Spatial constraints dictate glial territories at murine - - PowerPoint PPT Presentation



Spatial constraints dictate glial territories at murine neuromuscular junctions

Brill MS, Lichtman JW, Thompson W, Zuo Y, Misgeld T.

Sebastian Anastassiou

Introduction

 Glial cells have many functions around the nervous system.  Synaptic functions:



Monitor neurotransmission



Contain and clear released transmitters



Modulate synaptogenesis and plasticity

 Need a highly organized arrangement.  Many neurological diseases associated with altered glial

morphology and arrangement, e.g. ALS, HD.

 How glial cells establish and maintain their perisynaptic territories

is not known.

 NMJ ideal to study this due to accessibility and size.  Axonal and terminal Schwann cells (A/TSCs).  During development, SC are dynamic and proliferate.  During adulthood, SC numbers are stable.  Denervation causes reactive transformation of SC,

proliferation and growth of processes.

 Essential for guiding regenerating axons (bridge).

NMJ ideal for study

Aims

 What is the territory of individual terminal SC under

normal conditions.

 How is this territory established during development, and

what mechanisms maintain it.

 How single SC territories change after axonal degeneration,

and which signals drive these changes.

Methods

Transgenic Mice

Transgenic SC-GFP mice, express GFP in SCs. Crossbred with thy1-XFP mice to label axons. Transgenic ΔNLS mice – variation of Wlds protein to delay

axon fragmentation after axotomy.

Methods

Viewing individual SC territories

Sequential photobleaching of SCs.

 Confocal microscope  Laser

Sequential dye-filling of SCs.

 Rhodamine dextran

Methods

In-vivo NMJ imaging

Time-lapse confocal microscopy. Combined with photobleaching.

SC ablation

Laser pulse



Results

Experiment 1 and 2

 Used sequential photobleaching and dye filling methods to image

SCs.

 Aim: to compare morphologies of immature and mature TSCs.  Hypothesis 1: TSCs arrange themselves in a highly organized tile-

like manner.

 Hypothesis 2: Two processes by which this arrangement could

emerge:



SC territories are already segregated at immature NMJ as SCs emerge sequentially by local proliferation.



SC territories are initially intermingled and then segregate as development progresses.

 These morphological differences led to hypothesis that cell

dynamics were either involved in or even responsible for the remodeling.

 Used time-lapse imaging of the triangularis sterni muscles.

Experiment 3

Experiment 4

 What determines SC partitioning of the NMJ during these

different developmental stages?

 Hypothesis: Competition for perisynaptic space during SC

segregation determines SC partitioning of NMJ.

 Used the SC ablation method to destroy single SCs.

 Suggests mature terminal SCs’ lack of dynamism is due to

spatial competition.

 Axonal SCs are restricted by additional factors at the

heminode or by intrinsic factors as a result of their differentiation.

Experiment 5

 SCs could also be spatially constrained by axons.  Hypothesis: removing axons would result in SC dynamism

and intermingling.

 Axotomized motor neurons.

 Less dramatic than

SC ablation, but still showed fast volume expansion of SCs.

 Suggests also

underlying axon prevents SC intermingling.

 SCs explore vacated

gutter first then surrounding area following axon removal.

Experiment 6

 Axotomy leads to vacation of synaptic space as well as loss

• f neural activity.

 Axons maintain SC segregation via neural activity or axon

presence?

 Blocked NT with BoTX A.  Used the ΔNLS mice.

 SC segregation is independent of neuronal activity.

Conclusions

 Mature SCs are static and arranged in a tiled manner

around synapses.

 Immature SCs are dynamic, exploring synaptic and

extrasynaptic territory.

 The mature SC arrangement is maintained by competition

for perisynaptic space through axon-glial and glial-glial interactions.

Constraining SCs

 Basal lamina laterally constrains SCs.  Heminode prevents retrograde growth by TSCs.  None of these likely to constrain individual TSCs.  Space filling model rather than homotypic repulsion model:



No continuous expanding and retracting – TSCs in permanent contact.



Axon removal induces SC expansion.



Immature TSCs intermingle.

Big Burning Question

 What regulates the neonatal dynamism and adult

plasticity of terminal Schwann cells?

