Transcript Biodegradable scaffolds for spinal cord injury

oncomodulin
polyamines
include prostheses
go through Nogo etc more quickly
maybe a bit more matter of fact
highlight debate on gamma secretase
– have two papers in reading list
sema3a
- fred de winter, new nature medicine
paper. check NEW REVIEW folder
LIF CNTF
JAK STAT
future on key things for 2.1 and 2.2
candidates. what do I really want
them to know?
highlight stuff for firsts in reading
list
Degeneration and repair after
spinal cord injury
Dr Lawrence Moon
After this lecture and appropriate
reading you should be able to
1. Describe the neuropathology of spinal cord injury
2. Describe animal models of spinal cord injury
3. Describe possible mechanisms contributing to loss of function
4. Describe current treatments (pharmacological, rehabilitative) and their
shortcomings
5. Describe possible new therapies for spinal cord injury
Tips on answering exam questions
1. Answer the question, not just the part you revised!
2. Show evidence of additional reading and critical thought.
3. Use references (Bob et al., 2015)
Anatomy of
human spinal
cord
Pathology
Contusion
 Compression /
Maceration
 Laceration
 Solid core injuries

Animal models
Weight drop
 Clip, balloon
 Complete transection
 Partial section
 Dorsal
 Ventral (pyramid)
 Solid core injuries?

Spinal cord injury


Prevalence 250,000; incidence 11,000
SCI Information Network, U Alabama,USA
Few acute therapies
 steroids (SCI) – Hurlbert
 Few chronic therapies
 rehabilitation (locomotor)
 adaptation (sexual, bladder, bowel)
 None fully restorative – why is spont. recovery slight?

Why only some spontaneous recovery?
Very few new neurons are born (neurogenesis)
 Spontaneous failure of CNS axon regeneration
 Limited endogenous repair (adult vs neonate)
 Insufficient compensatory plasticity
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Poor intrinsic axon growth
 Pro-growth molecules down-regulated
 Anti-growth pathways switched on
Inhospitable extrinsic environment
 Cysts, cavities
 Fibrotic scar
 Growth-inhibitory molecules (intact & injured)
 Lack of growth factors, permissive substrates
Adult neurons grow very poorly

Goldberg et al., 2002
How might we repair the cord?
injured, some spontaneous changes
combination therapies
Neurotrophins
injured, some spontaneous changes
combination therapies
Neurotrophins promote axon growth
Nerve growth factor
 Brain derived neurotrophic factor
 NT-3
 NT-4/5

Deliver to cell body (Kwon et al,. 2002)
 or to injury site by
 Direct injection (Bradbury et al,. 1999)
 Osmotic minipump (Xu et al., 1995)
 Ex vivo genetically modified cells (Grill et al,. 1997)
 Viral vectors in vivo (Blits et al., 2004)

No studies in injured primates
 Some studies in Alzheimer’s disease
 Side effects (Apfel, 2002)
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Other growth factors
Glial-derived neurotrophic factor (GDNF)
Fibroblast growth factor (FGF)
LIF, CNTF, others...
How do neurotrophins signal?
Neurotrophins are 12kDa
 They form dimers
 p75 binds all four plus ->
 trkA binds NGF
 trkB binds BDNF and NT4
 trkC binds NT3

Classically trks considered high
affinity whereas actually
 NGF to trkA low affinity
 BDNF to trkB low affinity
 although co-expression of p75
increases trkA affinity for NGF

On binding, receptors dimerise and
signal intracellularly….


