The Neuromuscular Junction inHealth and Disease

Mini-Symposium V

Form and function of NMJ in animalmodels of Motor Neurone Disease

27.11.08

ANIMAL MODELS

• 5-10% of ALS is familial [90-95% sporadic]

– 20% of familial cases: SOD1 mutations

– Also: ALS2/alsin

SOD1 Mutants

• SOD1 is a Cu/Zn superoxide dismutase• Converts superoxide radicalH20 or H202

• >100 human SOD1 mutations ALS

• 3 well-studied in mouse models:– SOD1G85R; G37R; G93A

A Compensatory Subpopulationof Motor Neurons in a Mouse

Model of ALS.

Schaefer et al.2005

Background• Motor Neuron Disease is an “umbrella term” for a range of diseases

with MN pathology.• Most common disease within MND is Amyotrophic Lateral Sclerosis.• Incidence of ALS; 1/100,000• Pathology;

– Brain and spinal cord;– Loss of MNs in anterior horn, brainstem and motor

cortex.– Ubiquitin inclusions– Accumulations of Phosphorylated neurofilaments.

- Muscle;

•Atrophy of Muscle Fibres

•Denervation and re-innervation.

ALS Genetics• 10-20% of ALS cases are inherited/familial.

• At least 10 autosomal locations have been mapped;ALS1 – ALS10

• Most of these are mutations of proteins which areubiquitously/ widely expressed.

• 1 location on the X chromosome: very rare.

• Most families mutations are NOT linked to theselocations.

• Extensive allelic/locus heterogeneity.

Introduction to the paper• One of the main factors that is limiting treatment development for

ALS is lack of detailed knowledge of the disease progression on acellular level.

• A “dying back” phenomena was recently observed ( Fisher et al2004, Scaefer et al 2002):in ALS mouse model intact proximalaxons with absent Neuromuscular Junctions.

• This led to the hypothesis that some axonal branches are sparedwhile others degenerate.

• Two proposed theories of identity of these axons;– A process similar to development; retraction of some branches

with maintenance of others within the same motor unit.– A subset of MNs are more susceptible to the disease, and these

are seen to die back while other less susceptible MNscompensate the loss.

Main Aims• To find out which of the two theories of axon

degeneration is true.

• Using a common ALS mouse model: SOD1G93A

(also expressing YFP in all Motor Axons.)

• From the results (if a certain subset of MNs are refractory tothe disease) suggest investigations into identifyingwhy this subset is protected and provide astarting point for therapy development.

Methods 1.• Cross bred SOD1 G93A and YFP mice.

• For end-stage disease mice(P125-P144):– Mice killed under anaesthetic– Muscle dissections; Sternomastiod, cleidomastoid and

clavotrapezius, diaphragm, soleus and EDL.– Immuno-staining of Muscle with several primary and the

secondary anti-bodies: staining for the fluorescent protein,neurofilaments and Schwann Cells. (and AChRs)

– The samples were view and analysed using LaserScanning Microscopy and Monte Carlo Analysis (usingMATLAB software to compare against random data sets)

Results 1.• Main Finding;

– Denervating and Re-innervating NMJs seenin SOD1 transgenic mice.

• In the junctions analysed:

Some were indistinguishable from wild-type, age-mapped controls –Thick axons, Variegated in diameter, uniform width of terminal branches ,AChR richsites apposed to terminal branches

Vs.

Results 1 continued…

• Some Abnormal junctions were seen: theyfall into several categories:– 1. Completely Denervated

– A) 2/3 with normal postsynaptic specializations– B) 1/3 with few AChRs and disorganized shape,

suggesting denervation for several weeks.

Results 1 continued…– 2. Partially Innervated

– A) With fragmented axon» i) overlying the junction» Ii) nearby

-B) With abnormally thin axons

-i) apposing normal receptorsites

-Ii) apposing faint receptorsites

What this means• These two main morphological phenotypes of

abnormal axons may represent two types ofdegeneration:

– The fragmented axons may be undergoing WallerianDegeneration

– The abnormally thin axons may be engaging in a processsimilar to that seen in synapse elimination.

