

-Slit and Robo - slits are originally found in drosophila, secreted proteins, three versions Slit 1-3=4 receptors -Three vertebrate Slit ligands Slit1, Slit2 and Slit3 - Four Robo (roundabout) receptors Robo1, Robo2, Robo3 (Rig1) and Robo4 (magic roundabout)

Question 2

What is the slit downstream signalling?

Answer 2

• Slit:Robo downstream signalling
• robo= activates kinases= all leads down to DCC

Question 3

What do slit proteins do in vitro?

Answer 3

-Slit proteins repel axons in vitro, act as a chemorepellent -all slits: Slit 1, 2 and 3 act as a chemorepellent to the axons

Question 4

In commisural axon guidance once the axons get to the flooorplate, the midline, why don’t they stay there?

Answer 4

-something must force them out! -they don’t stay in the floor plate as the Slit 1,2,3 are there and the Robo receptors are on the growth cone of the commissural axon -in drodophila= the ventral midline= floor plate -Slits are crucial in the axon guidance across the midline of the spinal cord

Question 5

What happens in Slit mutants in Drosophila?

Answer 5

-the axons don’t exit the midline as there is nothing to push them out

Question 6

What happens in Robo mutants?

Answer 6

-the axons enter the ventral midline region and exit but come back in again and then repeat= roundabout= hence the name

Question 7

What is the normal movement of the commissural axons in Drosophila?

Answer 7

-

Question 8

Which specific Robos control the axon repulsion at the midline (Drosophila)?

Answer 8

-Robo1 and Robo2 jointly control axon repulsion at the midline -when knockout both robo1 and robo2 more severe than just one of them, similar to slit

Question 9

Is the repulsion of commisural axons at the midline the same in mice as in Drosophila?

Answer 9

-yes -Robo1 and Robo2 jointly control axon repulsion at the midline (mice)

Question 10

What happens in robo mutant embryos in vertebrates?

Answer 10

-Axons aberrantly cross the midline in vertebrate robo mutant embryos -Slit expressed in the midline binds to Robo expressed on the growth cone and repels the axons from the midline

Question 11

Why aren’t the axons stopped from entering the midline initially?

Answer 11

• 2 isoforms of Robo3 exist (Robo3A/3.1 and Robo3B/3.2)
• different functions
• Robo3A/3.1 is expressed before axons cross and desensitize axons to Slit allowing the axon to enter the midline
• Robo3B/3.2 is expressed as axons cross and allows Slit to activate Robo 1, which repels the axons from the midline

Question 12

What controls the midline crossing?

Answer 12

-Robo3 isoforms -Overexpression of Robo3A/3.1 allows axons to cross (and recross) -Overexpression Robo3B/3.2 repels axons -expression depends on what the axons are doing

Question 13

What are 3 more repulsive cues?

Answer 13

-Semaphorins (the ligand), -the receptors: Plexins and Neuropilin

Question 14

How many semaphorin ligands are there?

Answer 14

• 21 Semaphorin ligands (Sema3A-3G, Sema4A-4G, Sema5A-5B, Seman 6A-6D and Sema7A)

Question 15

Are semaphorins membrane bound or secreted?

Answer 15

-Semaphorins can be membrane bound or secreted

Question 16

What do the Semaphorins bind to?

Answer 16

-Semaphorins bind to receptors: Plexins (Plxn) (Plexin A,B,C,D) and Neuropilins (Nrp1 and Nrp2)=co-receptor= must be bound to plexin= then can signal, only sema3 that bind to it)

Question 17

What are the Neuropilin?

Answer 17

• Neuropilins act as co-receptors to plexin and only bind to class 3 Semaphorins (Sema3A-3G)

Question 18

What happens when axons are exposed to Sema3A?

Answer 18

-Exposure to Sema 3A causes growth cone retraction

Question 19

What are spinal cord axons repelled by?

Answer 19

-Spinal cord axons are repelled by Sema3B and Sema3F -Guidance of axons across the midline of the spinal cord – semaphorin is also involved

Question 20

What happens to neuropilin 2 knockout (-/-) animals?

Answer 20

-Axon growth across the midline is disrupted in Neuropilin 2 knockout (-/-) animals -Axon growth in other areas is altered in neuropilin knockout mice: trigeminal projections, spinal nerve projections, limb projections

Question 21

Are some molecules both chemoattractants and chemorepellents?

Answer 21

-yes -Netrin-1 can be attractive or repulsive depending on the type of neuron and the receptor expressed - DCC (Unc40) attractive signalling - Unc5 repulsive signalling

Question 22

How can netrin be repulsive?

