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future on key things for 2.1 and 2.2 candidates. what do I really want them to know? highlight stuff for firsts in read

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future on key things for 2.1 and 2.2 candidates. what do I really want them to know? highlight stuff for firsts in read

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  1. for future.cAMP / PKAmore on actin cytoskeleton and collapse!oncomodulinpolyaminesinclude prosthesesgo through Nogo etc more quickly maybe a bit more matter of facthighlight debate on gamma secretase – have two papers in reading listsema3a- fred de winter, new nature medicine paper. check NEW REVIEW folderLIF CNTFJAK STATgp130ATF3?conditioning lesion

  2. 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

  3. Degeneration and repair after spinal cord injury Dr Lawrence Moon

  4. 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 After this lecture and appropriate reading you should be able to 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)

  5. Anatomy of human spinal cord

  6. Contusion • Compression / Maceration • Laceration • Solid core injuries Pathology Animal models • Weight drop • Clip, balloon • Complete transection • Partial section • Dorsal • Ventral (pyramid) • Solid core injuries?

  7. 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? Spinal cord injury

  8. 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 Why only some spontaneous recovery?

  9. Adult neurons grow very poorly • Goldberg et al., 2002

  10. injured, some spontaneous changes combination therapies How might we repair the cord?

  11. injured, some spontaneous changes combination therapies Neurotrophins

  12. 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) Neurotrophins promote axon growth

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

  14. 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 neurotrophins signal?

  15. 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....

  16. injured, some spontaneous changes combination therapies Growth inhibitors

  17. 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 Inhibitors of growth

  18. 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.... Why is adult CNS inhibitory? What is mechanism?

  19. 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

  20. 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 Peptide against Nogo Receptor as a treatment for SCI

  21. 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? Nogo-A is a key inhibitor of axon growth in myelin

  22. 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) Myelin associated glycoprotein

  23. 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 Oligodendrocyte myelin glycoprotein (OMgp)

  24. 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 Chondroitin sulphate proteoglycans (CSPGs)

  25. CSPGs including versican Eph A4 EGF-like ligand? How are neurons inhibited by these molecules? Annexin as receptor for CSPGs? Ephrin B3 EGF receptor EGF R kinase phosphorylates EGF R Calcium increase

  26. 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 Remarkably, Nogo-A, MAG and OMgp all bind the same receptor complex

  27. 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? Raises more issues than it settles!

  28. 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? At least some CSPGs don’t signal through p75, NgR

  29. 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? More thorny issues for p75

  30. 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) TROY can substitute for p75

  31. 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 ? Given that Nogo-A binds this receptor complex, how does it signal intracellularly?

  32. 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?

  33. 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). Rho = No Grow PKC = Grow Free

  34. 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

  35. 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 Summary of mechanisms for axon growth

  36. Ephrin B3 in myelin inhibits axon growth ephrin b3 signals to CST neurons via binding to EphA4 receptor In vitro study – needs in vivo

  37. Screened 400 compounds Two inhibitors of EGF R kinases boosted neurite growth of DRGs and CGNs EGF receptor phosphorylation...

  38. CSPG Eph A4 EGF-like ligand? Summary Annexin as receptor for CSPGs? Ephrin B3 EGF receptor EGF R kinase phosphorylates EGF R Calcium increase

  39. Peripheral nerve • Schwann cells • Olfactory ensheathing glia • Macrophages • Stem cells • Embryonic • Adult • Progenitor cells • Many have been tried in various models of injury Cellular transplantation

  40. 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

  41. 500+ humans (Huang) • Fetal cells, largely uncharacterised – OEG? • No controls • Few follow-ups for safety or efficacy • Guest et al., (in press) Olfactory ensheathing glia

  42. 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? Conclusion

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

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