Exploratory behaviour of immature TSCs

 Immature axon terminals very dynamic:

 Synapse elimination frees up perisynaptic space.  Segregation of terminals from different motor axons may need

some compensatory glial dynamism.

 Seems to be a looser synaptic cell arrangement as axon

terminals extend and retract small processes readily.

 Gradual development of the basal lamina allows immature

axonal and glial processes to be sent outside synaptic boundaries.

Mature TSCs

 Spatial competition prevents mature TSC expansion.  Cannot exclude glial expansion induced by factors released

following cell ablation.

 Unpredicted discovery of phagocytic capacity.

TSCs as models for glial function

 Results show similarities between TSCs and CNS microglia

and astrocytes:

 Phagocytic capacity.  Static and dynamic states at immature and mature stages

respectively.

 Non-overlapping space filling arrangement.

Shortcomings

 Obtaining cell outlines by image subtraction is prone to

noise, creating uncertainty in fine detail.

 Possible phototoxicity by photobleaching (although

evidence suggests it does not occur).

Future Studies

 Use TSCs as disease models, e.g. ALS, HD.  Investigate TSC proliferation and expansion with more

chronic cell ablations.

 Investigate how this affects synaptic function.

 Investigate role of axon in SC segregation more since there

seems to be a slight delay before cell expansion occurs.

Reversing
the
outcome
of
synap4c Elimina4on
at
the
developing neuromuscular
junc4ons
in
vivo: evidence
for
synap4c
compe44on and
its
mechanism

Stephen
G.
Turney
and
Jeff
W.
Lichtman Presented
by:
Brodie
Ballan4ne

Background

• Prior
to
this
paper
lots
of
evidence
that
the

events
that
cause
mul4ple
synap4c
connec4ons to
just
a
single
connec4on
at
the
NMJ
is determined
by
local
mechanisms
at
each individual
NMJ.

• A
few
hypotheses
of
what
this
mechanisms
are:

1.Random
scale
back
of
arbors 2.Fate
predetermined
by
posi4onal
or
molecular cues 3.Compe44on

Aims

• Test
if
synap4c
elimina4on
is
driven
by
interaxonal

compe44on

• When
does
this
elimina4on
process
finally
become

irreversible? Method
–
abla4ng
axon
with
the
higher
chance
of
being maintained
and
seeing
if
fate
of
other
is
reversed.

Laser

• Diode‐pumped
mode
locked
Ti:sapphire
laser
oscillator
• Focused
laser
over
axon
using
scanning
microscope
system
• Axons
fluorescence
was
bleached,
causing
damage
to
one

axon
leaving
adjacent
undamaged.

• 30‐45
minutes
• Caused
die
back
similar
to
that
seen
in
acute
axon

degenera4on.

Experiment
1

• Using
mice
7‐8days
old
• They
located
NMJs
that
were
innervated
my
two
axons
• 87
experiments
failed
• 15
succeeded

10/15
larger
caliber
was
ablated
(easy
to
see
which
was had
larger
caliber)6
of
which
accompanied
less
than 5% 4/15
around
the
same
caliber
(based
of
appearance
and

• ther
factors
they
could
Iden4fy
the
one
that
had
less

territory 1/15
ablated
the
axon
with
the
small
caliber

Experiment
1
cont.

• How
long
did
remaining
axon
con4nue
to
lose

territory
for
un4l
changing
its
fate?

• They
reimaged
3
junc4ons
less
than
24
hours.
• One
at
6,
one
at
12
and
one
at
17h
• Even
at
6
hours
the
remaining
axon
had
lost

no
territory

Results

• In
12/13
axons
images
aeer
24
hours
the

remaining
axon
accompanied
the
en4re
synap4c site.

• The
other
accompanied
75%
• Along
with
thickening
of
the
caliber
of
its

preterminal
branch

Conclusion

Once
one
of
the
compe4ng
axons
(dominant
or not)
are
removed
the
remaining
changes
its
fate to
become
the
dominant
axon
with
no
lag
4me.

Experiment
2

• Abla4ng
of
singly
innervated
NMJ
while
other

axon
recently
eliminated
and
retrac4ng

• Are
the
retrac4ng
axons
irreversible
or
will
they

reverse
there
fate
again?

