Flashcards
BMS381 Questions - AF's lectures
Cards in this deck (96)
- 1What are the characteristics of axons?L1 surface adhesion molecule expressed
Polarised MTs
Tau crosslinking molecule - 2What are the characteristics of dendrites?GluR1 expression
Less ordered MTs
MAP2 - 3What does taxol do?It causes the artificial stabilisation of a neurite to become an axon
- 4What are the three theories on how only one neurite becomes an axon?1) A diffusible inhibitory molecule stops leading edge formation
2) limiting component - molecule inhibits by consuming essential substances
3) mechanical tension of the membrane and cytoskeleton at the leading edge prevents protrusion elsewhere - 5HRas <--> PI3KA positive feedback loop - they activate each other
High HRas concentration in the growth cone leads to extension
Overexpression of either leads to all of the neurites becoming axons - 6What is the role of PI3K?PI3K leads to increased levels of PIP3 which leads to the increased phosphorylation of Akt
Elevated PIP3 and pAkt levels are found in newly formed axons, but not in naive neurites - 7PIP3 inhibits GSK-3bGSK-3b inhibits microtubule stabilisation
Therefore inihibiting GSK-3b leads to multiple axons - 8What is lissencephaly?Smooth brain - lack of sulci or gyri
Disrupted layering of the cortex during development - due to defective neuronal migration
Pyschomotor retardation, failure to thrive and seizures - 9TGFb receptor mutationsTGFb expressed in VZ of the developing cortex
Initiates and promotes axon growth
KO = embryonic lethal
KD = neurones don't develop (in vivo or in vitro)
Milder mental retardation - 10SemaphorinsInhibitory
Expressed in basal to apical gradient
Attracts dendrites
Suppresses axons - 11What effect does Sema3A have on cGMP and cAMP?increases concentration of cGMP
decreases concentration of cAMP - 12Sema3A also inhibits the phosphorylation of GSK-3b and LKB1LKB1 regulates SAD kinases
axons contain phosphorylated LKB1
phosphorylated LKB1 activates SAD kinases
Loss of either SAD kinases or LKB1 leads to the loss of axons - 13How are the actin molecules arranged in the lamella and filopodia?Lamella - actin crosslinked into nets
Filopodia - actin polarised into actin bundles - 14Treadmilling of actinFilopodial F-actin treadmills in the resting growth cone
Actin is being recycled - added to one end and removed from the other - 15What happens when the growth cone comes across an attractive cue?1) F-actin treadmilling slows
2) F-actin accumulates - stabilising the filopodia
3) Microtubules are dragged into the filopodium - 16What are the two steps for how actin treadmilling may control filopodial extension?1) molecular clutch
when the molecular clutch is engaged, the treadmilling of actin slows
polymerisation of actin is still happening at the other side
forward movement of the filopodium
2) actin-tubulin link captures the MTs in the extending filopodium - 17What is the evidence for the molecular clutch?N-cadherin substrate put in spots on glass
use PALM (photoactivated location microscopy) to track the actin molecules
Slow movement of actin where the N-cadherin is --> arresting actin treadmilling - 18Clutch could be controlling actin crosslinking?Lamella - likely to be actin crosslinking
filopodia - likely to be substrate linking/molecular clutch - 19What are RhoGTPases?A family of molecules that are important regulators of the actin cytoskeleton
- 20What is a GAP?GTPase activating protein
Turns Rho-GTPase off
Activates cleavage of bound GTP to GDP - 21What is a GEF?Guanine neucleotide exchange factor
Puts RhoGTPase into on state by donating a phosphate to convert bound GDP to GTP - 22RhoAStress fibres
RhoGTP - inhibits axon growth
CA* = neurite retraction and collapse
DN- = blocks the collapse response - 23RacLamellopodia
RacGTP stimulates axon growth
DN- = blocks axon formation (but not dendrites)
CA* = also blocks axon growth - 24Cdc42Filopodia
Cdc42 GTP - stimulates axon growth
DN- = blocks formation of axons and dendrites
CA* = also blocks axon growth - 25What does Rac and Cdc42 CA unexpectedly inhibiting axon growth suggest?Suggests that the structures they form must be dismantled before being assembled for growth
e.g.