Chao, MV, 2003 Nat Neurosci Rev
How do neurotrophin receptors signal?
Dominant negative rhoA activity boosts neurite growth
Constitutively active rhoA blocks neurotrophin-induced growth
Gehler et al., 2004 J Neurosci ---- we’ll return to RhoA later....
Growth inhibitors
injured, some spontaneous changes
combination therapies
Inhibitors of growth
• Development – axon growth stops when
• synapses form
• myelin wraps axons
• extracellular matrix nets accumulate
• Critical window for regenerative success also closes
• Partly a geometrical issue, partly a molecular signaling event
• Pettigrew & Crutcher, 1999
Why is adult CNS inhibitory? What is mechanism?
What are the molecules?
How do neurons recognise them?
Extracellular receptors and co-receptors
How does the signal reach the intracellular region?
How does it prevent axon growth?
- linking to intracellular signalling pathways
- linking to axon cytoskeleton
- growth cone collapse
- slowing down / turning away
Thus, what pathways can be exploited to boost axon regrowth?
Translating basic science to the clinic....
Inhibitors of growth
• Purified myelin inhibits axon growth (Caroni & Schwab, 1989)
• Various myelin fractions contain growth-inhibitory molecules
• NI 35, 250 (Caroni & Schwab, 1989)
• IN-1 antibodies raised against NI 250
• promote spreading on myelin
• boost neurite outgrowth (Schwab & Caroni, 1989)
• IN-1 enhances axon regeneration of corticospinal tract
• Schnell et al 94; Bregman et al., 95
• ? proper controls in early studies ?
• confounded by spared axons, doesn’t work in transection (2 papers)
•CST growth after IN-1 treatment in 4 of 5 marmosets (Fouad et al 04)
• Need contusion studies, evaluation of pain
Peptide against Nogo Receptor as a
treatment for SCI
Dorsal hemisection, thoracic, rat
NEP 1-40 promotes axon regeneration (Grandpre et al., 2002)
NEP 1-40 subcutaneous and one week delayed (Li & Strittmatter, 2003)
CST and 5HT growth
Some locomotor benefits
NEP 1-40 intrathecal (Cao et al., 2004 SfN)
Rubrospinal axon growth
Some locomotor benefits
Nogo-A is a key inhibitor of axon growth in myelin
• Publication of partial sequence of peptide recognised by IN-1
• Spillmann et al., 1998
• Race is on! Cloning of Nogo
• rat nogo (Chen et al., 2000; GrandPre et al., 2000)
• human nogo (Prinjha et al., 2000)
• Three isoforms A,B,C. Nogo A is 200kDa, binds IN-1 IgM
• New antibodies 7B12, 11C7 IgG
• Three groups make Nogo-A knockout mice, variable results
• Names to know
• Stephen Strittmatter
• Marc Tessier-Lavigne
• Martin Schwab
• Are there other inhibitors in myelin?
Myelin associated glycoprotein
• Transmembrane and soluble forms
• Purified / recombinant MAG usually (but not always) inhibits
neurite growth (Mukhopadyay et al., 1994; McKerracher et al., 1994)
and depleting / neutralizing MAG improves axon growth.
• Overexpression of MAG in cells limits axon growth (Shen et al.,
1998)
• Name to know – Marie Filbin
Caveat.
• Axons do not regenerate appreciably better in MAG knockout mice
relative to wildtypes (Bartsch et al., 1995; Li et al., 1996)
Oligodendrocyte myelin glycoprotein (OMgp)
• GPi linked protein, 110 kDa
• Found in myelin
• Recombinant OMgp inhibits axon growth
• Wang et al., 2002
• Name to know - Zhigang He
• To my knowledge, knockout has not yet been tested
• All is in vitro
Chondroitin sulphate proteoglycans (CSPGs)
Family of proteins bearing CS glycosaminoglycan side chains
• Neurocan, versican, brevican, phosphacan, etc.
• CSPGs inhibit axon growth in vitro
• Degrading CS using chondroitinase ABC boosts axon growth
• in vitro McKeon et al., 1995 J Neurosci
• after penetrating brain injury (Moon et al., 2001)
and improves outcome following spinal cord injury
(Bradbury et al., 2002)
Other names to know – Jerry Silver, James Fawcett
How are neurons inhibited by these molecules?
CSPGs including versican
Eph A4
EGF-like ligand?
Annexin as receptor for CSPGs?
Ephrin B3
EGF receptor
EGF R kinase phosphorylates EGF R
Calcium increase
Remarkably, Nogo-A, MAG and OMgp all bind the
same receptor complex
• Nogo receptor (NgR1) binds Nogo-A (Fournier et al,. 2001)
• NgR1 binds OMgp (Wang et al., 2002a)
• NgR1 binds MAG (Liu et al., 2002; Domeniconi et al., 2002)
• GPi linked, lacks an intracellular domain, can’t signal on its own
• Nerve growth factor receptor (NGFR) interacts with NgR1 as a coreceptor for Nogo, MAG and OMgp (Wang et al., 2002b; Wong et al., 2002)
= p75
= tumour necrosis factor (TNF) receptor superfamily, member 16
• LINGO-1 (LRR and Ig domain containing, Nogo receptor interacting
protein; Mi et al., 2004)
Striking convergence of three anti-growth molecules with a pro-growth
receptor (NGF R). Chao, 2003 Nat Rev Neurosci 4:299-309
Raises more issues than it settles!
• Do all inhibitory molecules signal through this complex?
• all CSPGs?
• semaphorins?
• Are all parts of the complex necessary for all types of inhibitory
signaling?
• How does ligand / receptor complex binding transfer to an
intracellular signal and thus to the cytoskeleton?
At least some CSPGs don’t signal through p75, NgR
• Versican V2 inhibits neurite growth independent of p75 and NgR
(Schweigreiter et al., 2004)
• Neurons derived from p75 knockouts are inhibited by V2
• RhoA and rac1 are also modulated by V2
• Neurons from p75 knockout mice are still largely inhibited by
myelin – how can this be?
More thorny issues for p75
• Many adult mammalian neurons don’t express p75 yet they respond
to myelin inhibitors (Park et al., 2005 Neuron 45:345-351).
• p75 is not detectable on P8 cerebellar granule neurons by
immunolabeling (Moon, unpublished results).
• Myelin from p75 knockout mice still contains inhibitors of axon
growth in vitro and do not exhibit increased axon regeneration after
spinal cord injury (Song et al., 2004 J Neurosci 24:542-546).
• Is p75 really the key player? What else might act as a receptor for
myelin inhibitors?
TROY can substitute for p75
• Only one other TNFR superfamily member, TROY, binds NgR1
and forms a complex with LINGO-1 (and does so better than p75)
Park et al., 2005 Neuron 45:345-351
Shao et al., 2005 Neuron 45:353-359
• Overexpressing TROY in neurons retards axon growth on myelin
• Axon growth can be increased on myelin by interfering with TROY
(by providing truncated or soluble variants)
Given that Nogo-A binds this receptor
complex, how does it signal intracellularly?
• p75 and TROY both activate RhoA, a small GTPase (Park, Shao)
• p75 is needed to activate RhoA, at least for MAG, Nogo-66 and
OMgp (Yamashita et al,. 2002; Wang et al., 2002)
Rho kinase (Fournier et al., 2003 J Neurosci 23 1416-1423) activates
rho which in turn rigidifies the actin cytoskeleton, causing growth
cone collapse (Yamashita & Tohyama, 2003 Nat Neurosci 6:461467).
Inhibitors of rhoA and (downstream) rho kinase boost axon growth
and enhance axon sprouting and functional recovery after spinal cord
injury (Dergham et al., 2002; Fournier et al., 2003).
? sprouting of collaterals ?
MAG binding to p75 causes cleavage
First alpha then gamma. Blocking secretases reduces inhibition.
Intracellular fragment may be growth inhibitory
Domeniconi et al., 2005
How does this signal no-grow?
Rho = No Grow
PKC = Grow Free
Activation of small GTPase RhoA inhibits neurite
growth (Niederost et al,. 2002)
 After dorsal hemisection of thoracic spinal cord in adult
rats,
 inhibiting Rho using C3 botulinum toxin promotes
axon regeneration in vivo (Dubrueil et al,. 2003)
 inhibiting Rho kinase also promotes axon
regeneration in vivo (Fournier et al,. 2003)