• Of
the
18
examples
55%
of
the
retrac4ng
axons

reversed
their
fates,
grew
back
and
reinnervated the
whole
junc4on

• It
was
shown
however
that
the
further
away

from
the
junc4on
the
retrac4ng
axon
was
the less
chance
it
would
reinnervate.

Conclusion
–
retrac4ng
axon
can
reverse
their
fate depended
on
their
distance
from
the
junc4on Gives
the
impression
that
growth
s4mula4ng
signals
or stop
the
retrac4on
possess
signals
follow
abla4on

Experiment
3

• Does
this
method
of
axonal
takeover
take
place
in

natural
axonal
elimina4on? 2
Theories Does
one
axon
push
the
other
axon
off
its
site
and takeover Does
one
axon
takeover
in
response
to
one
other vaca4ng
the
site If
lafer
were
true
viewing
AChR
sites
occupied
then unoccupied
and
then
occupied
by
different
axon One
of
these
examples
was
seen
out
of
100s
of
afempts

Experiment
4

• Interested
in
if
small
denerva4on
of
target

cells
are
sufficient
to
s4mulate
a
regenera4on response.

• Carried
out
small
laser
axotomies
of
adult

NMJ,
which
denervated
small
isolated synap4c
boutons.

• 55%
of
these
synap4c
vacancies
were

innervated.
Delay
of
1d
and
complete
aeer
2d

Conclusions

• An
axon
whose
elimina4on
looks
certain
will
take
• ver
all
sites
when
its
compe44ve
neighbor
is

removed

• An
axon
that
is
in
the
process
of
retrac4on
will

grow
back
and
reinnervate
the
junc4on
if
its compe4tor
is
removed
(depending
on
its
distance from
the
junc4on)

• In
natural
axon
elimina4on
its
thought
that
one

axon
retracts
from
a
site
first,
which
causes
a signal
to
nearby
axons
to
takeover.

Further
Work

• Inves4gate
to
possible
signals
that
are

triggered
by
sites
being
vacant
and
cause takeover
by
other
axons A
few
possibili4es

• Schwann
cell
processes
become
ac4vated
and

signal
(GDNF
Strongest
known
motor
neuron s4mulus)

• Vacant
site
on
postsynap4c
cell
signals

Big
Burning
Ques4ons

• Are
the
triggers
for
elimina4on
and
takeover

dis4nct
and
different?

Bibliography

• Turney
SG,
Lichtman
JW.
Reversing
the
outcome
of
synap4c

Elimina4on
at
the
developing
neuromuscular
junc4ons
in vivo:
evidence
for
synap4c
compe44on
and
its
mechanism. PLoS
Biol.
2012
june;
10
(6)
e1001352

• Walsh
MK,
Lichtman
JW.
In
vivo
4me
lapse
imaging
of

synap4c
take
over
associated
with
naturally
occurring synapse
elimina4on.
Neuron.
2003
jan
9;37(1)87:73

Age‐dependent
synapse
withdrawal
at axotomised
neuromuscular
junc8ons
in Wld(s)
mutant
and
Ube4b/Nmnat transgenic
mice

Gillingwater
TH,
Thomson
D,
Mack
TG,
Soffin
EM,
MaKson
RJ,
Coleman
MP, Ribchester
RR.
J
Physiol.
2002
Sep
15;543(Pt
3):739‐55. Presented
by
CharloZe
Dewdney

Contents

• Introduc8on
• Aim
• Methods
• Results
• Discussion
• Big
Burning
Ques8on
• Cri8cal
Analysis
• Future
Work
• References

Introduc8on
to
the
Wlds
mouse

• Wallerian
degenera8on:
a`er
a
lesion
to
a
peripheral

nerve,
distal
axons
and
synap8c
terminals
degenerate first:
“dying
back”
phenomenon.

