1. Nerve injury & Motoneurons Core concepts: Loss of trophic support Excitotoxicity

2. Causes of motoneuron death NOCD Early postnatal injury Disease SMA ALS PMN

3. Motoneuron disease ~5000 affected in UK –1-2/100,000 new cases p.a. Affects men > women Onset >40 years of age –55-65 yrs old most commonly affected Genetic –Not possible to prevent onset Symptoms Progressive muscle weakness & wasting Hands, arms & legs usually affected first May get spasticity, painful cramps & loss of balance Affects vocal & respiratory muscles

4. Clinical syndromes: Spinal Muscular Atrophy Hereditary condition defect in SMN gene (Chromosome 5q12.2-q13) Insufficient peripheral motor sprouting Increased motoneuron activity – excitotoxicity?

5. Clinical syndromes: Age related motoneuron loss Senile Muscular Atrophy Affects 15% of elderly population 10-20% motoneuron loss Motoneurons show signs of damage –  CGRP, GAP43, p75 –  trkB, trkC;

6. ALS Affects UMN/LMNs Mechanisms – 10% Hereditary (SOD1) –Oxidative stress –EAA toxicity Glial EAAT2 abnormal  Glutamate in CNS  Glutamate in CSF NOS, Cox 2 induced Defect in RA pathway

7. Goals of neuromuscular disease research 1.Prevent death 2.Maintain phenotype 3.Repair neuronal damage

8. BDNF, NT3, NT4 GDNF LIF, CNTF FGF5, bFGF IGF1, IGF2 BDNF, NT3, NT4 GDNF, NTN, PSN LIF, CNTF, CT1 FGFsRA HGF IGF1 Trophic factors and motoneuron survival

9. NOCD & trophic factor knockout NGF- trk ANo motoneuron loss BDNF/NT4 - trk BNo motoneuron loss NT3 - trk CNo motoneuron loss p75No motoneuron loss BDNF-NT4 double KONo motoneuron loss trk B/C double KONo motoneuron loss CNTFNo motoneuron loss CNTFR  40-50% LOSS LIFR  LOSS gp13040% LOSS GDNF- GFR  1 20-40% LOSS GFR  2No motoneuron loss c-RETSignificant loss

10. Motoneuron survival depends on age and post-operative survival time P0 90% P3 80% P4 30% P5 0%

11.  YES  Neonatal motoneuron death depends on lesion site Yes Motoneuron loss also depends on duration of deprivation P0 axotomy: 1% survival P0 crush: 10-30% survival Delayed reinnervation P5 & P10 crush 60% survival

12. Is motoneuron death due to axon damage per se or target deprivation? Motoneuron loss is regulated by target deprivation can be induced by NMJ blockade at birth (maintains MN immaturity) injury induced release of glutamate (kills immature MNs) Can be mimicked by NMDA injection

13. Muscle induced neuro- transmitter release Motoneuron death is regulated by target contact Growing mode Transmitting mode Ach

14. Prevention of neuromuscular interaction No induced neuro- transmitter release Motoneuron death is regulated by target contact Growing mode Preserved immature state Death by glutamate excito- toxicity

15. Motoneuron maturation Growing neurone transmitting neurone

16. Neonatal axotomy: effects on surviving neurons Biochemical  expression, Reg2, HSP27, GFR  1, p75, CGRP, CB, gp130, trkC, CNTFR   expression GAP43, c-Jun, NOS, NR1, NR2B, GAL, mRNAs for LIF, trkB, c-RET Physiological  neuronal activity abnormal reflex patterns  dendrite number, altered morphology

17. Pharmacological manipulations that rescue dying motoneurons 1.Neurotrophic support 2.Preventing excitotoxicity Important Outcomes 1.Permanent survival 2.Rescued motoneurons must reinnervate muscles 3.Muscles must develop adequate force on reinnervation 4.Spinal circuits must be re-established

18. Neurotrophic support 1 week 2 weeks combination nerve + s.c P3 sciatic cut + single dose NTF treatment to injury site

19. Neonatal neurotrophic support Transient rescue BDNF < 3 weeks NT3 < 2 weeks NT4 < 1week CNTF < 2 weeks LIF < 2 weeks GDNF combinations Restore ChAT levels Permanent rescue GDNF (AAV) Deleterious NGF (activity dependent) High dose BDNF –receptor desensitisation or activity dependent

20.  NO/ YES -delayed YES  Adult motoneuron death NO

21. Adult nerve injury Loss of normal function Loss of reflex function Soma atrophy Motor c.v.   ChAT  transmitter receptors No cell death* –VRA –Repeated nerve injury Regeneration  GAP43, c-Jun  CGRP, GAL,  REG2  HSP27  c-Ret, GFR  1, LIFR, p75, CNTFR , trkB  gp130,  trkC

22.  trkB GFR  1 p75   NGF, BDNF, GDNF, NT4 * **  CNTF,  NT3 Regeneration GAP43, CGRP Tubulin  p75  apoptosis in Schwann cells   regeneration Exogenous NTFs reverse effects of injury & aid regeneration

23.  trkB GAP43 p75   BDNF, GDNF, NT4 * ** or  Schwann cell derived trophic factors Regeneration mode Adult injuries that kill:Repeated nerve injury  EAA toxicity  SA  Immature state

24.  trkB GFR  1 p75 NOS  Exogenous BDNF, GDNF prevents death * ** Regeneration possible  GAP43, CGRP, Tubulin Adult injuries that kill: Avulsion EAA toxicity No transport of Schwann cell derived NTFs No access to Schwann cells Activation of p75 death pathway?

25. Rescuing dying motoneurons Preventing excitotoxicity Riluzole NOS inhibitors Glutamate inhibitors MK801

27. Clinical trials in ALS CNTF severe side effects: fever, chest pains, muscle weakness, herpes virus activation BDNF – Major side effects. Pain. IGF1 – data not conclusive; drug well tolerated GDNF, NTN – not tested TCH346 (anti apoptotic) – failed phase 2 trial Riluzole – activity blocker Retinoic acid – RALD2 & RAR 

28. References Lowrie MB & Vrbova G 1992 TINS 15: 80- 84 Greensmith L & Vrbova G 1995 Neuro- muscular Disord 5: 359-69 Vejsada R et al 1995: EJN 7:108-115 Ma J et al. 2001 139: 216-223