Wallerian Degeneration ofInjured Axons and Synapses is

Delayed by a Ube4b/NmnatChimeric Gene

Mack T, Reiner M, Beirowski B, Weiqian M, Emanuelli M,Wagner D, Thomson D, Gillingwater T, Court F, Conforti L,

Fernando F. S, Tarlton A, Andressen C, Addicks K, Magni G,Ribchester R. R, Perry V. H and Coleman M. P.

Overview

Introduction Background Information Aims

Results Discussion Future Research Questions

Introduction - Background

Distal axons of injured neurons usually undergo Walleriandegeneration 24-48 hours after injury.

In C57BL/Wlds mice, injured axons can survive several weeksfollowing injury. This suggests that these mice have a protective mechanism specific to the

axons, and that this protective factor must be present in the axon prior to anyinjury occurring as the distal part of the axon is separated from the cell bodyand cannot benefit from new protein synthesis.

Wallerian degeneration is implicated in several humanneuropathologies. These include ALS, multiple sclerosis and traumatic disorders such as injury

to the spinal cord An understanding of the mechanisms controlling the slow

Wallerian degeneration exhibited by the C57BL/Wlds mousecould lead to the new therapeutic targets for such diseases.

Introduction - Background

A candidate Wlds gene was identified onchromosome 4 (mouse)

It is a chimeric gene containing codingregions for Ube4b/D4Cole1e in the mouse D4Cole1e was found to be equivalent to the

human enzyme Nmnat. Both proteins were found to be expressed in

Wlds mice, strongly suggesting that thischimeric gene is the Wlds gene

Introduction - Aims

Hypothesis: the Ube4b/Nmnat chimeric geneis the Wlds gene and produces the slowWallerian degeneration phenotype observedin the mutant mice.

To test this, the chimeric gene wasexpressed in transgenic mice. Several strains of mice were produced, each with

a different expression level of the chimeric gene.

Transgenic Mice Generated

4 lines of transgenic mice were generated forthis study: 4839, 4830, 4858 and 4836.

In each line, the expression level of thechimeric gene had been altered. The lowest level was expressed in 4839; 4830

and 4858 showed a medium level of geneexpression (only 4830 is further discussed in thepaper); 4836 had the strongest expression whichis almost identical to Wlds mouse.

Results

Structural Preservation of Transected Axons Structural preservation was investigated 3-5

days following a unilateral lesion to the sciaticnerve.

Electron microscopy showed that the vastmajority of axons in the 4836 mouse hadpreserved cytoskeletons 5 days after injury Successful replication of the Wlds phenotype.

WT mice showed clear signs of degeneration inmyelinated and unmyelinated axons.

Partial protection was observed as expected in4830 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 wererecorded

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 injurywere significantly lower than the 4836 mouse, and the durationafter injury which the axons and synapses appeared functionalwas also reduced.

Results

Results

Functional motor axons and synapses werevisualised using immunostaining methods

Homozygous 4836 mice demonstrated synapticvesicle recycling 5 days after injury, indicatingsynaptic transmission is still able to occur.

Homozygous 4836 mice also showed a highproportion of endplates fully occupied 5 daysafter injury.

Proportion of endplates occupied were reducedin 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 showeddose-dependent expression of Wld protein.

Homozygous 4836 mutant mice also demonstrated a higherproportion of intact axons 5 days after sciatic nerve lesioncompared to other stains, which were visualised using electronmicroscopy.

Results

Results

To further test how expression levels of Wldprotein protects axons from degeneration, thedegree of neurofilament degradation (measuredby 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 ofother strains.

In homozgous 4836 and Wlds mice, axonprotection was significantly better than that ofWT 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 notlocated in the nucleus of WT mice.

There was no detection of Wld protein in the axons of any mousestrain.

This study did not find evidence of Wld expression in glial cells,but previous studies have reported detecting Wld protein inSchwann 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 theactivity of the bacterial lysate.

In Wlds mice, Nmnat levels were measured in brainhomogenates and were found to be four timeshigher than the control.

Total NAD+ levels were not significantly increasedhowever, indicating that Wld protein increasesNmnat levels but not overall NAD+ levels.

Results

Conclusions

The paper concludes that the Ube4b/Nmnatchimeric gene has been successfully identifiedas the Wld gene.

The Wlds phenotype was successfully recreatedin the homozygous 4836 transgenic mouse.

It was also shown that the level of expression ofthe Wld protein directly relates to the level ofaxon protection.

Wld protein was found to be localised in thenucleus and that it protects axons through anindirect mechanism involving other factors.