• Wlds:
“slow
Wallerian
degenera8on
mutant
mouse”,[1]

delays
degenera8on
of
injured
axons
by
10‐fold

• Wlds
gene
mapped
to
chromosome
4,
triplica8on
of

the
exons
of
3
genes:
Ube4b,
Nmnat
&
Rbp7.
The fusion
of
two
genes
(Nmnat1
and
Ube4b)
creates
a chimera

• Axons
vs.
synapses
following
axotomy
in
Wlds
mice

The
effect
of
age
–
2
opinions

• Perry
et
al.
1992[2]
&
Ribchester
et
al.
1995[3]:

the
Wlds
phenotype
is
lost
as
mice
age,
so
by
6 months
of
age
mutant
mice
exhibit
normal rates
of
axon
degenera8on

• Crawford
et
al.
1995:
both
axons
and
synapses

are
equally
well
protected
from
axotomy induced
degenera8on
in
Wlds
mice
of
all ages[4]

The
evidence

Crawford
et
al:
%
innervated
NMJs
in 1
month
old
and
7
month
old
mice[4] Perry
et
al:
size
of
the
ac8on poten8al
compared
to
control
mouse at
given
ages
of
mice,
all
taken
5
days a`er
axotomy[2]

Aim

• “To
resolve
the
discrepancy
between
the

studies
of
Ribchester
et
al.
&
Crawford
et
al.”

• I.e.
is
axon
degenera8on
in
the
Wlds
mouse

dependent
on
age

Methods

• Mice:
mutant
mice
aged
1‐2
months
were
used,
some
were

maintained
un8l
they
reached
older
ages
(4,
7
and
12 months)
needed
for
the
age‐dependency
experiments

• Surgery:
the
scia8c
or
8bial
nerve
was
exposed
when
using

FDB
or
lumbrical
muscles,
the
intercostal
nerves
were lesioned
when
using
transversus
abdomius
muscles

• Electrophysiology:
intracellular
recordings
were
made

between
1
and
10
days
a`er
surgery
using
30
fibres
per muscle
(fibres
selected
at
random)

• Other
studies:
electron
microscopy,
axon
counts,
NMJ

staining,
fluorescence
imaging
and
analysis,
Western bloKng

Study
Design
Overview

• Used
the
methods
described
to
analyse
axon

preserva8on,
synapse
withdrawal
and electrical
ac8vity
at
certain
8me
intervals
post axotomy

• Compared
young
(2‐month
old)
and
mature
(4
• r
7
month
old)
Wlds
mice

Results

How
does
Wld
gene
expression
&
axon
protec8on
change
with
age?

A
‐
Western
blots
showed
that
Wlds protein
expression
didn’t
diminish
with
age B
–
No
difference
in
axon myelina8on
or
neurofilament preserva8on
in
these lesioned
nerves
4
days
post axotomy C
–
No
significant
difference in
number
of
axons
4
days post
axotomy

2
month‐old 7
month‐old

Figure
1

Results

There
is
a
progressive
loss
of
synap8c
terminals
in
2‐month
old Wlds
mice.
All
images
taken
3‐6
days
post
axotomy.

A:
Synapses
protected
from
degenera8on
3‐7 days
a`er
axotomy B:
Reten8on
of
lower
nerve
terminal
but
now

• nly
par/al
occupancy
of
the
upper
endplate

C:
Axon
termina8ng
in
a
retrac8on
bulb D:
2
endplates
on
the
le`
are
occupied,
the
2

• n
the
right
are
vacant

E:
Nerve
terminal
with
intact
mitochondria, synap8c
vesicles
&
membranes F:
Neurofilaments
accumulated
in
the
centre
of the
bouton. G:
Par8ally
occupied
NMJ H,I,J:
Graphs
–
intracellular
recordings
at
5
days post
axotomy. Figure
2

Results

Time
course
of
synapse
withdrawal
in
2‐month‐old
Wlds
mice

Figure
3

Results

What
was
the
effect
of
endplate
size
on
synap8c
withdrawal?

Figure
4

Overview
of
results
so
far
–
the
young Wlds
mouse

• Wld
gene
expression
is
independent
of
age
• Immunocytochemical
staining
showed
full
• ccupancy

par8al
occupancy

vacancy
• Young
Wlds
mice
progressively
withdrew
from

motor
endplates
following
axotomy.

• Many
similari8es
with
the
stages
of
synapse

elimina8on
that
occur
during
normal postnatal
development

Results

Degenera8on
of
synap8c
terminals
in
fully
mature
Wlds
mice compared
to
young
Wlds
mice

2
months 2
months Figure
5:
D
&
E

Results

Is
the
transforma8on
in
the
axotomy
reac8on
of
synap8c terminals
due
to
age
or
matura/onal
state
of
the
terminals?