•To obtain a clearer idea of what the cellularprocesses are in the progression of the disease theydecided to perform time-lapse imaging of thesternomastoid muscle.

Methods 2.• Mice were anaesthetised ,superficial incision was made

in the neck and approx 2% of AChRs in thesternomastoid were stained with labelled α-Bungarotoxin.

• Superficial junctions were imaged at 25x and 50x usingstandard epifluoresence microscopy.

• Animal the sutured and left to recover.

• This was repeated for subsequent imaging session: timeline not well defined.

Results 2…• Previous research has shown w/t NMJs are very stable.

(Balice-Gordon & Lichtman 1990)

• No changes in junctions of pre-symptomatic mice (P 41- P77)

• In symptomatic and end stage animals, 21/71 junctionsunderwent significant changes over time

Results 2…Summary of Alterations Seen

3IrregularCalibre

Thin SmoothAxon

3IncreasedOccupancy

Thin smoothaxon

1Thin Smoothaxon

Fragmented

8Thin smoothaxon

Normal4DenervatedNormal2FragmentedNormalN= (/21)AfterBefore •Of the 15 smooth

axons (out of 71) seenat 1st view, NONEwere fragmented orabsent at 2nd view.

• 9 remainedunchanged and 6changed as noted intable.

NB; the data collection ofthese alterations was nottaken until symptomaticstage the pre-symptomatic pathology wasmissed.

Conclusions from this• 1. Since there is no degeneration of thin axons, suggest

that these are in the process of re-innervation.

• 2. Junctions occupied by fragmented axons are in theprocess of being denervated.

• 3.Low incidence of fragmented axons suggests that re-innervation occurs quickly.

• This data was backed up by evidence gained throughsingle-time point imaging of sternomastoid muscle inPresymptomatic, Symptomatic and End-Stage mice.

Further Conclusions•Two observations were made that suggest that axondegeneration occurs before changed in Muscle Fibre orSchwann Cells:

1. There was never any evidence that abnormal AChRs wereinnervated by a normal axon, although the reverse was frequentlyseen.

2. No significant difference in number of terminal Schwann cellnuclei in end-stage SOD1 mice v.s. age matched controls( as shown my S100 immuno-staining)

This suggests that the earliest/primary changes occurring indisease happen at the terminal branches of the motor axonswhich leads to the sequential denervation and re-innervation ofthe junctions subsequent ALS pathology.

Distribution of Fragmenting and Re-growingterminal branches within Motor Units

• This still doesn’t answer how the denervation/reinnervation pattern is distrubuted within/ among MotorUnits.

• So SOD1G93A mice were bred to express YFP in a smallproportion of Motor Neurons.

• The YFP expression is random and has been observedto innervate both fast and slow-twitch muscles with equalfrequency.

• Entire Motor Units were then Reconstructed (methodpreviously outlined in other experiments)

Results of this..• Control; every terminal branch of the MU fully

innervated a receptor-rich post-synaptic site.• SOD1G93Amice MU’s fall into 3 categories;

1. Essentially normal.2. Fragmented Branches with no reinnervating

branches “winter tree” appearance3. Reinnervating axons with few/no fragmented

branches.

•Most of the MUs in category 3. were larger than those seen in wild type.

•Sprouting appears to arise from Nodes of Ranvier rather than terminals.

•These regenerating axons go on to develop into normal wild-type appearancejunctions.

Because the no. of whole motor units was limited, sections of motor units wereanalysed: 20 sets of 3-12 junctions could be assigned to a single motor unit.

8/20 contained fragmented axons, 12/20 contained thin axons but NONEcontained both.

Discussion: How this fits in withpast research

• Hypothesis that small calibre axons persist; these results show that axonspreviously thought to be inherently small calibre may be regeneratingaxons.

