Mature NMJ’s are mononeuronally innervated · 2012. 10. 1. · Mature NMJ’s are mononeuronally innervated Motor units are expanded and muscle fibres hyperinnervated in neonates

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Development and Degeneration of Neuromuscular Synapses

GillingwaterCourt

5 µm

Proliferation

Migration

Aggregation

Axonoutgrowth

Synapseformation

Cell Death SynapseElimination

InductionProgressive

Regressive

Apoptosis Wallerian Degeneration Synaptosis

+/+ neonate Axotomised Wld

x x

Neuropathy

~ ~

A

B

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The The ‘‘Life CycleLife Cycle’’ of Neuromuscular Synapses of Neuromuscular Synapses

500 µm

Mature NMJ’s are mononeuronally innervated

Motor units are expanded and muscle fibres hyperinnervated in neonates

50 µm

P5 DL

Teriakidis

3

1. Synapse formation

2. Synapse elimination

3. Competition & ‘intrinsic withdrawal’

4. Synaptic degeneration : the WldS mouse

5. Spinal Muscular Atrophy

Synapse formation

S. Ramon y Cajal, ca 1900, identifies neuronal growth cones

4

http://growthcones.neuroscience.umn.edu/Videos.html

Growth cones both extend and retract

Growth cone arrest and AChR clustering coincide

http://faculty.washington.edu/afolch/images/Concept_Synaptogen.jpg

Acetylcholine receptors cluster under theinfluence of Agrin

5

http://www.mun.ca/biology/desmid/brian/BIOL3530/DB_Ch11/fig11_36.jpg

Musk

AChR

NRG receptor (ErbB)

Agrin clusters ACh receptors via Muscle-Specific KinaseNeuregulin modulates AChR synthesis via ErbB receptors

Pun S, Sigrist M, Santos AF, Ruegg MA, Sanes JR, Jessell TM, Arber S, Caroni P.An intrinsic distinction inneuromuscular junction assembly and maintenance in different skeletal muscles. Neuron. 2002 Apr 25;34(3):357-70.

Synapses form and mature by two different pathways

The size and complexity of the motor endplate increase postnatally

“Plaque”

“Pretzel”

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Monoinnervated NMJ’s are stable throughout life

Balice-Gordon & Lichtman (1990) J Neurosci 10, 894

Quantal Content (variancemethod) at NMJ of rat HD

0 100 200 300 4000

50

100First EPPPlateau EPP (10 Hz)

Age

(Based on Kelly & Roberts, 1977 and Kelly, 1978)

Neonate: AChR - γ

Adult AChR - ε

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Missias et al. (1997) Development 124, 5075

AChR-ε are required for long-term maintenance of NMJ and survival

Age (days)

Witzemann et al. (1996) PNAS 93, 13286

F.Court et al. J Cell Sci 121,3901-3911

Four cell-types at mammalian NMJ

NFSV2/AChR S100/AChR

Kranocytes become restricted to the end plate region postnatally

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Synapse elimination


J.F. Tello Polyneuronal innervation in fetal human muscle

(1917)

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Man

Mouse/Rat

Birth

Birth

PI= [(A+B)-AB]/A

AB

AB

A B A BPI= AB/A

Physiological methods of measuring PI

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Walsh & Lichtman (2003). Neuron

Repeated visualisation of synapse elimination in vivo

Lichtman et al (1987) J Neurosci

Time lapse imaging of synapse elimination

Walsh & Lichtman (2003) Neuron 37,67-73

π

π

µ

µ

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Loss of motor neurones?Or elimination of connections?

Motor unit size can be estimated from theirisometric forces

Loss of motor neurones..XElimination of connections ✓

Motor unit size decreases postnatally

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Keller-Peck, C. et al.(2001) Neuron 31,381-394

Neurones retract some of their synapses while stabilising others

Tapia JC, et al. Pervasive synaptic branch removal in the mammalianneuromuscular system at birth. Neuron. 2012 Jun 7;74(5):816-29.

Motor neurones innervate massive numbers of muscle fibres prenatally

Synapse elimination is competitive: or is it?