Figure
6

Results

Age
dependence
of
synap8c
protec8on
in
Wld
mice.
2 transgenic
lines
of
Wld
mice:
4836
&
4830,
4836
expresses
Wld protein
more
strongly

Axon
preserva8on, measured
by
reten8on
of neurofilament,
was independent
of
age Synapses
s8ll
present
5
days a`er
axotomy,
2
month
old mouse,
implied
that
this
was age
independent Figure
7

Results

Age
dependence
of
synap8c
protec8on
in
Wld
mice

Homozygous
and heterozygous
lines showed
the
same age
dependence
in synap8c
response
to axotomy
as
seen
in Wlds
mice Figure
7

Results

How
the
Wld
gene
protects
axons
and
synapses
expressing fluorescent
protein

• Mice
can
express
fluorescent
protein

in
their
axons
and
synapses

axon and
synap8c
protec8on
by
the
Wld gene
can
be
visualised
in
living prepara8ons.

• Crossbred
Wlds
mice
with
thy‐1‐CFP

mice,
lesioned
the
8bial
nerve

• Results
show
that
the
fluorescent

protein
did
not
interfere
with protec8on
of
axons
&
synapses conferred
by
Wld
gene
in
young
mice.

• It
may
be
possible
to
visualise

axotomy‐induced
synapse
withdrawal in
real
/me

Figure
8

Summary
of
findings

Developmental synapse
elimina8on. Yellow:
par8ally innervated
synap8c terminals Axotomy
induces nerve
withdrawal similar
to
neonate Resembles
wild‐ type
i.e.
synap8c degenera8on
as

• pposed
to

withdrawal,
but axons
are protected Wallerian degenera8on: proximal
axon
&
cell body
in
tact Figure
9

Discussion

• Recap:

– Perry
et
al.
1992
&
Ribchester
et
al.
1995:
the
Wlds
phenotype
is lost
as
mice
age
so
by
6
months
of
age
mutant
mice
exhibit normal
rates
of
axon
degenera8on – Crawford
et
al.
1995:
both
axons
and
synapses
are
equally
well protected
from
axotomy
induced
degenera8on
in
Wlds
mice
of all
ages – Aim:
“To
resolve
the
discrepancy
between
the
studies
of Ribchester
et
al.
&
Crawford
et
al.”

• Age
has
no
effect
on
Wld
gene
expression
nor
on

preserva8on
of
Wld
axons

supports
Crawford
et
al.
1995

• BUT
as
Wld
mice
increase
in
age,
there
is
a
decrease
in

preserva8on
of
axotomised
synap/c
terminals

• Maturity
of
synapse
rather
than
age
of
motor
neuron

important

Discussion

• “Lesions
of
a
peripheral
nerve
induce
one
of
at

least
two
independent
modes
of
synap8c degenera8on
in
Wld‐expressing
mice”

– Progressive
vs.
synchronous

• Synapse
withdrawal
in
young
mice
parallels

developmental
synapse
elimina/on

– Par8al
occupancy
of
endplates – Forma8on
of
retrac8on
bulbs – Decline
in
quantal
content
that
precedes
loss
of presynap8c
terminals

• Supports
the
theory
of
compartmentalised
neurodegenera8on

Discussion

Figure
9

Discussion

• Synapse
elimina8on
occurs
in
reinnervated

muscle
i.e.
beyond
the
period
when
they
are normally
eliminated
as
part
of
development

Figure
6E

Big
Burning
Ques8on

Why
is
synap8c
protec8on
age‐dependent
in
WldS mice?

• Not
directly
answered
the
ques8on
but
have

shown
that
it
is
maturity
of
the
synapse
as
opposed to
age
that
is
important.

• Suggested
that
it
could
be:
Biochemical
state
of

regenerated
terminal,
local
regula8on
of
the response
to
axotomy/trafficking
of
maintenance factors,
change
in
gene
expression

Cri8cal
Appraisal

Strengths:

• Figures
very
clear
• Inven8ve
way
of
tes8ng
the
effect
of
maturity
• Study
supports
compartmentalised
neuron
theory
• Partly
solves
the
ques8on
as
to
the
effect
of
age
• Suggests
possibili8es
for
future
work

Weaknesses:

• Sample
size
for
ages
of
mice
not
stated
• Figure
7:
didn’t
show
%
fibres
showing
ac8vity
in
mature

homozygous
4836
mice,
mixed
homozygous
4830
& heterozygous
4836

• Lots
of
ques8ons
s8ll
to
answer!

