for future.for future.cAMP / PKAcAMP / PKA

more on actin cytoskeleton more on actin cytoskeleton and collapse!and collapse!oncomodulinoncomodulinpolyaminespolyamines

include prosthesesinclude prosthesesgo through Nogo etc more go through Nogo etc more

quickly quickly maybe a bit more matter of maybe a bit more matter of

factfacthighlight debate on gamma highlight debate on gamma secretase – have two papers secretase – have two papers

in reading listin reading listsema3asema3a

- fred de winter, new nature - fred de winter, new nature medicine paper. check NEW medicine paper. check NEW

REVIEW folderREVIEW folderLIF CNTFLIF CNTFJAK STATJAK STAT

gp130gp130ATF3?ATF3?

conditioning lesionconditioning lesion

future on key things for 2.1 future on key things for 2.1 and 2.2 candidates. what and 2.2 candidates. what do I really want them to do I really want them to

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reading listreading list

Degeneration and repair Degeneration and repair after spinal cord injuryafter spinal cord injury

Dr Lawrence Moon

After this lecture and After this lecture and appropriate reading you appropriate reading you should be able toshould be able to

1. Describe the neuropathology of spinal cord injury2. Describe animal models of spinal cord injury3. Describe possible mechanisms contributing to loss of function4. Describe current treatments (pharmacological, rehabilitative) and their shortcomings5. Describe possible new therapies for spinal cord injuryTips on answering exam Tips on answering exam questionsquestions1. 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 Anatomy of human human spinal cordspinal cord

PathologyPathology Contusion Compression / Maceration Laceration Solid core injuriesAnimal modelsAnimal models Weight drop Clip, balloon Complete transection Partial section

Dorsal Ventral (pyramid)

Solid core injuries?

Spinal cord injurySpinal 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 Why only some spontaneous recovery?recovery? Very few new neurons are born (neurogenesis) Spontaneous failure of CNS axon regeneration Limited endogenous repair (adult vs neonate) Insufficient compensatory plasticity

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 poorlyAdult neurons grow very poorly

Goldberg et al., 2002

How might we repair the How might we repair the cord?cord?

injured, some spontaneous changes combination therapies

NeurotrophinsNeurotrophinsinjured, some spontaneous changes combination therapies

Neurotrophins promote axon Neurotrophins promote axon growthgrowth 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)

Other growth factorsGlial-derived neurotrophic factor (GDNF)Fibroblast growth factor (FGF)LIF, CNTF, others...

How do neurotrophins signal?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 growthConstitutively active rhoA blocks neurotrophin-induced growthGehler et al., 2004 J Neurosci ---- we’ll return to RhoA later....

Growth inhibitorsGrowth inhibitorsinjured, 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-receptorsHow 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....

• 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

Inhibitors of growth

Peptide against Nogo Peptide against Nogo Receptor as a treatment for Receptor as a treatment for SCISCIDorsal 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 growthSome locomotor benefits

NEP 1-40 intrathecal (Cao et al., 2004 SfN)Rubrospinal axon growthSome 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 versicanEph A4EGF-like ligand?

Annexin as receptor for CSPGs?Ephrin B3EGF 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 co-receptor 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 p75TROY 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 45:345-351Shao 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 Given that Nogo-A binds this receptor complex, how does it receptor complex, how does it signal intracellularly?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 6:461-467).

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 MAG binding to p75 causes cleavagecleavage

First alpha then gamma. Blocking secretases reduces inhibition.Intracellular fragment may be growth inhibitoryDomeniconi et al., 2005

How does this signal no-grow?

Rho = No Grow PKC = Rho = No Grow PKC = Grow FreeGrow 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 ?How does rho = no grow ?

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

RhoGDPto

RhoGTP

Summary of mechanisms for Summary of mechanisms for axon growthaxon growth Some neurotrophins signal through p75 Some inhibitors in myelin signal through p75 … some convergence on p75 Does p75 “balance” or integrate Go and No-go signals? How? MAG-induced cleavage of p75 increases ratio of intracellular fragment…

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 Ephrin B3 in myelin inhibits axon growthgrowth

ephrin b3 signals to CST neurons via binding to EphA4 receptor

In vitro study – needs in vivo

EGF receptor phosphorylation...EGF receptor phosphorylation...Screened 400 compounds

Two inhibitors of EGF R kinases boosted neurite growth of DRGs and CGNs

SummaryCSPGEph A4EGF-like ligand?

Annexin as receptor for CSPGs?Ephrin B3EGF receptor

EGF R kinase phosphorylates EGF R

Calcium increase

Cellular transplantationCellular transplantation Peripheral nerve Schwann cells Olfactory ensheathing glia Macrophages Stem cells

Embryonic Adult

Progenitor cells

Many have been tried in various models of injury

Olfactory ensheathing gliaOlfactory ensheathing glia

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

Autologous transplants in dogs (Franklin) Naturally occurring injury Feasible, safe

Olfactory ensheathing gliaOlfactory ensheathing glia 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 primatesIs going straight to humans sensible?Does it have to be 100% safe?How much do we need to know?

Next stepsNext steps... using this new understanding of mechanism, test new therapeutics for SCI and stroke…