Regeneration in the optic nerve Flashcards
regeneration in the CNS vs PNS
CNS: poor intrinsic growth potential, inhibitory environment, olidodendrocytes (form myelin) causes inflammatory response (no regeneration), no genetic injury response
PNS: supportive environment, schwann cells form myelin (support regeneration), elevated growth potential, injury response genes
CNS vs PNS developmental differences
CNS: limited intrinsic growth capacity, developmental regenerative decline, polarised transport, poor intrinsic growth potential
PNS: high intrinsic growth capacity, regeneration maintained during development, efficient axon transport, good intrinsic growth potentials
glaucoma
neurodegenerative visual deterioration
synapse loss/axon degeneration/RGC loss
traditional therapy - lowering IOP (no treatment)
primary open area glaucoma (POAG) imbalance of input and output drainage - damages optic nerve axons loses RGCs
where are the optic nerve neurons located?
retina
extension of the CNS
models to study intrinsic control of axon regeneration
in-vitro: neuron specific, less extrinsic factors, less physiological relevance
in-vivo: neuron specific, more extrinsic factors (RGCs synapse elsewhere), more physiological relevance (eye)
cell cultures for the PNS/CNS
PNS - DRG
CNS - rodent brains
in-vitro models
laser axotomy
scratch assay - grow axons and scratch to induce injury
in-vivo models
rodents - cre recombinase, 2 loxP sites combined then the gene of interest is excised
c elegans - laser axotomy
sciatic nerve injury
optic nerve crush
adeno-associated virus (AAV) gene delivery
glaucoma model
AAV
cell specific gene delivery/editing
do not replicate
targets serotype/promoter provides specificity
benefits of using c elegans
contain few neurons, transparent, easy to manipulate
where does axon regeneration occur
axons need to connect with their targets to recover function
CNS - low intrinsic drive (retraction ball)
PNS - high intrinsic drive (growth cones)
growth cones
delineated by cytoskeleton: actin and microtubules
guided by surface receptors and activate signalling pathways, receptors internalised to deliver signals
where are growth cone receptors located
sit in membrane (transmembrane proteins)
growth cone receptor trafficking
ribosome (where protein is made) on ER
receptor inserted into ER
ER vesicle buds off
vesicle interacts with golgi
fuses with the membrane
what controls endosome trafficking
rab11
motor proteins
kinesins anterograde transport (- to +)
dyneins retrograde transport (+ to -)
important factors needed for axon transport
genetic factors
signalling pathways
axon transport
cytoskeleton
cycle of axon regeneration
1) transcription, translation and signalling
2) anterograde axonal transport of receptors
3) growth cone insertion and ligand binding
4) receptor activation
5) retrograde axonal signalling
6) effect of gene expression and transport
what do transcription factors and epigenetics control
RAG (regeneration associated genes)
RAG upregulated in response to injury
epigenetics
DNA wrapped around histones - nucleosomes - chromatin - chromosomes
methylation
tightly wound
RNA pol cannot bind
no gene transcription
histone acetylation
loosely bound
RNA pol binds
gene transcription occurs
PNS injury response
injury in peripheral branch
positive retrograde signalling to cell body
remove HDACs (histone deacetylases)
TFs which promote axon regeneration
CREB
SOX11
KLF7
downregulated during RGC development
TFs which inhibit axon regeneration
KLF4
KLF9
unregulated during RGC development
epigenetic factors which promote regeneration
oct4
sox2
KLF4 (OSK)
require demethylases TET1/2
restores vision loss in mouse glaucoma
ligands and their receptors in the PI3K pathway
NGF - TrkA
BDNF - TrkB
NT-3 - TrkC
PI3K pathway
integrins bind to ECM and promote axon regeneration
PIP2->PIP3 via PI3K
PIP3->PIP2 via PTEN (removes phosphate)
increased PIP3 delta expression in DRG increases axon regeneration
role of PIP3
promotes axon transport
AKT->mTOR
Risk of growth/neurotrophic factors
oncogenic
receptor down regulation/internalisation
not effective/long term
promising targets
glaucoma therapy
TrkB-BDNF AAV gene therapy
self activated TrkB approach - obtain TrkB R fused with farnesylated surface
protrudin
located in ER membrane
moves integrin into axons
mobilises RabII endosomes
it is a kinesin binding ER protein
kinesin I - axonal motor protein along MT
neuroprotective - prevents RGC death
role of the ER
protein synthesis
quality control
calcium regulation
contains protrudin
ARMCX1 role
increases mitochondrial transport to promote regeneration
links mitochondria to axonal motor protein (kinesins)
push/pull of growth cones
pCofilin (inactive) actin severing
cofilin (active) better regeneration
RhoA
small GTPase which opposes regeneration through growth cone actin
activates rho kinase = rigid actin
inhibit RhoA for MT protrusion
microtubules
polymerisation/rescue of tubulin (growth)
depolymerisation/catastrophe (shrinking of tubulin)
what prevents depolymerisation of tubulin
taxol (anti cancer treatment)
parthenolide
Epothilone B
axon morphology
uninjured - straight axons
regenerated axons - branching and turning
