Overview

 Introduction Wallerian Degeneration of  Background Information Injured Axons and Synapses is  Aims Delayed by a Ube4b/Nmnat  Results  Discussion Chimeric  Future Research Mack T, Reiner M, Beirowski B, Weiqian M, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L,  Questions Fernando F. S, Tarlton A, Andressen C, Addicks K, Magni G, Ribchester R. R, Perry V. H and Coleman M. P.

Introduction - Background Introduction - Background

 Distal axons of injured neurons usually undergo Wallerian  s degeneration 24-48 hours after injury. A candidate Wld gene was identified on  In C57BL/Wlds mice, injured axons can survive several weeks 4 (mouse) following injury.  This suggests that these mice have a protective mechanism specific to the  It is a chimeric gene containing coding axons, and that this protective factor must be present in the axon prior to any injury occurring as the distal part of the axon is separated from the cell body regions for Ube4b/D4Cole1e in the mouse and cannot benefit from new synthesis.  D4Cole1e was found to be equivalent to the  Wallerian degeneration is implicated in several human neuropathologies. human enzyme Nmnat.  These include ALS, multiple sclerosis and traumatic disorders such as injury to the spinal cord  Both were found to be expressed in  An understanding of the mechanisms controlling the slow Wlds mice, strongly suggesting that this Wallerian degeneration exhibited by the C57BL/Wlds mouse could lead to the new therapeutic targets for such diseases. chimeric gene is the Wlds gene

Introduction - Aims Transgenic Mice Generated

 Hypothesis: the Ube4b/Nmnat chimeric gene  4 lines of transgenic mice were generated for is the Wlds gene and produces the slow this study: 4839, 4830, 4858 and 4836. Wallerian degeneration phenotype observed  In each line, the expression level of the in the mutant mice. chimeric gene had been altered.  To test this, the chimeric gene was  The lowest level was expressed in 4839; 4830 expressed in transgenic mice. and 4858 showed a medium level of gene  Several strains of mice were produced, each with expression (only 4830 is further discussed in the a different expression level of the chimeric gene. paper); 4836 had the strongest expression which is almost identical to Wlds mouse. Results Results

Structural Preservation of Transected Axons  Structural preservation was investigated 3-5 days following a unilateral lesion to the sciatic nerve.  Electron microscopy showed that the vast majority of axons in the 4836 mouse had preserved cytoskeletons 5 days after injury  Successful replication of the Wlds phenotype.  WT mice showed clear signs of degeneration in myelinated and unmyelinated axons.  Partial protection was observed as expected in 4830 axons

Results Results

Functionally Competent Motor Axons and Synapses  Tested whether the motor axons were still functional as well as structurally preserved.  Using intracellular recordings, the response of the muscle to nerve stimulation and the generation of action potentials were recorded  No evidence of motor axons still functioning in WT mice.  In transgenic mice, there was evidence of functioning motor axons and synapses for at least three days after injury.  In homozygous 4836 mice, nerve stimulation led to a functional response in 80% of muscle fibres 3 days after injury.  In heterozygous 4836 mice and 4830 mice, the percentage of muscle fibres which responded to nerve stimulation after injury were significantly lower than the 4836 mouse, and the duration after injury which the axons and synapses appeared functional was also reduced.

Results Results

 Functional motor axons and synapses were visualised using immunostaining methods  Homozygous 4836 mice demonstrated synaptic vesicle recycling 5 days after injury, indicating synaptic transmission is still able to occur.  Homozygous 4836 mice also showed a high proportion of endplates fully occupied 5 days after injury.  Proportion of endplates occupied were reduced in hemizygous 4836 mice and in 4830 mice. Results Results

Protection Depends on Wld Protein Expression Levels  The level of Wld protein expression in the mutant strains was quantified using a Western blot analysis.  Homozygous 4836 mutant mice showed similar expression of the protein to the Wlds mouse, again confirming that the Wlds phenotype was successfully recreated. Other strains showed dose-dependent expression of Wld protein.  Homozygous 4836 mutant mice also demonstrated a higher proportion of intact axons 5 days after sciatic nerve lesion compared to other stains, which were visualised using electron microscopy.

