for future. cAMP / PKA more on actin cytoskeleton and collapse! 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 gp130 ATF3? conditioning lesion

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 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 changescombination therapies

Neurotrophins injured, some spontaneous changescombination 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)

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 changescombination 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....

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 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 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:

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: ). 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: ). 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: Shao et al., 2005 Neuron 45: 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 ) activates rho which in turn rigidifies the actin cytoskeleton, causing growth cone collapse (Yamashita & Tohyama, 2003 Nat Neurosci 6: ). 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 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 ? 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 RhoGDP to RhoGTP

Summary of mechanisms for axon 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 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 Many have been tried in various models of injury

Olfactory 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 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 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…