CA* Rac blocks disassembly - leaving filopodia stabilised and growth is blocked
DN- Rac blocks assembly - preventing growth - 26Are the GTPases instructive or permissive?instructive - would expect cues to turn them on or off directly
(balance of Rac/Rho determines actin cytoskeleton and they 'sit' between the cues and cytoskeleton)
permissive - just have a general effect on axon growth
Accumulating evidence suggests they are instructive - 27semas in fly musclesin flies, semaphorins are expressed in certain muscles to guide innervation by motor neurones
Motor neurones avoid the area that semaphorins are in - 28How can guidance factors effect RhoGTPases?guidance factors can either:
1) bind and modulate the RhoGTPases directly
(e.g. Plexin B directly binds to Rac)
2) bind to the GAPs/GEFs that regulate the Rho GTPases - 29What are plexins?The receptors for semaphorins
- 30Changing the relative levels of Plexin B, RhoA and Rac changes the sensitivity of the motor axons to semaphorinsAxons become more sensitive to semas:
- Increase plexin
- Remove Rac (positive regualtor)
Increasing Rac can make the axons less sensitive. Can restore the wildtype phenotype - 31The balance between free Rac and RhoA determine semaphorin signalling
- 32Regulation of RhoGTPasesRho GTPase family is small and broadly expressed
Whereas the GAP/GEF family is much larger and more restricted in their expression - gives much more specific regualtion - 33Localised calcium influx turns GCsRyanodine induced release of calcium from ER stores can attract growth cones
Ryanodine activates Rac/Cdc42 and suppresses RhoA
Ca2+ affects the RhoA/Rac balance:
Increased Rac + decreased Rho = attraction - 34Artificial induction of local Ca2+ transients can turn a growth coneCan use light sensitive Ca2+ cage
when a light is shone on it, the cage will break releasing the Ca2+ (can see using fluo-3 dye)
initiates GC - 35Ways to modulate the actin cytoskeleton1) filament disassembly
2) branching
3) termination of branch extension via capping proteins
4) filament assembly
5) actomyosin contractility
all of these are potential points of regulation by guidance cues - 36Microtubule invasion occurs before the actin gets there (in response to attractive cue)invasion of MT to contact site happens before cessation of actin retrograde flow
- 37When MTs move in they are thought to bring goodies with them into the filopodiaRho GEFs that interact with MT plus end tracking proteins
Monomeric actin to help with the polymerisation
CRMP - promotes MT polymerisation. and drives WAVE complex proteins into filopodia in kinesin-dependent manner - 38What is netrin?A chemoattractant that floorplate cells of the spinal cord secrete
It has a diffusion length of 0.5mm - 39In mammals, commissural axons tend to turn anteriorly after crossing the midine (floorplate)Could be an AP gradient of an attractant or repellent
Could be either long range diffusible or short range non-diffusible cue
(determined it was a long range diffusible cue) - 40Evidence for diffusible factor:Large explant - still get the anterior turn
Small explant - guidance disrupted, axon stalling - 41Evidence for attractant:In large explants the anterior axons turned posteriorly
(because the cue diffuses into the collagen) - 42WntsDiffusible signalling molecules associated with patterning
Bind to frizzled receptors
Act over distances of ~4mm
(spinal cord at the time of C axon crossing in mice = ~7mm) - 43Wnt4Expressed in an AP gradient in the floorplate
Wnt4-transfected cells turn post-crossing axons in either direction
Receptor KO mice have confused turns post floorplate - axons can no longer decide which way to turn
Wnt KO is embryonic lethal - used earlier on - 44Temporal detectionchange of concentration over time
cells must be able to compare the amount of ligand at two different time points - 45Spatial detectionChange of concentration across the cell (or growth cone)
cells must be able to compare the amount of ligand at two different points on its surface
more important it eukaryotic cells - 46Accuracy of orientationLarger cells can orient more accurately in shallower gradients - it is easier to compare two points if they are further away from each other
- 47What two factors does the accuracy of orientation rely on?1) absolute concentration of the chemoattractant
(most accurate closest to the KD - the concentration where half of the receptors are occupied - gives the greatest capacity for being able to detect changes to concentration)
2) steepness of the gradient - 48what are the two methods to enhance gradient detection?Amplification - to enhance sensitivity of GCs to gradients
Adaptation - to stretch gradients - 49Amplification: Clustering of componentsNeed local enhancement of signal along with inhibition of signal reception in other parts of the cell
Transport of receptors and signalling components to the area where receptors have been activated from other areas
may be mediated by the cytoskeleton (e.g. MTs)