Results Results

 To further test how expression levels of Wld protein protects axons from degeneration, the degree of neurofilament degradation (measured by Western blot) and the number of intact axons (electron microscopy) were measured after 10- 14 days.  In Wlds and homozygous 4836 mice, neurofilament degradation was less than that of other strains.  In homozgous 4836 and Wlds mice, axon protection was significantly better than that of WT and other mutant strains.

Results Results

Wld is a Predominantly Nuclear Protein  Wld protein was found to be localised in the nucleus of Wlds and transgenic mice, using immunostaining methods. It was not located in the nucleus of WT mice.  There was no detection of Wld protein in the axons of any mouse strain.  This study did not find evidence of Wld expression in glial cells, but previous studies have reported detecting Wld protein in Schwann cells using RT-PCR  Wld may have roles other than axon protection in other cell types Results Results

The Wld protein has Nmnat Enzyme Activity  Intrinsic Nmnat activity was initially measured using recombinant protein expression and measuring the activity of the bacterial lysate.  In Wlds mice, Nmnat levels were measured in brain homogenates and were found to be four times higher than the control.  Total NAD+ levels were not significantly increased however, indicating that Wld protein increases Nmnat levels but not overall NAD+ levels.

Discussion And Further Conclusions Research

 The paper concludes that the Ube4b/Nmnat  The role of Ube4b and Nmnat chimeric gene has been successfully identified  As the Wld protein is located in the nucleus, the actions as the Wld gene. of Ube4b and Nmnat may be influence downstream s regulatory pathways, which can then go on to directly  The Wld phenotype was successfully recreated influence axon protective mechanisms. in the homozygous 4836 transgenic mouse.  Ube4b is involved in ubiquitination processes may  It was also shown that the level of expression of produce protective effects by influencing the stability of the Wld protein directly relates to the level of protein-protein interactions or RNA, or by affecting axon protection. nuclear transport.  Nmnat levels in Wlds mice is increased, but NAD+ levels  Wld protein was found to be localised in the remain similar to that of WT. This suggests that the nucleus and that it protects axons through an NAD+ is being metabolised. These metabolites may indirect mechanism involving other factors. produce neuroprotective effects.

Discussion And Further Questions Research

 Wld Protein as a Therapeutic Target  How could nuclear Wlds protect severed axons? What is the s significance of constant levels of NAD+? Is the -proteasome  Wld phenotype is known to protect axons from system involved? toxic effects of vincristine.  Nuclear Wlds most likely protects severed axons by altering  Studies have also indicated that Wlds protects regulatory pathways prior to the axon being severed. against a mouse model of motor neurone disease.  The constant NAD+ levels observed in Wlds mice indicate that a significant proportion of NAD+ is being metabolised. Metabolites  From the identification of the Wld gene, and of NAD+ are known to exert both neuroprotective and neurotoxic through investigation into the mechanisms effects. through which Wld protein exerts its  Ube4b is a multi-ubiquitinating enzyme. Although the full Ube4b neuroprotective effects, therapeutic targets for gene is not expressed in the chimeric gene, it is likely that the several neuropathologies could be identified. role of Ube4b in Wlds is likely to be similar to that of the regular enzyme. Therefore, there is a strong possibility that the ubiquitin- proteasome pathway is involved in the protection of axons; possibly through degrading proteins which produce or enhance the process of Wallerian degeneration Age-dependent synapse Outline withdrawal at axotomised

neuromuscular junctions in  Intro/background Wlds mutant and  Aims/hypothesis  Methods Ube4b/Nmnat  Results transgenic mice  Conclusion

Thomas H. Gillingwater*†, Derek Thomson*†, Till G. A.  Strengths/weaknesses Mack‡, Ellen M. Soffin*, Richard J. Mattison*, Michael P. Coleman‡ and Richard R. Ribchester*  BBQs Journal of Physiology (2002), 543.3, pp. 739–755 DOI:  10.1113/jphysiol.2002.022343 Summary © The Physiological Society 2002

Mairi Laverty

Background Aims and Hypothesis

 Wallerian degeneration: the molecular and cellular responses that are involved in the degeneration of distal axons and synaptic terminals after  Previous controversy whether age affected lesion or injury the neuroprotective role of the Wlds gene

 Wlds mutation: overexpression of a chimeric Ube4b/Nmnat (Wld) gene that protects axons from Wallerian degeneration  Aim: “to resolve the discrepancy between the  Wlds mutation is inherited as a single autosomal dominant characteristic, by a gene located on the distal end of chromosome 4. studies of Ribchester et al. (1995) and