• Fits in with previous findings of enlarged motor units.

• Identifies “Intermediate” junctions as those which aredegenerating/regenerating, and further distinction of these has led toidentification of vulnerable/refractory motor unit groups.

• Motivate research into intrinsic distinction among MNs as well as targetmuscle; in light of certain muscles being spared in ALS.

• Does not contradict (but cannot support) theory that slow-twitch MNs arepreserved in favour to fast-twitch MNs: does define that if this were to betrue, slow twitch MNs would need to be refractory to degeneration ratherthan just degenerating on a slower time scale.

Discussion; Proposed Mechanisms

1.Random fluctuations of some quality (e.g. toxic gainfunction) may reach a threshold value starts off thedegeneration in certain MNs.

2. There are extra-neuronal factors that influence who is“Loser” and “compensator”; subject to differentinfluences from glial cells etc. or vasculature.

3. MNs may vary in level of SOD1 they express.

4. There is some endogenous quality of a certain subset ofMNs that make them more susceptible to pathology; MUsize, fibre type, trophic factor receptors etc. or someunknown quality.

Take-Home Messages• In ALS there are essentially two subsets of MNs:

Degenerating Axons and Compensatory Axons.• Either one or the other, seldom both.

• Prior to disease-onset all MNs appear healthy andindistinct.

• Some intrinsic property of MNs governs theirsusceptibility.

• ALS is a distal axonopathy: degenerating axons havewinter-tree appearance and axon degeneration occursbefore changes in glial cells or post-synaptic site.

Critical Analyses

• Good Points:• Used a well Characterized ALS Model• Compared results with past research and added

information to the field.• Used in vivo time-lapse imaging to gain insight into

disease progression.

•Bad Points:•Time intervals for in vivo imaging were veryill-defined.

•No recognition of pathology beforesymptom onset.

Further Work• Perform experiments that can confirm or deny

that slow-twitch are refractory to diseasecompared to fast twitch.

• Select candidate trophic factors/receptors thatmay have an influence on pathology (e.g. axonaltransport, hypoxic response) and investigate anyintrinsic differences between subsets of MNs ina disease model.

• Repeat time lapse imaging and characterise thepresymptomatic pathology of ALS: may provideclues in to molecular mechanisms

Wlds mice

• Spontaneous mutant• “Slow Wallerian degeneration”

• Novel chimeric gene from splicing offragments of Ube4b and Nmnat1 on Ch. 4

• Novel gene product

Wlds mice

• Transected axons survive 4 weeks post-transection, functionally for 2 weeks

• Also protects against toxin-induced axondegeneration

• Mechanisms not known

Wlds mice• Also protect against genetic axonal degen. –

Pmn mouse model– Protein-zero-deficient mouse model

• Prevents MN loss, slow disease progression

• ↓ no. of axonal spheroids (common to mouseand human ALS)

Wlds mice in ALS – SOD1 Models

• SOD1-mediated MN loss:– Distal proximal

• Many NMJ denervated before noticeablechange to ventral root/lumbar MNs.

• Can Wlds genotype offer protection?

Breeding

• Wanted to compare mice carrying SODG93A

mutations and wereWlds -/+/++

Methods• Rotarod testing, weekly

• Measure of disease onset

• hSOD1 quantification• Anaesthetisation• Killing

• Once ‘dead’ = can’t right themselves in 30s when placed ontheir back

• Fixing• Microscopy

• Microscopy– End plate/NMJ– Bungarotoxin– Monoclonal ABs to neurofilament NF160– 2o AB– Fluorescent microscopy

Endplates deemed:‘innervated’, ‘denervated’ or ‘intermediate’

• Microscopy– Investigated nerve root– Transectioned sciatic nerve

– Distal stumps then examined 5d later

RESULTS

• ROTAROD testing:

No difference betweenSOD/WT and SOD/Wlds

Insignificant delay inSOD/Wlds

Progressive ↓ in aptitude (timespent on rotarod)

RESULTS - SURVIVAL

SOD/WT 131.0 ± 3.7 days

SOD/Wlds 141.9 ± 8.9 days

SOD/Wlds/Wlds 140.3 ± 5.0 days

Wlds gene prolongs survival for ~ 10 days,irrespective of homo/heterozygosity.