way of steepening the signal - 50experimental evidence in the Dictyosteliuma chemoattractant is detected by a GPCR and activates PI3K --> PIP3 --> PH domain docking site
1) GFP fused to the PH domain proteins
2) can pipette chemoattractant
3) see bands of GFP at the leading edge near the source - 51other evidence of amplification: asymmetrical vesicle transport in GCsfollow the vesicles that are labelled with membrane dye
can see them being moved towards the point of contact with the cues - where the receptors are being activated
vesicles tightly associated with MTs - 52What happens when a cue is found?1) cue encountered by filopodium
2) activates PI3K - increase in cAMP and Ca2+ concentration
3) MTs are stabilised and captured
4) MTs ship in Rho GTPases which determine whether the cue is right for the growth cone
5) Depending on the cue and set of Rho GTPases activated - growth cone extension (or collapse) may follow - 53Evidence for adaptation in response to gradients Frog axon turning assayIsolated neurones from the early frog spinal cord
Netrin was pulsed in to the environment to maintain conc gradient
The neurones were wiggly
The attractive and repulsive turning suggests cycle of desensitisation and sensitisation of the growth cone to netrin - 54To determine whether growth cones adapt to increasing netrin concentrations the axons where pretreated with differing concentrations of netrinLow level of netrin in bath before pulsing
Axon had prior exposure and doesn't turn at 30 minutes
But axon does turn at 120 minutes - resensitised - 55Point source gradientTheoretical range of ~1mm
Exponential gradient
Shape depends on:
1) limited diffusion rate of the molecule
2) a sink - mechanism for removing the molecule at a distance - 56Linear gradientLinear substrate bound gradients longer - ~1cm
normally used in mapping - e.g. retinotectal system - 57Ectopic floorplateAfter commissural axons have crossed the midline, they lose responsiveness to netrin
Axons exposed to ectopic FP after crossing don't respond to it
Axons exposed to ectopic floorplate before crossing the midline respond by turning - 58After crossing:The C axons become sensitive to the semas and slits in the floorplate
The semas and slits are also expressed in the ventral spinal cord - creates a channel that the axons follow - which is why they turn after crossing - 59Robo and Comm ventral nerve mutantsMutants identified in mutant screens
roundabout - no longitudinals
commissureless - no commissures - 60RoboRobo is the receptor for the inhibitory protein slit
The levels of Robo at the cell surface determine whether or not axons cross the midline - 61Robo mutantRobo protein is expressed at high levels in axons that don't cross the midline (ipsilateral neurones)
Robo mutant - slit is no longer detected so the axons go back and forth over the midline - forms roundabout of axons - 62Comm mutantComm is expressed in midline cells and axons that cross the midline
Mutant - Robo is expressed in cells that normally cross the midline - but now they don't - they extend their axons longitudinally
Forced expression - Robo is lost everywhere so results in a Robo-mutant like phenotype - 63Comm's control of RoboComm regulates Robo expression
But it is NOT a TF
It is a membrane-associated protein
In both models, Comm prevents Robo interaction with slit - 64Comm's control of Robo Theory 1) SORTINGPresence of Comm forces the sorting of Robo into late endosomes - Robo is prevents from going into the axon/growth cone
When the axon is over the midline, Comm is turned off and Robo can get to the cell surface - 65Comm's control of Robo Theory 2) CLEARANCEHomophilic interactions between Comm on the midline cells and the GC somehow prevents Robo getting to the surface
- 66In mammalsRobo is expressed at low levels before crossing and high levels afterwards - correlating with high slit sensitivity in post crossing neurones
however no Comm homologue has been found in vertebrates - 67Rig1A 2nd Robo like receptor
Expressed on pre-crossing axons
KO - stops C axons crossing the flooplate - 68What are the two possibilities of Rig1 function?1) Rig1 is a receptor for a floorplate attractant
2) Rig1 prevents premature sensitivity to a floorplate repellent - 69How to differentiate between the two Rig1 models:Precrossing C axons lacking Rig1 are not attracted to the floorplate
But they are attracted to the floorplate if slit function is removed (using soluble Robo) - 70Netrin overrides the effect of slit, but only if Rig1 is presentWT precrossing C axons ignore slit in the presence of netrin
Axons lacking Rig1 are repelled - no longer ignore the slits - 71So what is the function of Rig1?To prevent premature slit sensitivity
- 72Rig1 analogous to CommPrevents Robo (slit receptor) from signalling before the midline - preventing the premature slit response
But do so via different methods
The loss of Rig1 doesn't lead to the upregulation of Robo in precrossing C axons - 73How is the netrin response lost?Xenopus SC turning assay
The axons turn towards netrin