 Protection differs in axons and synapses after axotomy: in Wlds mice Crawford et al. (1995) using a combined the motor nerve terminals persist for only 4-10 days while distal axons persist up to 3 weeks genetic, biochemical, morphological and electrophysiological approach.”  Thought to be differences in protection for age as well

Methods

 Mice: natural mutant Wlds mice, some used at around 1-2 months old, whereas others were kept to older ages (4,7 and 12 months)

 Surgery: FBD or lumbrical muscles=either sciatic or tibial nerve exposed and partly removed (denervating the majority of muscles in hind foot); TA= intercostal nerves were similarly exposed and lesioned. Results  Electrophysiology: intracellular recordings were made 1-10 days after surgery

 Electron Microscopy: special preparations then viewed through Phillips CM12 TEM.

 Axon Counts: cross sections were cut, stained and examined. Total number of myelinated axon profiles were recorded in 6 cross-sections

 NMJ staining: preparations were fixed then acetylcholine receptors were labelled,

 Fluorescence imaging and analysis: used standard fluorescence microscope or laser scanning confocal microscope

 Western Blotting of mouse brains Age-independence of axon protection and Wld gene Results Sections expression

Figure 1:  Age-independence of axon protection and Wld A: Western Blots= age has no  Progressive loss of synaptic terminals in juvenile Wlds mice effect on Wlds gene expression  Rapid degeneration of Wlds synatpic terminals in mature mice  Recapitulation of synaptic withdrawal at reinnverated Wlds muscles B: shows qualitative  Age dependence of synaptic protection in Wld transgenic mice preservation of disconnected  Protection of axons and synapses expressing fluorescent protein by axons; no difference in age for the Wld gene axon loss or degeneration after axotomy

C: no significant difference in numbers of axon profiles between proximal and distal nerve stumps at either age

Data shows that both Wld gene expression and distal axon preservation are largely independent of age in Wlds mice.

Progressive loss of synaptic terminals in juvenile Wlds Figure 3: time course of withdrawal in 2 month old Wlds mice mice Figure 2: Axotomized nerve terminals retract from endplate in A: synapses protected from degeneration, 3 days after young adult Wlds mice -Measured B: complete retention of lower nerve terminal but partial morphologically and occupancy of the upper endplate, 6 days post-axotomy electrophysiologically

C: retraction bulb= also found in synapse elimination -80% of synapses were retained 3 days after D: 2 vacant endplates and 2 fully occupied, 6 days after axotomy but by 5 days this dropped to 60% E: EM of nerve terminal bouton then 30-50% by 7 days. F: retained good synaptic ultrastructure but neurofilaments are accumulated in the centre of the bouton

G: partially occupied NMJ, neighbours re unoccupied and covered by the nucleus of a terminal Schwann cell

H-J: intracellular recordings: robust transmission, weak transmision and loss of transmission

Figure 4: Effect of endplate size and occupancy on synaptic withdrawal Rapid degeneration of Wlds synaptic terminals in mature mice A: Withdrawal of synaptic boutons was Figure 5: Degeneration of synaptic asynchronous and terminals in fully mature Wlds mice independent of endplate size -no correlation between endplate area and fractional occupancy ⇒Onset of synapse withdrawal occurred randomly but proceeds at a constant rate once started B: Depression of s transmitter release •Synaptic terminals in the young Wld mice preceeded structural progressively withdrew from motor endplates withdrawal following axotomy Similar to synapse elimination that occurs during ⇒Still occupied but low ⇒ Axons are removed synchronously in old mice compared to progressively in quantal content normal post-natal development younger mice, therefore axotomy-induced synaptic response in Wlds mice ⇒Time course is also similar changes systematically; from withdrawal to degeneration as these mice mature. Figure 6 D and E: Recapitulation of synaptic withdrawal at reinnervated Wlds muscles

Figure 6: Synaptic protection in Wlds mice depends on synaptic maturity and not the age of the animal

B: 7 month old 3 days post axotomy: one of the few remaining terminals next to 3 vacant endplates

C: 14 month old s regenerated synapse 5 “new” synapses in old Wld mice are better protected form days post axotomy: all degeneration than the mature synapses innervating muscles endplates are occupied without a prior, conditioning lesion applied to the nerve -Therefore it is the maturity of the nerve not the age.