Answer 22

-Expression of Unc-5 protein converts netrin-1 attraction to repulsion

Question 23

Where does netrin-1 act as a repulsive cue?

Answer 23

-Netrin-1 repels trochlear ventral hindbrain motor neurons from the indbrain -netrin as a repulsive cue in vivo -this is in the hindbrain, floor plate still there -trochlear motor neurons from the ventral part of the hindbrain section -axons move dorsally and cross over -the netrin in the floor plate is pushing them away from the floor plate

Question 24

How did they check that it was Unc5 involved in the repelling behaviour in motor neurons?

Answer 24

-Motor neurons are repelled by Netrin-1 released from the vertebrate floor plate -to check that it is via Unc5 that netrin has the repulsive activity -figured it out via putting in an Unc5 antibody -Addition of Unc5 blocking antibody inhibits the ability of netrin-1 to activate Unc5 and therefore motor neuron axons grow towards the floorplate

Question 25

What does the neuron do when expressing DCC and when expressing Unc5?

Answer 25

• if have a DCC expressed (also called Unc40) then will grow to the source of Netrin
• if have Unc5 and DCC will grow away from the netrin

Question 26

How do Unc5 and DCC interact?

Answer 26

• Unc5 and DCC form a receptor complex that mediates Netrin repulsion
• the receptors form dimers
• when netrin binds to DCC will form a homodimer= havce 2 receptors bindibg to each other
• Unc5 =will form a dimer with DCC that results in repulsion
• if lot of unc5= can dimer with itslef= and also repulsion

Question 27

Does netrin-mediated repulsion only require Unc5 in some cases?

Answer 27

-yes

Question 28

What is the Netrin downstream signalling?

Answer 28

• the Unc5 signalling pathway is not know as well -things in the growth cone also extremely important
• PKA (protein kinase A)- lot of roles, with netrin and DCC= promotes teh traficking of the DCC receptor to the membrane= puts more DCC on the surface of the cell= more likely for the growth cone to be attracted to the netrin
• PKA can also activate cAMP amd cGMP= lead to changes in calcium levels in the cell= that leads to changes in the cytoskeleton
• if have high levels of calcium in a cell= then will get growth cone atttraction
• low levels of calcium= repuslion
• it is key the amount of cGMP and cAMP in a cell

Question 29

Can cyclic AMP affect axon growth?

Answer 29

-Cyclic AMP levels in the growth cone can also alter its response to Netrin-1 -high cAMP= lot of calcium -on right= low cAMP= low lwvels of calcium= the growth cone is repelled from the netrin -so it is not just the extracellular cues or the receptors, it is the intracellular protein that matters as well - Netrin-1 acts as an attractive cue when cAMP levels are high in the growth cone - Netrin-1 is repellent when cAMP levels are low in the growth cone

Question 30

How can Eph-ephrin signalling be repulsive?

Answer 30

- activation of Eph receptor results in receptor phosphorylation, Rho GTPase activation and remodeling of the actin cytoskeleton (forward signalling) - ephrin’s are also able to signal (reverse signalling) (kinase activation and other signalling pathways) - both signalling events can happen simultaneously -forward= repulsive

Question 31

How can Eph-ephrin affect the growth cone in different ways?

Answer 31

-Bi-directional signalling by Eph-ephrin activation can have different affects on the growth cone -Addition of EphA7-Fc to neurons in culture activates ephrin on the growth cone and causes growth cone spreading Addition of ephrinA-Fc to neurons activates EphA receptors on the growth cone and causes growth cone retraction

Question 32

How do retinal ganglion cell axons react to ephrinA5?

Answer 32

-Retinal ganglion cell axons are repulsed by ephrinA5 in a protein stripe assay -stripe assays= stripes= of different proteins, tests what they will grow on

Question 33

What is the general rule with Ephrin-Eph signalling?

Answer 33

• if have ephrin on one cell you cause it to be attracted to that cell
• if you activate eph= make it repulse from the cell

Question 34

How do retinal ganglion cells react to EphA7?

Answer 34

-EphA7 activation of ephrin also repulses retinal ganglion cells -Eph-ephrin signalling can be attractive or repulsive depending on the cell type and the receptor/ligands present -epha5 is repulsive here -it’s not just cut and dry, that one cue will cause a response, it depends on the cell itself, where the cell is etc…

Question 35

What is the summary?

Answer 35

• Slit/Robo signalling and Semaphorin/Neuropilin signalling are chemorepulsive
• Some environmental cues may be chemoattractive and chemorepulsive depending on the cell type and the ligands/receptors present (eg. Netrin/Unc5, Eph/ephrin)