Discussion And FurtherResearch The role of Ube4b and Nmnat

As the Wld protein is located in the nucleus, the actionsof Ube4b and Nmnat may be influence downstreamregulatory pathways, which can then go on to directlyinfluence axon protective mechanisms.

Ube4b is involved in ubiquitination processes mayproduce protective effects by influencing the stability ofprotein-protein interactions or RNA, or by affectingnuclear transport.

Nmnat levels in Wlds mice is increased, but NAD+ levelsremain similar to that of WT. This suggests that theNAD+ is being metabolised. These metabolites mayproduce neuroprotective effects.

Discussion And FurtherResearch Wld Protein as a Therapeutic Target

Wlds phenotype is known to protect axons fromtoxic effects of vincristine.

Studies have also indicated that Wlds protectsagainst a mouse model of motor neurone disease.

From the identification of the Wld gene, andthrough investigation into the mechanismsthrough which Wld protein exerts itsneuroprotective effects, therapeutic targets forseveral neuropathologies could be identified.

Questions

How could nuclear Wlds protect severed axons? What is thesignificance of constant levels of NAD+? Is the ubiquitin-proteasomesystem involved? Nuclear Wlds most likely protects severed axons by altering

regulatory pathways prior to the axon being severed. The constant NAD+ levels observed in Wlds mice indicate that a

significant proportion of NAD+ is being metabolised. Metabolitesof NAD+ are known to exert both neuroprotective and neurotoxiceffects.

Ube4b is a multi-ubiquitinating enzyme. Although the full Ube4bgene is not expressed in the chimeric gene, it is likely that therole of Ube4b in Wlds is likely to be similar to that of the regularenzyme. 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 enhancethe process of Wallerian degeneration

Age-dependent synapsewithdrawal at axotomised

neuromuscular junctions inWlds mutant and

Ube4b/Nmnattransgenic mice

Thomas H. Gillingwater*†, Derek Thomson*†, Till G. A.Mack‡, Ellen M. Soffin*, Richard J. Mattison*, Michael P.

Coleman‡ and Richard R. Ribchester*

Journal of Physiology (2002), 543.3, pp. 739–755 DOI:10.1113/jphysiol.2002.022343

© The Physiological Society 2002

Mairi Laverty

Outline

Intro/background Aims/hypothesis Methods Results Conclusion Strengths/weaknesses BBQs Summary

Background

Wallerian degeneration: the molecular and cellular responses that areinvolved in the degeneration of distal axons and synaptic terminals afterlesion or injury

Wlds mutation: overexpression of a chimeric Ube4b/Nmnat (Wld) genethat protects axons from Wallerian degeneration

Wlds mutation is inherited as a single autosomal dominantcharacteristic, by a gene located on the distal end of chromosome 4.

Protection differs in axons and synapses after axotomy: in Wlds micethe motor nerve terminals persist for only 4-10 days while distal axonspersist up to 3 weeks

Thought to be differences in protection for age as well

Aims and Hypothesis

Previous controversy whether age affectedthe neuroprotective role of the Wlds gene

Aim: “to resolve the discrepancy between thestudies of Ribchester et al. (1995) andCrawford et al. (1995) using a combinedgenetic, biochemical, morphological andelectrophysiological approach.”

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 partlyremoved (denervating the majority of muscles in hind foot); TA= intercostal nerves weresimilarly exposed and lesioned.

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 myelinatedaxon 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 laserscanning confocal microscope

Western Blotting of mouse brains

Results

Results Sections

Age-independence of axon protection and Wld gene expression Progressive loss of synaptic terminals in juvenile Wlds mice Rapid degeneration of Wlds synatpic terminals in mature mice Recapitulation of synaptic withdrawal at reinnverated Wlds muscles Age dependence of synaptic protection in Wld transgenic mice Protection of axons and synapses expressing fluorescent protein by

the Wld gene

Age-independence of axon protection and Wld geneexpression

Figure 1:

A: Western Blots= age has noeffect on Wlds gene expression

B: shows qualitativepreservation of disconnectedaxons; no difference in age foraxon loss or degeneration afteraxotomy

C: no significant difference innumbers of axon profilesbetween proximal and distalnerve stumps at either age

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

Progressive loss of synaptic terminals in juvenile Wlds

mice

A: synapses protected from degeneration, 3 days after

B: complete retention of lower nerve terminal but partialoccupancy of the upper endplate, 6 days post-axotomy

C: retraction bulb= also found in synapse elimination

D: 2 vacant endplates and 2 fully occupied, 6 days after

E: EM of nerve terminal bouton

F: retained good synaptic ultrastructure butneurofilaments are accumulated in the centre of thebouton

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

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

Figure 2: Axotomized nerveterminals retract from endplate inyoung adult Wlds mice

Figure 3: time course of withdrawal in 2 month old Wlds mice

-Measuredmorphologically andelectrophysiologically

-80% of synapses wereretained 3 days afteraxotomy but by 5 daysthis dropped to 60%then 30-50% by 7 days.