Protein kinase C (PKC) activation is required for MAG
and Nogo to activate Rho and inhibit growth
(Sivasankaran et al., 2004).

How does rho = no grow ?
RhoGDP
to
RhoGTP
MAG binds to p75 and causes activation of Rho (Yamashita et al 2002)
 Gamma secretase requires protein kinase C activation (Domeniconi…)
 Cytoplasmic p75 activates RhoA and results in axon growth inhibition
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Summary of mechanisms for axon growth
Some neurotrophins signal through p75
 Some inhibitors in myelin signal through p75
 … some convergence on p75
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Does p75 “balance” or integrate Go and No-go signals?
 How?
 MAG-induced cleavage of p75 increases ratio of intracellular
fragment…
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Other mechanisms less well understood
 EGF receptor and EGF receptor kinases
 role of p75-like receptors
 role of cyclic AMP
Ephrin B3 in myelin inhibits axon growth
ephrin b3 signals to CST neurons via binding to EphA4 receptor
In vitro study – needs in vivo
EGF receptor phosphorylation...
Screened 400 compounds
Two inhibitors of EGF R kinases
boosted neurite growth of DRGs and
CGNs
Summary
CSPG
Eph A4
EGF-like ligand?
Annexin as receptor for CSPGs?
Ephrin B3
EGF receptor
EGF R kinase phosphorylates EGF R
Calcium increase
Cellular transplantation
Peripheral nerve
 Schwann cells
 Olfactory ensheathing glia
 Macrophages
 Stem cells
 Embryonic
 Adult
 Progenitor cells
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Many have been tried in various models of injury
Olfactory ensheathing glia
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Transection, OEG (Ramon-Cueto et al., 2000)
 Improved climbing
 Serotonergic growth distally
 Not reproduced
Cervical lateral hemisection, acute or delayed
transplant of OEG (Raisman)
 Improved respiration and climbing
 CST growth
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Autologous transplants in dogs (Franklin)
 Naturally occurring injury
 Feasible, safe
Olfactory ensheathing glia
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500+ humans (Huang)
 Fetal cells, largely uncharacterised – OEG?
 No controls
 Few follow-ups for safety or efficacy
 Guest et al., (in press)
Conclusion
Transection + any therapy
Weight bearing stepping on hindlimbs is the exception
Contusion + any therapy
Few studies have been reproduced independently
Things to think about
Very few safety or efficacy studies in primates
Is going straight to humans sensible?
Does it have to be 100% safe?
How much do we need to know?
Next steps
... using this new understanding of mechanism, test new therapeutics
for SCI and stroke…