Age dependence of synaptic Protection of axons and synapses protection in Wld transgenic mice expressing fluorescent protein by the Wld gene

  2 transgenic lines of Wld mice: lines 4836 and 4830 which show Available mice that can express fluorescent protein in the Wld phenotype their axons and synapses under control of a thy1  Examined the age dependence of synapse loss: Wlds expression promoter. and axon preservation independent of age  Allows us to see axon and synaptic protection in living  intracellular recordings show the same age dependence in preparations synaptic response to axotomy as seen in Wlds mice, with a  Crossbred Wlds mice and thy1-CFP mice similar time constant  Fluorescent protein expression did not interfere with the protection of axons and synapses presented by the Wld  Overall similar to natural mutant Wlds mice gene in young mice.  Useful for future studies  Degree of protection was similar to Wlds mice not expressing CFP  Shows future potential

Main Findings/Discussion Conclusions

 “the main finding of the present study is that lesions of peripheral  Wld gene expression and axon protection is age independent nerve induce one of at least two independent modes of synaptic  Loss of synaptic terminals is progressive in juvenile Wlds mice degeneration in Wld-expressing mice, depending on the maturity of and similar to synapse elimination the synapses that are axotomised.”  Degeneration of Wlds synaptic terminals in mature mice is rapid and not progressive  Support Crawford et al., 1995: axonal loss is independent of age s BUT preservation of axotomised Wlds nerve terminal is strongly  “new” synapses in old Wld mice are better protected after age-dependent. conditioning lesion= it is maturity of the synapse rather than age of the motor neuron or mouse  Transgenic mice show same age dependence as natural mutant  Axons are protected from degeneration at all ages but synapses are not which suggests that both mechanisms of synaptic degeneration Wlds mice occur independently of axonal degeneration.  Fluorescent protein expressing mice also show the same protection of axons and synapses  Further support that the neurones are compartmentalised with respect to the mechanisms they contain for bringing about degeneration Strengths and Weaknesses

 Strengths: BBQs  Discovered useful methods for future research: transgenic and fluorescent mice  solve controversies over previous thoughts on age Why does the Wlds phenotype decline with dependency of synapse withdrawal age? What mechanisms might produce recapitulation of the juvenile Wlds phenotype  Weaknesses: at regenerating synapses in old mice?  Did not mention sample size

Why does the Wlds phenotype decline What mechanisms might produce recapitulation of the juvenile Wlds phenotype at regenerating synapses in old mice? with age?  Axonal and synaptic protection is an indirect mechanism and does not interact with other that are uniquely expressed in axons and  It is actually the maturity of the synapse not the age that synapses, as the Wld protein is localised to the cell nuclei seems to play the important role  incorporation of a ubiquitination cofactor (the N-terminal 70 amino acids  Reasons are unknown but perhaps: of Ube4b) in the Wld gene could play an important role in degeneration and response to synaptic axotomy.  Biochemical state of the regenerated terminal and local regulation of the response to axotomy,  One hypothetical link: perhaps selective physiological trafficking of maintenance factors during early postnatal development (or after  or to recapitulation of patterns of gene expression in motor reinnervation) results in the same withdrawal response as that induced neurone nuclei by surgical axotomy in young Wlds mice.

 Could have molecular mechanisms or physiological trafficking when the synapse or axon is newly innervated compared to when it has been present for a while such as in a mature mouse.

Summary: Take home Future Studies message

 The use of thy1-CFP mice could be very useful in future studies to visualise axotomy-induced synapse withdrawal in real-time

 Transgenic mice also hold significant value for future studies  2 modes of synaptic degeneration depending on maturity of the synapses that are  More studies into the role of protein ubiquitination in synapse withdrawal axotomised:  The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: Insights from Wld(s) and other mutant  Progressive withdrawal in young and Wallerian- mice: Thomas H. Gillingwater and Richard R. Ribchester like in mature mice

 Could it relate to humans and have therapeutic potential for neurodegenerative diseases References

 This article: http://onlinelibrary.wiley.com.ezproxy.webfeat.lib.ed. ac.uk/doi/10.1113/jphysiol.2002.022343/abstract  Subsequent study: Questions? http://www.springerlink.com/content/mj0426238011n 282/  Crawford’s conflicting study: http://www.springerlink.com/content/n5v0451vwq58 6v13/