Figure 4: Effect of endplate size and occupancy on synaptic withdrawal

A: Withdrawal ofsynaptic boutons wasasynchronous andindependent of endplatesize-no correlation betweenendplate area andfractional occupancy⇒Onset of synapsewithdrawal occurredrandomly but proceedsat a constant rate oncestartedB: Depression oftransmitter releasepreceeded structuralwithdrawal⇒Still occupied but lowquantal content

•Synaptic terminals in the young Wlds miceprogressively withdrew from motor endplatesfollowing axotomy⇒Similar to synapse elimination that occurs duringnormal post-natal development⇒Time course is also similar

Rapid degeneration of Wlds

synaptic terminals in mature miceFigure 5: Degeneration of synapticterminals in fully mature Wlds mice

Axons are removed synchronously in old mice compared to progressively inyounger mice, therefore axotomy-induced synaptic response in Wlds micechanges systematically; from withdrawal to degeneration as these mice mature.

Recapitulation of synaptic withdrawal at reinnervatedWlds muscles

Figure 6: Synapticprotection in Wlds micedepends on synapticmaturity and not the age ofthe animal

B: 7 month old 3 dayspost axotomy: one ofthe few remainingterminals next to 3vacant endplates

C: 14 month oldregenerated synapse 5days post axotomy: allendplates are occupied

Figure 6 D and E:

“new” synapses in old Wlds mice are better protected formdegeneration than the mature synapses innervating muscleswithout a prior, conditioning lesion applied to the nerve -Therefore it is the maturity of the nerve not the age.

Age dependence of synapticprotection in Wld transgenic mice

2 transgenic lines of Wld mice: lines 4836 and 4830 which showthe Wld phenotype

Examined the age dependence of synapse loss: Wlds expressionand axon preservation independent of age

intracellular recordings show the same age dependence insynaptic response to axotomy as seen in Wlds mice, with asimilar time constant

Overall similar to natural mutant Wlds mice Useful for future studies

Protection of axons and synapsesexpressing fluorescent protein by theWld gene Available mice that can express fluorescent protein in

their axons and synapses under control of a thy1promoter.

Allows us to see axon and synaptic protection in livingpreparations

Crossbred Wlds mice and thy1-CFP mice Fluorescent protein expression did not interfere with the

protection of axons and synapses presented by the Wldgene in young mice.

Degree of protection was similar to Wlds mice notexpressing CFP

Shows future potential

Main Findings/Discussion

“the main finding of the present study is that lesions of peripheralnerve induce one of at least two independent modes of synapticdegeneration in Wld-expressing mice, depending on the maturity ofthe synapses that are axotomised.”

Support Crawford et al., 1995: axonal loss is independent of ageBUT preservation of axotomised Wlds nerve terminal is stronglyage-dependent.

Axons are protected from degeneration at all ages but synapses arenot which suggests that both mechanisms of synaptic degenerationoccur independently of axonal degeneration.

Further support that the neurones are compartmentalised withrespect to the mechanisms they contain for bringing aboutdegeneration

Conclusions

Wld gene expression and axon protection is age independent Loss of synaptic terminals is progressive in juvenile Wlds mice

and similar to synapse elimination Degeneration of Wlds synaptic terminals in mature mice is rapid

and not progressive “new” synapses in old Wlds mice are better protected after

conditioning lesion= it is maturity of the synapse rather than ageof the motor neuron or mouse

Transgenic mice show same age dependence as natural mutantWlds mice

Fluorescent protein expressing mice also show the sameprotection of axons and synapses

Strengths and Weaknesses

Strengths: Discovered useful methods for future research: transgenic

and fluorescent mice solve controversies over previous thoughts on age

dependency of synapse withdrawal

Weaknesses: Did not mention sample size

BBQsWhy does the Wlds phenotype decline with

age? What mechanisms might producerecapitulation of the juvenile Wlds phenotype

at regenerating synapses in old mice?

Why does the Wlds phenotype declinewith age?

