1
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
2
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
The The ‘‘Life CycleLife Cycle’’ of Neuromuscular Synapses of Neuromuscular Synapses
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
16
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
The The ‘‘Life CycleLife Cycle’’ of Neuromuscular Synapses of Neuromuscular Synapses
“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
20
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
Apoptosis Wallerian Degeneration Synaptosis
Gillingwater & Ribchester 2001 J Physiol. 534:627-39Gillingwater & Ribchester 2003 J Neurocytol. 32:863-81
X
bcl2 WldS [Gene X]
“Compartmental Neurodegeneration” Hypothesis
SUMMARY
1. Neonatal synapse elimination results from amostly-competitive local interaction betweenmotor nerve terminals at endplates, reducingmotor unit size postnatally.
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
Apoptosis Wallerian Degeneration Synaptosis
+/+ neonate Axotomised Wld
x x
Neuropathy
~ ~
A
B
29
SUMMARY
1. Neonatal synapse elimination results from amostly-competitive local interaction betweenmotor nerve terminals at endplates, reducingmotor unit size postnatally.
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.