Non-Nuclear Wlds Determines its  Introduction Neuroprotective Efficacy for  Aim Axons and Synapses In  Methods  Results Beirowski et al, 2009 Vivo  Conclusions  Big Burning Question Presented by Jen Sturgess  Strengths/Weaknesses  Future work

Intro Intro  Wlds delays axon degeneration  trying to discover mechanisms  Important point - study is in vivo  Wlds found to be abundant in only cell nuclei, suggesting indirect axonal effects of the protein  Some in vitro studies show overexpressed  However, absence in other structures not proven Nmnat has similar neuroprotective effects to experimentally due to detection limits in subcellular Wlds, however in vivo this is not the case compartments, therefore may be present  Differences between in vivo and in vitro – in s  Where are the Wld subcellular sites of action? vivo makes more clinically relevant Aim Methods

 Created ΔNLS Wlds mice widespread Wlds axon  Create transgenic mice with reduced nuclear targeting and cytoplasmic redistribution of Wlds  show differences distribution between WT, native WldS and ΔNLSWldS mice after nerve  2 point mutation (R213A, R215A) within the NLS of the lesion Nmnat1 domain  (Δ NLS = deleted nuclear localisation sequence)

 To find the subcellular location of Wlds action in vivo  Tested in vitro and in vivo for expression of Wlds variants S  Investigate neuroprotective effects of extranuclear Wld  Confirmed reduction of Wlds protein in the nucleus through cell culture of hippocampal and dorsal root ganglion, and HeLa and PC12 cells – showed almost complete exclusion of Wlds from the nucleus

Methods Methods

Biochemical assessment of variant Wlds protein levels in transgenic mice Assessment of axon preservation  Segments from brain, lumbar spinal cord and sciatic nerve were  Right sciatic nerves transected or crushed in sampled s s  Tissue was homogenized and centrifuged wild type, Wld native and ΔNLS Wld mice  Levels of protein were measured in –  Tested structural preservation using confocal

•Nuclear fraction •Nuclear and postnuclear fraction microscopy of a YFP-labelled axon subset •Cytoplasmic fraction •Cytoplasmic and mitochondria-enriched fraction  •Cytosolic fraction •Microsome-enriched and cytosolic fraction Also tested using light/electron microscopy  Evaluated axonal integrity • Measurement were quantified using integrated optical density (OD) of bands from 3 blots per experimental group

Methods Methods Electrophysiology and vital labeling of NMJs  To test whether NMJs were preserved after axotomy, they Immunocytochemistry and immunohistochemistry recorded muscle contractions, electromyography and vital  Immunofluorescence detection of WldS variant expression labeling of synaptic terminals  Special techniques for high-sensitivity detection of low S  Preparations of tibial nerve flexor digitorum brevis (FBD) abundance Wld protein variants used  Nonfluorescent immunohistochemically stained tissues were imaged  FBD preparations also stained and stimulated for morphological quantification of functionally preserved NMJs Confocal imaging and fluorescence intensity  Acetylcholine receptors stained and endplate occupancy quantification quantified  Immunostained tissue sections and vital dye-labelled muscle preparations were imaged •Lots of tests to make sure they had created the neuroprotective phenotype they wanted Results •Breeding to homozygosity elevated ΔNLSWldS protein expression in brain by approx twofold compared to Results hemizygosity Figure 1 •Expressed full Nmnat enzyme activity  The strength of axon protection is closely related S •Significantly reduced nuclear targeting and relative with levels of Wld cytoplasmic redistribution of WldS protein  If WldS works through a nuclear mechanism, then hypothesised that reduction of nuclear WldS would decrease axon protection  However the opposite happens – redistribution away from nucleus increases axonal protection  ΔNLSWldS delays wallerian degeneration more robustly than native WldS

Figure 2

Figure 4 Results

Figure 3. Time course of Wallerian degeneration at 3, 5 and 14 days following sciatic nerve transection

•Confocal images of lesioned sciatic/tibial nerves •WldS heterozygotes = pronounced degradation from 21 days •WldS homozygotes = pronounced degradation from 35 days •ΔNLSWldS heterozygotes = uninterrupted axons up to 35 days •ΔNLSWldS homozygotes = uninterrupted axons up to 49 days