It is actually the maturity of the synapse not the age thatseems to play the important role

Reasons are unknown but perhaps: Biochemical state of the regenerated terminal and local

regulation of the response to axotomy, or to recapitulation of patterns of gene expression in motor

neurone nuclei

Could have molecular mechanisms or physiologicaltrafficking when the synapse or axon is newly innervatedcompared to when it has been present for a while suchas in a mature mouse.

What mechanisms might produce recapitulation of the juvenileWlds phenotype at regenerating synapses in old mice?

Axonal and synaptic protection is an indirect mechanism and does notinteract with other genes that are uniquely expressed in axons andsynapses, as the Wld protein is localised to the cell nuclei

incorporation of a ubiquitination cofactor (the N-terminal 70 amino acidsof Ube4b) in the Wld gene could play an important role in degenerationand response to synaptic axotomy.

One hypothetical link: perhaps selective physiological trafficking ofmaintenance factors during early postnatal development (or afterreinnervation) results in the same withdrawal response as that inducedby surgical axotomy in young Wlds mice.

Future Studies

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

Transgenic mice also hold significant value for future studies

More studies into the role of protein ubiquitination in synapse withdrawal

The relationship of neuromuscular synapse elimination to synapticdegeneration and pathology: Insights from Wld(s) and other mutantmice: Thomas H. Gillingwater and Richard R. Ribchester

Could it relate to humans and have therapeutic potential forneurodegenerative diseases

Summary: Take homemessage

2 modes of synaptic degeneration dependingon maturity of the synapses that areaxotomised: Progressive withdrawal in young and Wallerian-

like in mature mice

References

This article:http://onlinelibrary.wiley.com.ezproxy.webfeat.lib.ed.ac.uk/doi/10.1113/jphysiol.2002.022343/abstract

Subsequent study:http://www.springerlink.com/content/mj0426238011n282/

Crawford’s conflicting study:http://www.springerlink.com/content/n5v0451vwq586v13/

Questions?

Non-Nuclear Wlds

Determines itsNeuroprotective Efficacy for

Axons and Synapses InVivoBeirowski et al, 2009

Presented by Jen Sturgess

Introduction Aim Methods Results Conclusions Big Burning Question Strengths/Weaknesses Future work

Intro Wlds delays axon degeneration trying to discover

mechanisms Wlds found to be abundant in only cell nuclei,

suggesting indirect axonal effects of the protein However, absence in other structures not proven

experimentally due to detection limits in subcellularcompartments, therefore may be present

Where are the Wlds subcellular sites of action?

Intro

Important point - study is in vivo Some in vitro studies show overexpressed

Nmnat has similar neuroprotective effects toWlds, however in vivo this is not the case

Differences between in vivo and in vitro – invivo makes more clinically relevant

Aim Create transgenic mice with reduced nuclear targeting and

cytoplasmic redistribution of Wlds show differencesbetween WT, native WldS and ΔNLSWldS mice after nervelesion

To find the subcellular location of Wlds action in vivo

Investigate neuroprotective effects of extranuclear WldS

Methods Created ΔNLS Wlds mice widespread Wlds axon

distribution 2 point mutation (R213A, R215A) within the NLS of the

Nmnat1 domain (Δ NLS = deleted nuclear localisation sequence)

Tested in vitro and in vivo for expression of Wlds

variants Confirmed reduction of Wlds protein in the nucleus through

cell culture of hippocampal and dorsal root ganglion, andHeLa and PC12 cells – showed almost complete exclusionof Wlds from the nucleus

MethodsBiochemical assessment of variant Wlds protein levels intransgenic mice

Segments from brain, lumbar spinal cord and sciatic nerve weresampled

Tissue was homogenized and centrifuged Levels of protein were measured in –

•Nuclear fraction•Cytoplasmic fraction•Cytosolic fraction

•Nuclear and postnuclear fraction•Cytoplasmic and mitochondria-enriched fraction•Microsome-enriched and cytosolic fraction

• Measurement were quantified using integratedoptical density (OD) of bands from 3 blots perexperimental group

MethodsAssessment of axon preservation Right sciatic nerves transected or crushed in

wild type, Wlds native and ΔNLS Wlds mice Tested structural preservation using confocal

microscopy of a YFP-labelled axon subset Also tested using light/electron microscopy Evaluated axonal integrity

MethodsElectrophysiology and vital labeling of NMJs To test whether NMJs were preserved after axotomy, they

recorded muscle contractions, electromyography and vitallabeling of synaptic terminals