Future
work

• It
may
be
possible
to
visualise
axotomy‐

induced
synapse
withdrawal
in
real
8me
using thy‐1‐CFP
Wlds
mice
and
to
compare
to synapse
elimina8on
in
more
detail

• Eludes
to
further
studies
of
axotomy‐induced

retrac8on
of
synapses
in
Wld‐expressing
mice

– Where
is
synapse
withdrawal
ini8ated?
Studies
by Teriakidis
et
al.
suggest
that
only
larger
motor units
undergo
intrinsic
withdrawal

Future
work

• Manufacture
other
genes
that
more
directly

protect
synapses
from
the
consequences
of nerve
lesions

• How
does
the
Wld
gene
ini8ally
protect
axons

and
synapses
from
degenera8on?

– Fluorescence
studies
suggest
that
the
mutant
gene doesn’t
interact
with
other
genes – Study
the
role
of
Ube4b,
a
ubiqui8na8on
cofactor
in the
Wld
gene,
in
synap8c
response
to
axotomy? – Determine
the
loca8on
of
ac8on
of
the
Wlds
gene

References

1:
Lunn
ER,
Perry
VH,
Brown
MC,
Rosen
H,
Gordon
S.
1989.
Absence
of Wallerian
degenera8on
does
not
hinder
regenera8on
in
peripheral
nerve. Eur.
J.
Neurosci.
1:27–33

2:
PERRY,
V.
H.,
BROWN,
M.
C.
&
TSAO,
J.
W.
(1992).
The
effec8veness
of
the

gene
which
slows
the
rate
of
Wallerian
degenera8on
in
C57Bl/Ola
mice declines
with
age.
European
Journal
of
Neuroscience
4,
1000–1002. 3:
Ribchester
RR,
Tsao
JW,
Barry
JA,
Asgari‐Jirhandeh
N,
Perry
VH,
Brown
MC. 1995.
Persistence
of
neuromuscular
junc8ons
a`er
axotomy
in
mice
with slow
Wallerian
degenera8on
(C57BL/WldS).
Eur.
J.
Neurosci.
7:1641–50 4:
CRAWFORD,
T.
O.,
HSIEH,
S.‐T.,
SCHRYER,
B.
L.
&
GLASS,
J.
D.
(1995). Prolonged
axonal
survival
in
transected
nerves
of
C57Bl/Ola
mice
is independent
of
age.
Journal
of
Neurocytology
24,
333–340.

Severe
neuromuscular
denerva/on
of clinically
relevant
muscles
in
a
mouse model
of
spinal
muscular
atrophy

Karen
K.
Y.
Ling{,
Rebecca
M.
Gibbs{,
Zhihua
Feng
and
Chien‐Ping
Ko∗

Background

• Spinal
muscular
atrophy
(SMA),
a
motor
neuron
disease

caused
by
a
deficiency
of
the
survival
of
motor
neuron
(SMN) protein,
is
characterized
by
motor
neuron
loss
and
muscle weakness.

• Affects
1
in
6000–10,000
live
births

Aims

• The
main
aim
of
this
study
was
to
understand
if
the
widespread

loss
of
NMJs
is
involved
in
SMA
pathogenesis
and
to
provide
a systema/c
inves/ga/on
of
NMJs
in
a
wide
range
of
axial
(head, neck
and
trunk)
and
appendicular
(controlling
the
limb)
muscles that
are
relevant
to
SMA
symptomatology.

• Secondary
Aims:
• 1. To
understand
if
failure
of
synap/c
maintenance
or
a
lack
of
ini/al

nerve‐muscle
contact
is
responsible
for
NMJ
Denerva/on.

• 2. Provide
a
therapeu/c
target
for
SMA.