RESULTS - SURVIVAL

Sex-dependent survivalin mice with Wlds gene

RESULTS-SCIATICTRANSECTIONS

Wlds phenotype retained in SOD/Wlds mice

RESULTS-AXONAL PATHOLOGY

Wlds mice have a ahigher % ofinnervated motorendplates at 80d,100d and death

The only significantdifference is at 80d

RESULTS-SENSORYNEUROPATHY

RESULTS-SENSORY

NEUROPATHY

CONCLUSIONS

• Wlds modestly ↑ survivial in SOD1g93A mice

• Greater protection in females-males

• No increased homozygote protection

CONCLUSIONS

• Evidence that MN pathology in SOD is a‘dying back’ process, beginning at NMJ?

• Age-dependent Wlds synapse protection

• Non-synaptic events of other models.

CONCLUSIONS

Action of Wlds

• Not by ↓ copy number/expression of SOD• Sex-dependece

– Androgens known to support MN growth and regeneration– Low serum [testosterone] in many ALS patients– No other known Wlds sex-dependence

CONCLUSIONS

SENSORY DEFICITS

• New finding in this model– Non-progressive degeneration

– Eventual attempts at regeneration?

• Matches human ALS clinical picture

Selective vulnerability and pruning of phasic motoneuron axons in motoneuron diseasealleviated by CNTF.

San Pun1, 2, Alexandre Ferrão Santos1, 2, Smita Saxena1, 2, Lan Xu1 & Pico Caroni1

Burning Questions• 1.What mechanisms make fast motor units more

vulnerable than slow units to degeneration in SOD1transgenic mice?

• 2.How (and why) does the mechanism of degenerationof motor terminals differ in response to hypoxia(ischemia/reperfusion), compared with nerve injury(Wallerian degeneration)?

• 3. How could attempts to use the NMJ as a model ofdisease be improved?

• 4. What other animal models of MND are there?

Selective vulnerability andpruning of phasic motoneurondisease alleviated by CNTF

Pun et al. 2006

Background

• G93A SOD1 mice have a predictable pattern of disease progression– Show 1st clinical signs at P80-90– But NMJs are lost from P50 onwards

• Loss due to difference between motor neurons and/or muscle fibres

• 3 types of motor neurons; FF, FR and S – establish motor units with distinctfatigue and force properties

• Characteristic pattern of denervation may reflect selective vulnerabilities ofsubtypes of motor neurons, muscles and/or motor units

Aims

• To quantitatively map the innervation of hindlimb musclecompartments by motor neurons and their functionalsubtypes

• Explore principles and mechanisms behind early diseaseprogression

A quantitative map ofgastrocnemius innervation by MNs

• LGC divided into 3 nonoverlappingcompartments

• Branch selection by MNs definedsubcompartments of musclesinnervated by nonoverlapping sets ofMNs

• 8 subcompartments

• Numbers of large myelinated axonswithin intramuscular nerve brancheswere stereotyped

• Number of motor units persubcompartment highly reproducibleacross different mice

3 subcompartment classes withrespect to denervation

• 2 temporally well-definedepisodes of axon loss inmuscle

• 3 distinct classes ofmuscle subcompartments

• Classes dramatically differin patterns of denervation

Analyse denervation patterns in G93A SOD1 mice as a function of age:

3 subcompartment classes withrespect to denervation

• Class 1: all NMJs abruptly &permanently denervated between P48-52

• All large MN axons lost between P50-55

• Class 2: some intramuscular nerveslost between P50-55

• Denervated NMJs (P48-55) - partlyreinnervated at P60 but not stable

• 2nd phase of abrupt denervation(P80-90) – permanent loss

• Class 3: fractions of axons lost ineach episode

• Hardly any denervated NMJs fromP60-120

• Resisting axons reinnervated NMJsthrough collateral sprouts

Synchronous pruning of FF, andthen FR motoneuron axons

AIM: To determine whether motor unit subtypes might reflect known motor neuronsubtypes

• Each subtype of motor neurons exclusively innervates muscle fibres of their ownsubtype - FF: type-IIb; FR: type-IIa; S: type-I

• Class 1: exclusively innervated by FFmotor neurons

• Class 2: innervated by FF and FRmotor neurons

• Class 3: innervated by FF, FR and Smotor neurons

Synchronous pruning of FF, andthen FR motoneuron axons

FF motor neurons: (i) abruptly disconnect peripheral synapses at P48-52

(ii) lose intramuscular nerve branches at P50-55 (1st loss episode)

FR motor neurons: (i) initially sprout to reinnervate NMJs on type-IIb muscle fibers

(ii) Become increasingly incapable of maintaining additional NMJs

(iii) Prune intramuscular nerve branches at P80-90 (2nd loss episode)

S motor neurons: (i) compensate efficiently through sprouting

(ii) Continue to maintain greatly expanded motor units until death

Conclusions:

Selective vulnerabilities to diseaseof FF and FR motor neuron axons

Crush-reinnervation experiments before denervation episodes:

• P30 – reinnervation of wild-type and G93A

• P38 – no axons reinnervate Class-1 NMJs but did reinnervate Class-2

•Therefore selective reinnervation deficit in FF axons after P38-45 – beforedenervation

Forced resistant soleus motor neurons to innervate vulnerable tibialis anteriormuscle

Selective vulnerabilities to diseaseof FF and FR motor neuron axons

• delay in nerve regeneration

• reduced reoccupation of NMJs by regenerating axons

Conclusion:

• patterns of denervation reflect selective vulnerability properties of thecorresponding presynaptic motor neuron axons

Synaptic vesicle stalling anddepletion in axonal vulnerability

To determine how early and selective defects in axonal synaptic vesicle transportrelate to selective losses of peripheral synapses and intramuscular axonarborization

• complete synaptic vesicle loss in Class-1subcompartments between P38 and P46

• synaptic vesicle loss precedes axon loss

• “Dying back” process in FALS specifically associatedwith selective vulnerability of axonal transport – 1st inFF, then FR axons – leading to synaptic vesiclestalling and loss from NMJs

• FF motor neurons vulnerable from P38-38

• FR motor neurons vulnerable from P50-55

• No synaptic vesicle loss seen before then

Comparable axonal vulnerabilityphases in FALS models

Do these characteristic patterns and time courses of axonal vulnerability in G93ASOD1 mice generalise to mouse models of FALS? If so, to what extent?

Analysed muscle innervation in G85R SOD1 transgenic mice – differs to G93A:

• disease caused by SOD1 mutant with different stability and toxicity properties

• disease onset greatly delayed

Comparable axonal vulnerabilityphases in FALS models

• Peripheral pruning of FF motoneuronaxons in G85R mice

• Axons lost at 6.2 months

• Similar to G93A at P85-90

• Compared innervation patterns

Conclusion:

• very similar patterns and time coursesof selective motor neuron axonvulnerabilities

To see if observed axonal vulnerability patterns in G93A mice can be generalisedto mouse models of FALS

CNTF specifically alleviates axonalvulnerability in FALS

Daily applications of CNTF fromP38-68 protected FF axons from:

(i) synaptic vesicle loss

(ii) denervation and axon pruning

CNTF applications from P55-80:

Protected FR motor units bymaintaining expanded size andoccupancy state of NMJs

• CNTF preserves retrograde transport and delays disease progression

CNTF protects against several aspects of disease-associated vulnerability

By activating processes that counteract the mechanism of axonal vulnerability

CNTF specifically alleviates axonalvulnerability in FALS

Analysed organisation of neurofilaments to investigate candidate targets ofCNTF protection

Electron microscopy: average densities similar in wild-type

• No differences at P30

• P45 – greatly reduced in SOL, slightly elevated in FF axons

• +CNTF – reduction in neurofilaments in both

Therefore, CNTF boosts axonal resistance to disease

CNTF specifically alleviates axonalvulnerability in FALS

Searched for molecular markers upregulated in motor neurons at the time of peripheralaxon pruning.