Preparations of tibial nerve flexor digitorum brevis (FBD)used

FBD preparations also stained and stimulated formorphological quantification of functionally preserved NMJs

Acetylcholine receptors stained and endplate occupancyquantified

MethodsImmunocytochemistry and immunohistochemistry Immunofluorescence detection of WldS variant expression Special techniques for high-sensitivity detection of low

abundance WldS protein variants Nonfluorescent immunohistochemically stained tissues were

imaged

Confocal imaging and fluorescence intensityquantification

Immunostained tissue sections and vital dye-labelled musclepreparations were imaged

ResultsFigure 1

•Lots of tests to make sure they had created theneuroprotective phenotype they wanted

•Breeding to homozygosity elevated ΔNLSWldS proteinexpression in brain by approx twofold compared tohemizygosity

•Expressed full Nmnat enzyme activity

•Significantly reduced nuclear targeting and relativecytoplasmic redistribution of WldS protein

Figure 2

Results The strength of axon protection is closely related

with levels of WldS

If WldS works through a nuclear mechanism, thenhypothesised that reduction of nuclear WldS woulddecrease axon protection

However the opposite happens – redistribution awayfrom nucleus increases axonal protection

ΔNLSWldS delays wallerian degeneration morerobustly than native WldS

ResultsFigure 3. Time course of

Wallerian degeneration at 3, 5and 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

Figure 4

Results

• Transmission electronmicroscopy

•Preservation ofultrastructure - ΔNLS Wlds

mice have intact myelinsheaths, regularly spacedcytoskeleton and normalappearing mitochondriaafter 49 days

Figure 4

Results Protective WldS phenotype effective in young

mice but is almost completely lost in oldermice

ΔNLSWldS phenotype shows strongersynaptic protection in young (100% intactNMJs 6 days after transection compared with~50% in WldS)

So will strong ΔNLSWldS protection of NMJstill be present in older mice?

Figure 5

•Electrophysiology of mice aged7.5 months (old), 6 days aftersciatic nerve lesion

•A and B - amplitude ofisometric force generated bynerve stimulation in ΔNLDWldS

is ~50 times that of native WldS

•C and D – amplitude of theevoked EMG response inΔNLDWldS is ~10 fold greaterthan native WldS

•Native WldS mice almostcompletely lose synapticprotection – only weak or nocontraction upon stimulation

• Robust contractions ofΔNLDWldS indistinguishablefrom nonaxotomised muscles

Figure 5 Confocal imaging confirms

loss of occupied NMJs inold WldS native mice

In NLS mice, 95% NMJoccupancy even in 12month old mice

Decline of synapticprotection with age isreduced when WldS proteinis extranuclear

ResultsDectection of WldS protein variants in axons WldS protein was found to be present in the axoplasm

following axotomy, which supports a direct axonal role ratherthan 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 ofaxonal transport of WldS protein

WldS signal much stronger in NLS mice on both sides of anerve crush – suggests that ΔNLSWldS protein is transportedboth anterogradely and retrogradely

More direct evidence needed to confirm this

Figure 7

control

Results The association of WldS with organelles in subcellar

fractionation of brain tissue and culture was studied WldS and NLSWldS were present in mitochondria

and microsomes (absent in WT). Similar data fromrats and mice.

~85% of extracellular native/ΔNLS WldS waslocalised to mitochondria, although manymitochondria were WldS free

•Blue stains nucleus

•Green stains WldS protein

•Red stains mitochondria

•Merge shows the overlap

•In native WldS mice, WldS proteinassociated with nucleus

•In ΔNLSWldS mice, WldS proteinassociated with mitochondria

Superior cervical ganglionFigure 9

•Same as above but with E.R

Summary graph

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

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 anddecline of protection with age is significantly reduced

Strong synaptic protection Need to investigate exact mechanisms further

Conclusions

ΔNLS Wlds transgenic mice considerablyenhance axon and synapse protection

Therefore ΔNLS Wlds transgenic miceshould show more effective axon andsynapse protection in neurodegenerativedisease models

“BBQ” What are the potential therapeutic implications of cytoplasmic WldS fractionating

with mitochondria and microsomes?

Mitochondria require NAD+ to synthesis ATP and regulate cell signallingpathways

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

(ROS) from mitochondria

Microsomes = fragmented E.R – just stated that found WldS protein inmicrosomes (restricted subdomains) …didn’t suggest any mechanism of action.Affects synthesis of proteins involved in axon protection?

