In vivo Time-Lapse imaging of synapticIn vivo Time-Lapse imaging of synaptictakeover associated with naturallytakeover associated with naturally

occurring synapse eliminationoccurring synapse eliminationIn this presentation we will analysis the findings of Walsh and Lichtman to

show:

• In vivo observation of synaptic takeover at immature poly innervatedneuromuscular junctions.

• the unpredictable and not always monotonic shift of competitiveaxons in favour of one and then the other, referred to as flip-flop.

• The dynamics of axons during neuromuscular junction developmentand the impact this may have on synaptic elimination

• The unexpected occurrence of synaptic elimination without synaptictakeover

• And finally how all of these lead to the stabilisation and observedmorphology of the mature neuromuscular junctions

So...So...WHYWHY try to observe synaptic try to observe synaptictakeover during development?takeover during development?

The authors of this paper ‘sought to directly image thiscompetitive process to determine the time course andstructural changes that accompany axon eliminationand see if the structural alterations in the remaininginput might accompany the elimination of acompetitor.’

HOWHOW did they observe synapse did they observe synapsekinetics in vivo?kinetics in vivo?

GFP subset expressorCFP 100% expressor

Double transgenic mouse

In vivo time lapse imaging

WatchingWatchingsynapsesynapse

elimination inelimination invivovivo

Synaptic takeoverSynaptic takeover

The competitive process by which one input wouldwithdraw completely and another would elaborate into theunoccupied space could take several days.

Flip flop Flip flop

•A process by which inputslost territory and regained it,was observed in 3 junctions.

•Synapses taken over in thesecond week in all cases werestill present in adulthood.

Dynamism in singly innervatedDynamism in singly innervatedjunctionsjunctions

•Singly innervatedjunctions in the secondpostnatal week exhibitedprocess extensions,retractions, and terminalcaliber changes.

•2 weeks later thisdynamics was lost andjunctions presented thestability seen in themature adult junction.

Synapse elimination withoutSynapse elimination withouttakeovertakeover

•In 4/28 junctions the vacated site was not reoccupiedby the surviving axon. AChR labelling of the eliminatedaxon is rapidly and completely lost.

SummarySummary• In vivo observation of synaptic takeover at

immature poly innervated neuromuscular junctions.

• the unpredictable and not always monotonic shift ofcompetitive axons in favour of one and then theother, referred to as flip-flop.

• The dynamics of axons during neuromuscularjunction development compared to at maturejunctions

• The unexpected occurrence of synaptic eliminationwithout synaptic takeover

Biological implicationsBiological implications

• AChR are lost if there is no takeover and therefore some kind ofdismantling mechanism is caused by the presence of anoccupied synaptic site at the same junction.

• It has been suggested that axons takeover neighbouringterritory in an opportune way, as the other axon withdraws thecompeting axon grows into the space in response to theavailability of secreted growth factor. Or alternatively thoseaxons with the highest intrinsic dynamics are more available toreoccupy space that becomes available.

• Synaptic flip flop may also be explained by a change inresources available at the junction of interest due to competitiveoutcomes at other branches of the neurons.

Future studiesFuture studies

• What are these possible growth factors? What causes inputs to be eliminated???

• Compare competition between junctions tosee if takeover at one branch effectsresults at another.

• What causes AchR site dissembling?

• Why do inputs become stable?

LimitationsLimitations• The re-entry of junctions

• The waste of mice, small sample sizebrainbow?

• The inability to gain temporalinformation.

• Relabeling of AChR sites

Big burning questions!!!Big burning questions!!!

• Do the methods justify the conclusionsof this paper? How do they giveinsight into pre and postsynapticchanges during synapse elimination?

Age-dependent synapsewithdrawal at axotomised

neuromuscular junctions in Wlds

mutant and Ube4b/Nmnattransgenic mice

Thomas H Gillingwater, DerekThomson, Till G A Mack, Ellen M Soffin,

Richard J Mattison, Michael PColeman, and Richard R Ribchester

• In normal neurons Wallerian Degenerationoccurs after injury/damage.

• This does not occur in the axons of Wlds

mutant mice and synapse elimination alsooccurs differently.

• There is a view that different degenerationmechanisms following never damage arecompartmentalised within neurons.

• This paper aims find out whether there is arole for age in synapse withdrawal anddegeneration in Wlds mice.

Intro, background and aimsThe findings

• Age had no effect on Wld expressioneven when different promoters areused.

After axotomy:• Preservation of Wld axons is unaffected

by age.• Preservation of Wld synaptic terminals

IS affected by age.

Methods• Wlds mice and transgenic mice were used• Surgery: nerves innervating FDB,

Lumberical, TA muscles• Electrophysiology• Electron microscopy• Axon counts• NMJ staining• Fluorescence imaging• Western Blotting

Results

TheWldsmuta-onshowsitsneuro‐protec-veeffec-venessonaxons,independentofage.