Results Results

 Protective WldS phenotype effective in young Figure 4 mice but is almost completely lost in older • Transmission electron microscopy mice •Preservation of  ΔNLSWldS phenotype shows stronger ultrastructure - ΔNLS Wlds mice have intact myelin synaptic protection in young (100% intact sheaths, regularly spaced NMJs 6 days after transection compared with cytoskeleton and normal S appearing mitochondria ~50% in Wld ) after 49 days  So will strong ΔNLSWldS protection of NMJ still be present in older mice? Figure 5 •Electrophysiology of mice aged 7.5 months (old), 6 days after Figure 5 sciatic nerve lesion  Confocal imaging confirms •A and B - amplitude of loss of occupied NMJs in isometric force generated by old WldS native mice nerve stimulation in ΔNLDWldS is ~50 times that of native WldS  In NLS mice, 95% NMJ •C and D – amplitude of the occupancy even in 12 evoked EMG response in month old mice ΔNLDWldS is ~10 fold greater than native WldS  Decline of synaptic S •Native Wld mice almost protection with age is completely lose synaptic reduced when WldS protein protection – only weak or no is extranuclear contraction upon stimulation • Robust contractions of ΔNLDWldS indistinguishable from nonaxotomised muscles

Figure 7 Results Dectection of WldS protein variants in axons  WldS protein was found to be present in the axoplasm following axotomy, which supports a direct axonal role rather than an indirect nuclear role of WldS  Increased detection sensitivity showed presence of WldS protein in native WldS mice axons also  Results suggest presence of extranuclear WldS in axons (higher levels in NLS than native WldS mice) and possibility of axonal transport of WldS protein  WldS signal much stronger in NLS mice on both sides of a nerve crush – suggests that ΔNLSWldS protein is transported both anterogradely and retrogradely  More direct evidence needed to confirm this

control

Figure 9 Superior cervical ganglion

Results •Blue stains nucleus •Green stains WldS protein S  The association of Wld with organelles in subcellar •Red stains mitochondria fractionation of brain tissue and culture was studied •Merge shows the overlap S S  Wld and NLSWld were present in mitochondria •In native WldS mice, WldS protein and microsomes (absent in WT). Similar data from associated with nucleus rats and mice. •In ΔNLSWldS mice, WldS protein associated with mitochondria  ~85% of extracellular native/ΔNLS WldS was localised to mitochondria, although many mitochondria were WldS free

•Same as above but with E.R Summary graph Conclusions

 WldS more effective when extranuclear  Suggests cytoplasmic/direct axonal route of protein action  Subcellular localisation to mitochondria and E.R (microsomes)  Extracellular WldS slows Wallerian degeneration – axon survival extended from 4 to 7 weeks and decline of protection with age is significantly reduced  Strong synaptic protection  Need to investigate exact mechanisms further

• Shows there is non-nuclear medicated delay of Wallerian degeneration

Conclusions “BBQ”  What are the potential therapeutic implications of cytoplasmic WldS fractionating with mitochondria and microsomes? s  ΔNLS Wld transgenic mice considerably  Mitochondria require NAD+ to synthesis ATP and regulate cell signalling pathways enhance axon and synapse protection  WldS  overexpression of Nmnat1  increased NAD synthesis  Therefore WldS axons can maintain higher levels NAD+ and consequently ATP levels after axon lesion better than wild type axons  Alternative mechanism = Nmnat blocks production of reactive oxygen species  Therefore ΔNLS Wlds transgenic mice (ROS) from mitochondria

 Microsomes = fragmented E.R – just stated that found WldS protein in should show more effective axon and microsomes (restricted subdomains) …didn’t suggest any mechanism of action. synapse protection in neurodegenerative Affects synthesis of proteins involved in axon protection? disease models  Implications = study has brought us closer to understanding protective WldS mechanism although more research is need to know exact mechanisms that would be therapeutic targets