Bc12a1-a = antiapoptotic protein, regulated by stress in motor neurons

• upregulation from P45 onwards

• daily applications of CNTF prevented upregulation of Bc12a1-a

Upregulation of Bc12a1-a at P45 reflects onset of stress response to axonal vulnerability inFF motor neurons

Downregulation by CNTF indicates CNTF specifically alleviates axonal vulnerability in FALS

From axonal pruning to lactacystin-sensitivepruning

To investigate the relationship between axonal vulnerability and NMJ denervation andperipheral axon pruning

CNTF applications from P45 onwards were ineffective in preventing the loss ofperipheral innervation and intramuscular axons at FF motor units

CNTF still effective in preventing Bc12a1-a upregulation at motoneuron cellbodies at P55

From axonal pruning to lactacystin-sensitivepruning

Daily applications of lactacystin from P45 onward prevented peripheral pruningand denervation up to P54

Application of lactacystin from P35 or P45 was ineffective in preventing stallingand depletion of synaptic vesicles from NMJs or Bc12a1-a upregulation in MNs.

Conclusion: CNTF specifically alleviates axonal vulnerability in FALS

BUT does not prevent lactacystin-sensitive peripheral pruning of NMJs andintramuscular nerves late in the vulnerability process

Summary1. Produced quantitative topographic account of motor units innervating the LGC in the

mouse

2. Disease induced 2 episodes of axon loss – P50-55 and P80-90

3 distinct classes of muscle subcompartments

3. FF MNs abruptly disconnect peripheral synapses at P48-52; lose intramuscular nervebranches at P50-55 (1st loss episode)

FR MNs sprout to partially reinnervate NMJs but prune intramuscular nerve branchesat P80-90 (2nd loss episode)

S MNs compensate efficiently through sprouting

4. Patterns of denervation in FALS mice reflect selective vulnerability properties ofcorresponding presynaptic MN axons

5. “Dying back” process in FALS specifically associated with selective vulnerability ofaxonal transport – 1st in FF, then FR axons – leading to synaptic vesicle stalling andloss from NMJs

6. Similar axon vulnerability phases in FALS models

7. CNTF specifically alleviates axonal vulnerability in FALS

8. CNTF does not prevent lactacystin-sensitive peripheral pruning of NMJs andintramuscular nerves late in vulnerability process

Conclusions• Axons of FF and then FR motoneurons are selectively vulnerable at well-defined

times early in the disease

• S motor neuron axons resist and compensate through sprouting and reinnervation

• Axonal vulnerability involves: absence of neurofilament adjustments, synaptic vesiclestalling, loss of synaptic vesicles from all peripheral synapses and upregulation ofBc12a1-a in motor neurons

• Followed by lactacystin-sensitive pruning of all peripheral synapses and terminalaxon branches

• Axonal vulnerability alleviated by applications of CNTF

Discussion• Axonal protection delays but does not prevent disease progression - other

disease-related influences impairing MN function?

• FF axon vulnerability useful as a diagnostic tool?