Fluorescence Imaging. 2 months old

Theseimagesshowexamplesoffullyoccupied,par-allyoccupiedandvacantNMJsinWlds

mouseof2months.

There was no relationship between thesize of the endplate and synapse

withdrawal

NMJs:Wlds

mouseof4monthspostaxotomy.

2months

Synapseswereregeneratedandaxotomisedagain

NaturalWldsmutantmice(fig5)

Main findings• Main finding – There are at least two modes ofsynaptic degeneration in Wld expressing mice and thefactor that decides which mode occurs is the maturityof the synapse that is axotomised.

• Axons are protected from degeneration at all ages bythe Wld gene.

• In young Wlds mice, synaptic withdrawal resemblesthat which occurs in neonatal development as a resultof competition .

• In mature synapses the neuro-protective effects of theWlds mutation appears reduced.

Future studies

• Continuous visualisation studies ofsynapse withdrawal in real time. To sethe pattern of synapse withdrawal.

• Mechanism by which the Wld geneinitially protects axons and synapsesfrom degeneration. A role forubiquitination in regulating synapticform/ function?

Wlds protein requires Nmnat activityand a short N-terminal sequence to

protect axons in mice.

Wlds

VCP binding domain Catalytic domain

Ube4b

Wld-18

Nmnat

N C

Nmnat

Nicotinamide mononucleotideadenylyltransferase 1

Catalyses the final step in the salvagepathway of the synthesis of NAD+

Ube4b

Ubiquitin conjugation factor E4 b

Binds to valosin-containing protein (VCP)

No other biochemical properties known

Controversy Nmnat alone is sufficient to protect axons

in vitro, but not in vivo.

Some or all of the N-terminal domains arenecessary - which?

Other forms of neurodegeneration blockedby enzyme dead Wlds in vivo. Is Nmnat achaperone?

What question does this paperask?

Is the VCP binding domain is required?

Is the catalytic activity of Nmnat required?

The answer?

• Wlds protein requires Nnmatactivity and a short N-terminalsequence to protect axons in mice.

ΔN16WldS

VCP binding domain absent

Distal sciatic nerve 5d afterlesion ATX3Wlds

Different VCP binding domain (only 5amino acid residues the same)

Distal sciatic nerve 5d afterlesion

Distal sciatic nerve 14d afterlesion

Preservation of neuromuscularfunction 3d after lesion

ATX3WldS, flexor digitorum brevis

Isometric single twitch20 Hz tetanic tension

Preservation of neuromuscularfunction 3d after lesion

ATX3Wlds Wlds

6ms

200µV

Confocal microscopy W258AWlds

Enzyme dead Wlds (no catalytic site)

Distal sciatic nerve 72h (3d) afterlesion Summary

Big Burning Question How strong is the residual phenotype when

either one of the two components of WldS isinactivated?

Indistinguishable from wild-type.

What are the alternative mechanisms for itsprotective effects?

Nmnat may act as a chaperone, but it is notsufficient for protection.

For the future

Blocking the rate limiting step in NAD+ salvagepartially reversed the Wlds phenotype.

Are there other roles for Nmnat? Are there other roles for the N terminal

domains? Do other metabolites play a role in

degeneration or protection?

For the future

Why does Nmnat need to be targeted toa specific site in the cytoplasm?

How does it act there?

Wlds protein requires Nnmat activityand a short N-terminal sequence to

protect axons in mice.

Identity, developmental restrictionand reactivity of extralaminar

cells capping mammalianneuromuscular junctions.

F.A Court, T.H Gillingwater, S Melrose, D.L Sherman, K.N.Greenshields, A.J Morton, J.B Harris, H.J Willison and R.R

Ribchester

PresentedbyKa-eHoban

Background Information

•NMJ – made up of 3 components: 1)muscle fibres2) neuron terminals and 3) perisynaptic terminalSchwann cells.

•These constituents are essential for intracellularsignalling that control motor function and behaviour.

•Previous suggestions of ‘another cell type’, but nostudies to confirm this.

• ELECTRONMICROGRAPH

IMAGE SHOWINGTHE NMJ

STRUCTURE

Motornerveterminal–green

Musclefibre–brown

TerminalSchwannCell–yellow

Aim and Hypothesis

• Investigate further, and establish therole this ‘new cell type’ has to play atthe NMJ.

• Authors hypothesise that these ‘NMJ-capping cells’ should be considered asan integral part of the NMJ, making itthe 4th cell type present at NMJs.

Study Design and Methods

• The experiments used the Triangularis sterni’ muscleeither from transgenic or mutant generated mice .