Strengths/Weaknesses Future Work

Strengths Weaknesses  Prove mechanism of WldS in mitochondria, and  Very well illustrated  Last few experiments of investigate action of WldS in E.R  Very thorough and convincing paper were not described  Use results from other studies to very clearly  Address roles of NAD+ synthesis and VCP binding support their results well  Some of the imaging in axons and synapses  Created ΔNLS Wlds mouse and did figures did not explain S sufficient tests to prove the very well what the  Is the critical location of Wld axonal or cytoplasmic? phenotype is correct different stains were  Can the enhanced protection offered by the ΔNLS  Positive step forward to finding a showing Wlds variant be developed into more effective therapeutic for neurodegenerative  Inconsistent use of diseases – points to more effective different lines of therapy for axonopathies? therapeutic strategies based around transgenic mice  Can ΔNLS Wlds overcome the age dependent S Wld  Small sample size – only weakening of Wlds neuroprotection?  Suggests what to investigate next 2/3 mice of each different after each of their conclusions line/age  Discusses what other studies have found with respect to WldS mechanism  Introduction Axonal and neuromuscular  Aims synaptic phenotypes in Wlds,  Methods SOD1G93A and ostes mutant mice  Results identified by fiber-optic confocal  Conclusions microendoscopy  BBQ Wong et al 2009  The future

Introduction Aims

 Discovery of neuroprotection in animal models is  Find genetic modifiers that would enhance synaptic important for identifying new targets for treatment of disease protection in Wlds mice   Slow Wallerian degeneration in spontaneous To find an effective method to recognize these mutant Wlds mice delays degeneration of distal phenodeviants which may have no behavioural axons signs  This phenotype has no behavioural signs  To prove that CME and genomic mutagenesis can  Current screening methods may not be effective be combined to investigate neuromuscular in finding neuroprotective mutations pathology in vivo  Better methods need to be developed

Methods Methods

 CME: Experimenters used a 1.5mm optical fiber probe to visualize the neuromuscular junctions  ENU mutagenesis in BALB/c mice cross bred with thy1.2-yfp16/Wlds double homozygotes  Sciatic nerve cut at 1-2 months in F1 generation  Outcome assessed 3 days later using CME  219 mice were screened in this way Results Results  ENU induces modifiers of axonal and synaptic degeneration in Wlds mice  CME can be  7/219 mice showed variations in synaptic or axonal used to phenotype  2 of these phenodeviants (CEMOP_S2 and S5) showed distinguish the most protection between intact axons from those undergoing Wallerian degeneration

Results Results

 CME can be used in longitudinal studies to monitor the degeneration of motor units  Tests also carried out on SOD1 to prove that degeneration was not due  Inheritance tests provide evidence that line to the CME method itself CEMOP_S5 carries an autosomal-dominant, ENU  Show that CME can be used to induced mutation that delays synaptic degeneration detect and monitor pathological  Other lines also showed signs of inheritance but signs of spontaneous synaptic more tests will be needed degeneration

Results Conclusions

 CME is also sensitive enough to monitor  Combining ENU mutagenesis with CME is an differences in peripheral effective tool for studying the rate of synaptic nerve regeneration in degeneration and regeneration ostes mice  It identifies phenodeviants that are undetectable  Successfully confirmed delayed axonal using conventional screening methods regeneration in  Ability to do longitudinal studies - gives evidence for homozygous and potential diagnostic value heterozygous ostes mice Conclusions Future studies

 CME has a lower spatial resolution than other  Further developments of CME technology methods and identification of the mechanisms in  However, it is faster, less invasive and more versatile mutants identified may be used for routine examination and monitoring of treatment from  A variant of the CME method may be suitable for longitudinal examination of NMJs in humans early stages in progression of neuromuscular  This could lead to more effective monitoring and disease treatment of neuromuscular disease

Future studies Big Burning Question

 Find beneficial modifier of early  What molecular effects does the CEMOP_S5 have and how do these suppress synaptic degeneration? Do they also affect axons? How would neuromuscular synaptic and axonal we find out? degeneration in SOD1G93A mutant mice  Could involve modification of transcription or translation of other genes  Or by direct modification of intracellular signaling  The CME could be used to overcome the  Or by targeting enzymic activity to intracellular compartments involved difficulties in studying SOD1G93A in energy metabolism  Need to establish the gene mutations to identify mechanism of action  Did not show protection of axons in this study but could repeat to check for axon protection

Strengths Summary

 Very convincing findings  ENU was used to induce genomic mutations in mice  CME was then used to visualize the synaptic  All in vivo phenotypes in these mutant mice  Large sample size  When combined these tools can be a powerful  Easy to read and understand approach to investigation of neuromuscular pathology  Backed up there findings with more  These findings may lead to better treatment of experiments neuromuscular disease