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Competition

The negative effect that one neurone or synapsehas on others by consuming, or controllingaccess to, resources that are limited inavailability.(Based on a definition by Keddy, P.(1989) Competition. Chapman & Hall)

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Nguyen QT, Parsadanian AS, Snider WD, Lichtman JW (1998) Science 279:1725–1729.

Transgenic expression of a growth factor, GDNF, delays elimination

MPN LPN SN

4DL

X

Betz,W.J.,Caldwell,J.H. & Ribchester,R.R.(1979). The size of motor units during postnataldevelopment of rat lumbrical muscle. J.Physiol. 297,463-478.

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Evidence forcompetiton: nochange in size ofsurviving motor unitsafter partialdenervation at birth

Evidence againstcompetiton: survivingmotor unit sizecontinues to declineafter partialdenervation at birth

Fladby & Jansen (1987) Acta Physiol Scand. 129,239-246.

Betz, Caldwell & Ribchester (1980) J.Physiol. 303,265-279

“Competition” or “Intrinsic Withdrawal”?

We can determine motor unit size in thy1.2-YFP mice bycounting the number of muscle fibres that are innervated

Adrianna Teriakidis

Neonate~ 1 month after

partial denervation

Adrianna Teriakidis

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Only large motor units undergo intrinsic withdrawal

Adrianna Teriakidis

Kasthuri & Lichtman (2003) Nature

Is every one of a dominant motorneurone’s synapses a winner?

Motor units may compete in a “dominance hierarchy”

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Birth-20 days +30 days

MN Form

MN Die

MF Form

NMJ Form

Myelin Form

NMJ Elim

AChR γ->εNMJ Reshape

NMJ Expand

Summary of key stages in the development of rodent NMJ’s

Progressive

RegressiveRemodel

SUMMARY

1. Neonatal synapse elimination results from amostly-competitive local interaction betweenmotor nerve terminals at endplates, reducingmotor unit size postnatally.

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Synapse degeneration


“Wallerian” Degeneration

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Axons degenerate after a latent period of about 36 hours

Beirowski et al. 2004

Slater, 1966, Nature 209,305-307.

Synaptic failure precedes axon degeneration

6h axotomy 15h axotomy

Winlow & Usherwood (1975) J Neurocytol 4,377-394

Synaptic vesicles are degraded within 6-15 h

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24hrs axotomy: remnants of terminal engulfed by Schwann cell

Gillingwater

1 µm

★ NT

★ SC

Enter the WldS mouse…

Hint: It’s on a C57Bl/6 background.…

Axons are protected from degeneration in WldS mutant mice

M.C. Brown, V.H. Perry et al. (1989) Eur J Neurosci 6,420-428

UnoperatedWt 7d x WldS7d x

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0+/+3d X

WldS/WldS3d X

Axonal and synaptic degeneration/protection in thy1.2 -YFP16 /WldS mice

YFP16:WldS - 5d axotomy

Gillingwater/ D.Thomson

Ch.2

5 mV

10.00 ms

Ch.2

5 mV

10.00 ms

Ch.2

5 mV

10.00 ms

Synaptic degeneration precedes axonal degeneration in WldS mice

Synapses retract asynchronously within motor units: Thy1-YFP:H/WldS 5 Days Post Sciatic Lesion

Gillingwater

22

Axotomised WldS synapses remain functional for several days

D. Thomson

2

2

2 Month Wlds

Days Post Axotomy

0 2 4 6 8 10

% F

ibre

s S

ho

win

g

Ev

ok

ed

Ac

tivity

0

20

40

60

80

100Mean Data

Best Sigmoidal Fit to Mean Data

2

2

2

WT

% fi

bres

with

act

ivity

Nmnat-1 Rbp7 Ube4b

N C

N70-Ube4bWld-18

Nmnat-1

Genomic DNA

Chimeric Wlds Protein

VCP binding domain

Michael Coleman/Laura Conforti/Bogdan Beirowski

NAD catalytic domainNLS domain

The WldS neuroprotective gene is a Ube4b-Nmnat1 chimera

+

WldS

Tg-4836

A

5 mN

5 s

ΔNLS(R213A/R215A)WldS- Line 3

B

0.1 mN

5 s

WldS

Exclusion of WldS from the nucleus strengthens the neuromuscular synaptic protection phenotype

Beirowski et al (2009) J Neurosci 29: 653-638

merge

AM1-43/BTX

WldS

merge

AM1-43/BTX

ΔNLS(R213A/R215A)

WldS- Line 3

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The NAD biosynthesis pathways from nicotinamide , based on Revolla et al, 2004Mack et al demonstrated a 4 fold increase in Nmnat1 enzymatic activity but no increase inthe amount of NAD in the nervous system in WldS mice. The theory that WldS protection ismediated by increased Nmnat1 activity, argues that the increased Nmnat1 activity results inan increase in flux through the Sir2 pathway known to regulate survival during nutrientrestriction in yeast.