• Consistent with previous work – slow type NMJs more resistant todenervation in FALS

• Showed that it’s motor neuron axons – not NMJs or muscles that areselectively vulnerable

• Disease progression involves abrupt pruning events – not gradual loss ofsynapses

• Disease processes in different forms of ALS converge onto shared finalpathway of selective axon vulnerabilities

Discussion

• Findings may apply to other major neurodegenerativediseases

• Therapeutic strategies aimed at boosting resistance andrepair potential of more resistant axons during earlyclinical phases – using axon protecting factors

Early vulnerability toischemia/reperfusion injury in

motorterminals innervating fast

muscles of SOD1-G93A miceExp Neurol. 2007 March ; 204(1):

411–420.Gavriel David, Khanh Nguyen, and

Ellen F. Barrett,

Background

• Much previous research focused on changes in thespinal cord.

• Several studies suggest changes in axons and nerveterminals may precede symptoms.

Evidence• Fisher et al (2004) - more peripheral damage than

central in ALS patient after premature death.• Rich et al (2004) - evidence for distal before central

defects in canine ALS.• Mariotti et al (2002) – mechanical injury of peripheral

nerves can accelerate disease progression• Previous research indicates motor nerve terminals are

effected by I/R• Axons, schwann cells and muscle fibres show no

change.

Questions

• Are nerve terminals in G93A-SOD1 mousemodel of ALS more vulnerable comparedto WT and when this occurs.

• Do nerve terminals innervating differentmuscle types are differentially sensitive toI/R injury.

Are Motor Terminals inSOD1 mutant moresensitive to I/R thanWT?

•Extensive de-nervationseen in SOD1 mutantsafter I/R injury.

•No change in Wild Type

Fig1

Are fast twitchmuscles Morevulnerable than slowtwitch?

•Fast twitch muscleswere significantly morevulnerable to I/R thanslow twich.

•Decline seen by P31 –before symptom onset.

•Result reproduced inplantaris

Fig2

How does duration of ischemia affectdenervation?

Fig3

Is denervation correlatedwith end plate area?Could it be caused bydamage to neuron duringI/R

•Endplate occupancy notcorrelated with endplatearea.

•Mechanical injury doesnot cause denervation.

Fig4

Post I/R denervation ofEDL SOD1-G93Aendplates developsrapidly

•Images showed thatdennervation occurredbetween 35 and 100minutes ofreoxygenation.•Fluorescencemeasurements indicategreatest loss between 80and 120 minutes.

EDL motor terminals inSOD1- G93A mice can re-sprout following I/Rinjury.

•Following I/R stress EDLmuscles able to re-sprout(P 65 and P89)•Results show slightly lessre-innervation in the P89mice.

Summary of Findings• G93A SOD1 mice are more susceptible to I/R injury

that WT at ages precede symptom onset.• Fast twitch muscle fibres (EDL) = more vulnerable

than slow twitch (plantaris and soleus).• This process is rapid, not affected by endplate area

and not an artefact of axon damage.• Post I/R axons in G93A SOD1 mice are capable of re-

sprouting even at quite late ages.• Results support observations of selective decline of

type IIb motor units in G93a- SOD1 mice (Hegedus etal)

• Finding of atrophy in EDL but not soleus in SOD1-G93A mice (Derave et al)

Interpretations

• Although re-sprouting occurs,regenerating axons are particularlysensitive to stress.

• Even if they don’t die, regenerating axonsmay fail to re-innervate all endplates –disease acceleration.

• Supports evidence of disease progressionafter intensive exercise in disease mice.

• Mice lacking VEGF crossed with SOD1speeds disease progression.

Implications• Results implicate motor nerve terminal as

significant sight of nerve damage in mouse modelsof ALS and possibly in humans.

• If so then protecting is might slow diseaseprogression.

• Neurotrophic factors are neuroprotective – thoughtto be due to retrograde transport, may be due toactions at the nerve terminal.

• Support – VEGF = neuroprotective but is nottransported.

• Neurotrophic factors released from muscle = moreeffective than released from glial cells.

Limitations and Future Work

• Could it be to do with a lack ofnutrient/other factors delivered in theblood not just oxidative stress?

• Although there are suggestions as to whythey are more vulnerable it is not knownexactly why.