• The design of the study meant that each subsequentmethod built upon findings from the previousexperiment.Techniquesused:

•Immuno‐staining•Confocalmicroscopy•Electronmicrography•3Dreconstruc-on•BrdUlabelling•X‐Galstaining

Thewiderangesofdifferenttechniquesused,complementedtheinterpreta-onofeachoftherespec-veresults.

Results1) NMJCappingCellshaveadis-nctImmunocytochemical

Profile

2) Postnatally,NMJCappingCellsarerestrictedtotheNMJ

3) NMJCappingCellsspreadaZerdenerva-on,paralysisormuscularatrophy.

NMJ Capping Cells have adistinct Immunocytochemical

Profile Figure1:showsimmuno‐stainingoftheTSmusclewith2166an:body.

A–showsthelabellingoftheNMJcappingcell(green)

B–showsthestainingextendsbeyondthemotorendplateboundary,bylabellingtheAChRs.

C‐E–moreNMJcappingcellexpression

F–thenumberof‘NMJcappingcells’thatcaptheNMJ

Green=2166an-bodylabels‘newcelltype’=NMJCAPPINGCELLS

Figure2B:showsNMJcappingcellsareanindependentcelltype.

B–showsrPHandAChRlabelling.Ifstainwith2166too–seenooverlap.

Figure3:showstheNMJcappingcellsareCD34andCTB+veandaresituatedoutsidethesynap:cbasallamina.

A–showstheuniformdistribu-onforCD34,inlinewith2166expression.

B–showsop-calcutsofanareawithintheCD34expressionzone.

C–showsthecelltypeliesoutsidethebasallamina.

D–showstheplasmamembraneoftheNMJcappingcellusingCTB(conjugateofcholeratoxinB)

NMJ capping cells areunderstated in current literature

Figure4:showsNMJcappingcellscovermotorendplatesand3Dimageconstruc:onsconfirmthis.

A‐B–showthe4differentcelltypesattheNMJ.

C–3DimagesproveNMJcappingcellscover60%ofthenerveterminalatthisNMJ.

D–EMofaratNMJ.ArrowsshowtheareacoveredbytheNMJcappingcells.

Postnatally, NMJ Capping Cells arerestricted to the NMJ

• This was shown using 2166 antibodyimmunostaining measured at P1->P28.

Figure5A/B/C:showsaNerbirth,NMJcappingcellsbecomemorelocalisedtoNMJ.Asseenbythegreen2166whichbecomesprogressivelyclearer.

NMJ Capping Cells spread afterdenervation, paralysis or muscle

atrophy.

Figure5D/E/F:showsstainingresultsaNerdenerva:onoftheTSmuscle.

Thesefindingsillustrate2newtheories:

1)NMJcappingcellsproliferateaZernerveinjury

2)Thesecellsac-vateterminalSchwanncellsandguidesproutstodenervatedendplates.

Testing Hypothesis 1

BrdU labelling

NeuromuscularParalysis or

AtrophyFigure6:showsNMJcappingcellsspreadinparalysedandatrophicmuscles.

A‐B–showthestaining1dand6daZerparalysisusingbotulinumtoxintypeA.

C–WTTSmusclestaining.

D–R6/2mutantmouseTSmusclestainingshowingsimilarappearancetodenervatedandparalysedmuscle.

Molecules in NMJ Capping CellReaction?

• Adhesion molecules e.g. tenascin-C, N-CAM

Figure7:showsNMJcappingcellsarelooselyassociatedwithtenascin‐Cbutnotneededtolocalise,reactorspreadfromNMJs.

A–no+veimmunostainingfortenascin‐CinWTTSmuscle

B‐C–showsimmunostaining3+6daysaZerdenerva-onrespec-vely.

D‐E–XGalstainingwithlacZreportergene5daysaZerdenerva-on.See+vestainingattheintramuscularaxonandmotorendplate.

Conclusions• New ‘NMJ Capping Cell’ established, in

addition to 3 main cell types at NMJ• NMJ Capping cells are restricted to the

endplate postnatally• These cells react to denervation or paralysis by

proliferation and spreading.• They do not depend on a molecular marker

such as tenascin-C• Termed this new NMJ Capping Cell a

‘Kranocyte’• Kranocytes have a distinct

immunocytochemical profile and this study hasshown they are important in development,maintenance and repair of NMJ

Future Work• Evident link between injury, paralysis and

neuromuscular dieseases – look atKranocyte plasticity

• Potential targets for neuroprotection at theNMJ

BBQ

• If not due to tenascin-C, how dokranocytes associate and disperse inrelation to the nerve terminal? Whatmight be the crucial molecular markers?

KranocytesproliferateanddisperseaZerdenerva-onandparalysis,thisdoesn’tdependontenascin‐C.

Perhapsthereisinvolvementofothermolecularmarkersoracombina-onofmoleculesworkingtogethertomediatethiseffect?