Sirtinolinhibits

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Axonal Nmnat-2 levels decline rapidly after nerve injury

Gilley & Coleman (2010) PLoS Biol

Axonal Nmnat-2 knockdown simulates Wallerian degeneration

Figure I.3

The NAD biosynthesis pathways from nicotinamide , based on Revolla et al, 2004Mack et al demonstrated a 4 fold increase in Nmnat1 enzymatic activity but no increase inthe amount of NAD in the nervous system in WldS mice. The theory that WldS protection ismediated by increased Nmnat1 activity, argues that the increased Nmnat1 activity results inan increase in flux through the Sir2 pathway known to regulate survival during nutrientrestriction in yeast.

Sirtinolinhibits

FK866

25

0

20

40

60

80

100

Act

ive

fibre

s at

16h

ex

vivo

(%)

WldS 0 1 10

WT+FK866 (µM)

Rosalind Brown/Laura Conforti

NAMPT-inhibitor FK866 preserves wild-type axons


Gillingwater & Ribchester 2001 J Physiol. 534:627-39Gillingwater & Ribchester 2003 J Neurocytol. 32:863-81

X

bcl2 WldS [Gene X]

“Compartmental Neurodegeneration” Hypothesis

SUMMARY


2. Synaptic degeneration after axotomy in adultmuscle is triggered by a fall in Nmnat-2 inaxons. The WldS mutant protein substitutesfor this enzyme, keeping NMN substratebelow toxic levels, and slowing synapticdegeneration.

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Spinal Muscular Atrophy (SMA)

Spinal Muscular Atrophy• Neurodegenerative disorder with autosomal recessive genetic heredity in 95% of cases.

• Degeneration of α-motor neurons of the spinal cord, resulting in muscle weakness and progressive paralysis.

• Incidence about 5-7 per 100,000 live births. The prevalence of individuals with the carrier state is 1 in 80.

• The most common degenerative disease of the nervous system in children and the leading heritable cause of infant mortality

• Caused by a homozygous deletion of the survival motor neuron (SMN1) gene on chromosome 5.

• SMN2 has reduced stability due to C-to-T transition in exon 7 (--> SMNΔ7 protein)

• Onset/severity of SMA varies depending on number of SMN2 gene copies (up to 8) Type I (Werdnig-Hoffman Disease) terminal in neonates; Type IV - adult onset.

• Normal function of SMN protein is unknown. It is expressed in many cell types, and has been implicated in a range of cellular functions, including small nuclear ribonucleoprotein (snRNP) assembly.

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Mouse Models of SMA

• Mice possess a single Smn gene, which has 82% amino acid identity with its human homolog and a similar expression pattern

• Homozygous Smn deletion results in massive embryonic cell death and lethality at birth

• Expression of a human SMN2 transgene on the Smn-null background rescues lethality and transgene copy number modifies severity

• Introduction of a second transgene, containing human SMNΔ7 extends the lifespan from 6 to 13 days

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2010


+/+ neonate Axotomised Wld

x x

Neuropathy

~ ~

A

B

29

SUMMARY


2. Synaptic degeneration after axotomy in adultmuscle is triggered by a fall in Nmnat-2 inaxons. The WldS mutant protein substitutesfor this enzyme, keeping NMN substratebelow toxic levels, and slowing synapticdegeneration.

3. Synapse degenerate before axons inneonatal spinal muscular atrophy.

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Mature NMJ’s are mononeuronally innervated · 2012. 10. 1. · Mature NMJ’s are mononeuronally innervated Motor units are expanded and muscle fibres hyperinnervated in neonates