But when slit is in the medium, then stop turning
Specific to netrin - BDNF (diff receptor) is not affected by slit - 74what is DCC?the netrin receptor
- 75how is DCC affected by slit? two models1) ligand-ligand interaction
Slit is competing to stop netrin from binding to DCC
2) receptor mediated signalling
Robo/slit silence DCC/netrin - 76which of the models is correct?The second one -
The silencing of the netrin response (by slit) is due to an interaction between the cytoplasmic domains of DCC and Robo - 77How can you show that it is the receptor interaction that is causing the loss of netrin response via slit?Make a hybrid version of Robo with the same intracellular domains but the extracellular domain for NGFR
now find that NGF can substitute for slit and block the netrin turning response
Because NGF doesn't normally do this - favour of the second model - 78SummaryBefore crossing the midline Rig1 silences Robo - therefore netrin can attract the C axons
After crossing Robo interferes with DCC/netrin
Axons become repelled by the Slit in the midline cells - 79Responses to guidance cues are affected by the concentrations of cAMP/cGMPIncreased cAMP = attraction
Increased cGMP = repulsion
Reversal occurs over such a narrow range that it is essentially a switch rather than a graded response - 80Some repulsive cues Nogo and MAG work by...Inhibiting cAMP rather than increasing cGMP
- 81Where is the highest calcium concentration seen?On the side of the growth cone that has experienced the cue - regardless of whether it is a attractive or repulsive cue
- 82What happens when an attractive cue is encountered?1) increase [cAMP]
TRPC channels let in Ca2+ or IP3 levels elevated
2) Ca2+ and IP3 trigger further Ca2+ release from the IC stores
3) Store release of Ca2+ feeds back to further elevate the levels of cAMP and Ca2+
4) this positive feedback loop leads to high Ca2+ levels which activates growth cone extension - 83What happens when an repulsive cue is encountered?1) increase [cGMP]
2) cGMP inhibits Ca2+ release from IC stores
3) cAMP levels don't get amplified
4) Low amplitude Ca2+ activates growth cone repulsion and collapse - 84What are the factors that contribute to CNS axon not regenerating?- Presence of inhibitory factors in the myelin (Nogo and MAG)
- Takes longer for the macrophages to clear away the debris
- Failure to activate the growth promoting program in injured neurone
- Formation of a glial scar (e.g. astrocytes) - physical barrier to axon growth - 85Can CNS axons regenerate?CNS axons do have the capacity to regenerate if:
1) they are given the appropriate substrate
2) the right genes are activated - 86Cut the peripheral branch of sensory axons a few days earlier than cutting the central branch of the dorsal columnsubstrate/factors in the periphery can activate a regrowth program that can ignore CNS inhibition
Leads to central and peripheral branch regeneration - 87What are the factors in the CNS myelin that inhibit growth?MAG
OMgp
Nogo
activate RhoA
inhibit GC growth - 88RhoA leads to growth cone collapseCan try to develop therapeutic strategy to suppress the inhibitory effects of myelination during regeneration
- 89What are the three strategies?1) Db-cAMP - on cAMP levels
2) ROCK inhibitor
3) Ibuprofen - on RhoA activation - 901) Db-cAMP - cell permeable cAMP analoguePromotes the regrowth of central sensory afferents
1) preconditioning peripheral lesion elevates cAMP levels
2) Db-cAMP allows the growth of sensory fibres in vitro in the presence of inhibitory CNS myelin
3) Db-cAMP injection into the DRG prior to lesion mimics the peripheral lesion - can enhance regrowth through subsequent dorsal column lesion - 912) ROCK inhibitorAdministration at the same time as lesion to dorsal column promotes regrowth
Claim to see motor control recovery
Controversy:
animals with saline recovered after 30 days,
lesion relatively mild - 923) ibuprofen - RhoA inhibitorMild enhanced recovery of motor function in mice with lesion
increased BBB score, longer stride lengths and shorter stride widths compared to injured but not treated mice
Naproxen doesn't work - highly specific - not to do with the COX enzyme pathway
Activates a TF that inhibits RhoGEF so suppresses RhoA activation - 93PTENDid conditional knockouts of major growth genes
Only PTEN KO significantly enhanced neuronal survival and allowed retinal ganglion cell regeneration in mice - 94PTEN regulates the mTOR pathwaymammalian target of rapamycin
Rapamycin is immunosuppressant for organ transplant and is an anti-proliferative
Rapamycin inhibits mTOR function
Want to turn on the mTOR pathway to regrow axons - 95PTEN deletion enhances spinal axon regrowthsome of these regenerated axons formed synapses - but no functional recovery was reported
Need a drug that inhibits PTEN - 96—
Information
- Description
- Flashcards on BMS381 Questions - AF's lectures, created by rachel-chads on 01/02/2016.
- Created by
- rachel-chads
- Views
- 0
- Rating
More on Axon growth and guidance