Method

• examined
NMJ
innerva/on
paZerns
in
.20
different

axial
muscles
(head,
neck
and
trunk)
and
proximal and
distal
appendicular
muscles
(controlling
the limbs)
from
SMND7
mice
from
P1
to
P14

• Labelled
endplates
with
α
bungarotoxin
(red)
• Immunostained
nerve
terminals
with
an/‐

synaptophysin
an/bodies
expressing
YPF
(green)

Severe
denerva/on
of
proximal
and
distal
muscles
in
end‐ stage
SMND7
mice.

Axial
muscles
[serratus
posterior
superior (SPS),
SPI,
trapezius
and
splenius]
and appendicular
muscles
(FDB‐4
and
‐2)
of
YFP‐ SMA
mice
were
immunostained
for
nerve terminals
with
an/‐synaptophysin
[syn]
(in green)
and
motor
endplates
with
a‐ bungarotoxin
(in
red). Arrows
show
dispersing
AChR
clusters
a denervated
endplate.

Results

Quan/ta/ve
analyses
of
innerva/on
in
end‐ staged
(P12–14)
SMND7
mice
and
age‐ matched
control
mice. Innerva/on
%
expressed
as
number
of
fully innervated
endplates/total
number
of analysed
endplates
(.200
NMJs)
in
each animal. Avg
%
obtained
from
three
to
five
pairs
of animals.

Results

NMJs
are
formed
but
not
maintained
in
the
vulnerable
axial
muscle,
SPI,
in SMND7
mice In
the
SPI
muscles
at
embryonic
day
17.5
almost
all
endplates
are
fully innervated. This
suggests
that
failure
of
synap/c
maintenance
is
responsible
for
denerva/on.

Results


NMJs
are
formed
but
not
maintained
in
the
vulnerable
appendicular
muscle, FDB‐2/3,
in
SMND7
mice.

Neurofilament
Accumula/on

Neurofilament
accumula/on
in
the
axonal swellings
and
at
the
presynap/c
terminal
is considered
one
of
the
morphological hallmarks
of
NMJs
in
SMA inves/gated
whether
or
not
this
pre‐ synap/c
pathology
predicts
NMJ vulnerability
to
denerva/on,
percentage
of NMJs
with
neurofilament
accumula/on
in vulnerable
(FDB‐2)
and
resistant
(FDB‐4) muscles
before
and
afer
denerva/on Neurofilament
accumula/on
is
observed
in both
vulnerable
and
resistant
muscles
in SMND7
mice
at
P1
and
P14

SMA
treatment
with
TSA

inves/gated
whether
denerva/on
in vulnerable
muscles
can
serve
as
a
reliable marker
for
evalua/ng
drug
efficacy
at
the NMJ. treated
SMND7
mice
from
P1
to
P12
with daily
intraperitoneal
(IP)
injec/ons
(10mg/kg body
weight)
of
TSA,
a
histone
deacetylase inhibitor
that
has
previously
been
shown
to upregulate
SMN
expression,
improve
motor deficits
and
increase
lifespan
in
SMND7
mice 
injected
SMND7
mice
with
DMSO
as
a control

Conclusions

• 1. uncovered
severe
denerva/on
in
many
muscles
involved
in

vital
motor
func/ons

• 2. found
that
denerva/on
is
more
prominent
in
muscles

located
in
the
head
and
trunk,
but
denerva/on
also
occurred in
proximal
and
distal
limb
muscles.
Showing
NMJ vulnerability
is
not
solely
determined
by
muscle
loca/on.

• 3. loss
of
innerva/on
likely
results
from
defects
in
synapse

maintenance
rather
than
ini/al
forma/on
of
nerve‐muscle contacts.

• 4. Neurofilament
accumula/on
does
not
correlate
with
NMJ

vulnerability
to
denerva/on

• 5. demonstrated
that
severe
denerva/on
in
clinically
relevant

muscles
is
amendable
by
postnatal
TSA
treatment
in
SMND7 mice

Further
Inves/ga/ons

• whether or not there is a selective degeneration of

motor neurones within the same spinal segments that may account for selective NMJ

• Whether NMJ denervation occurs in a dying back

fashion.

• In ALS fast twitch muscles are more vulnerable… most
• f the muscles in this experiment were fast twitch
• Are there other physiological defects that could serve

as a marker preceding loss of NMJ’s?

Big
Burning
Ques/ons

• How
will
we
find
out
whether
SMN
is

specifically
involved
in
maintenance
of
some MN/synapses
not
others?