Implications = study has brought us closer to understanding protective WldS

mechanism although more research is need to know exact mechanisms thatwould be therapeutic targets

Strengths/WeaknessesStrengths Very well illustrated Very thorough and convincing Use results from other studies to

support their results well Created ΔNLS Wlds mouse and did

sufficient tests to prove thephenotype is correct

Positive step forward to finding atherapeutic for neurodegenerativediseases – points to more effectivetherapeutic strategies based aroundWldS

Suggests what to investigate nextafter each of their conclusions

Discusses what other studies havefound with respect to WldS

mechanism

Weaknesses Last few experiments of

paper were not describedvery clearly

Some of the imagingfigures did not explainvery well what thedifferent stains wereshowing

Inconsistent use ofdifferent lines oftransgenic mice

Small sample size – only2/3 mice of each differentline/age

Future Work Prove mechanism of WldS in mitochondria, and

investigate action of WldS in E.R Address roles of NAD+ synthesis and VCP binding

in axons and synapses Is the critical location of WldS axonal or cytoplasmic? Can the enhanced protection offered by the ΔNLS

Wlds variant be developed into more effectivetherapy for axonopathies?

Can ΔNLS Wlds overcome the age dependentweakening of Wlds neuroprotection?

Axonal and neuromuscularsynaptic phenotypes in Wlds,

SOD1G93A and ostes mutant miceidentified by fiber-optic confocal

microendoscopyWong et al 2009

Introduction Aims Methods Results Conclusions BBQ The future

Introduction Discovery of neuroprotection in animal models is

important for identifying new targets for treatmentof disease

Slow Wallerian degeneration in spontaneousmutant Wlds mice delays degeneration of distalaxons

This phenotype has no behavioural signs Current screening methods may not be effective

in finding neuroprotective mutations Better methods need to be developed

Aims Find genetic modifiers that would enhance synaptic

protection in Wlds mice To find an effective method to recognize these

phenodeviants which may have no behaviouralsigns

To prove that CME and genomic mutagenesis canbe combined to investigate neuromuscularpathology in vivo

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

Methods

Results

CME can beused todistinguishbetween intactaxons fromthoseundergoingWalleriandegeneration

Results ENU induces modifiers of axonal and synaptic

degeneration in Wlds mice 7/219 mice showed variations in synaptic or axonal

phenotype 2 of these phenodeviants (CEMOP_S2 and S5) showed

the most protection

Results

Inheritance tests provide evidence that lineCEMOP_S5 carries an autosomal-dominant, ENUinduced mutation that delays synaptic degeneration

Other lines also showed signs of inheritance butmore tests will be needed

Results CME can be used in longitudinal

studies to monitor the degenerationof motor units

Tests also carried out on SOD1 toprove that degeneration was not dueto the CME method itself

Show that CME can be used todetect and monitor pathologicalsigns of spontaneous synapticdegeneration

Results CME is also sensitive

enough to monitordifferences in peripheralnerve regeneration inostes mice

Successfully confirmeddelayed axonalregeneration inhomozygous andheterozygous ostesmice

Conclusions Combining ENU mutagenesis with CME is an

effective tool for studying the rate of synapticdegeneration and regeneration

It identifies phenodeviants that are undetectableusing conventional screening methods

Ability to do longitudinal studies - gives evidence forpotential diagnostic value

Conclusions CME has a lower spatial resolution than other

methods However, it is faster, less invasive and more

versatile A variant of the CME method may be suitable for

longitudinal examination of NMJs in humans This could lead to more effective monitoring and

treatment of neuromuscular disease

Future studies

Further developments of CME technologyand identification of the mechanisms inmutants identified may be used for routineexamination and monitoring of treatment fromearly stages in progression of neuromusculardisease

Future studies

Find beneficial modifier of earlyneuromuscular synaptic and axonaldegeneration in SOD1G93A mutant mice

The CME could be used to overcome thedifficulties in studying SOD1G93A

Big Burning Question What molecular effects does the CEMOP_S5 have and how do these

suppress synaptic degeneration? Do they also affect axons? How wouldwe find out?

Could involve modification of transcription or translation of other genes Or by direct modification of intracellular signaling Or by targeting enzymic activity to intracellular compartments involved

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

Very convincing findings All in vivo Large sample size Easy to read and understand Backed up there findings with more

experiments

Summary ENU was used to induce genomic mutations in mice CME was then used to visualize the synaptic

phenotypes in these mutant mice When combined these tools can be a powerful

approach to investigation of neuromuscularpathology

These findings may lead to better treatment ofneuromuscular disease