1. Hannah
Shapero
Research
Project
Final
Document
The
Effects
of
sut-‐2
Mutation
on
Caenorhabditis
elegans
Expressing
Human
TDP-‐43
ABSTRACT
Amyotrophic
Lateral
Sclerosis
(ALS)
is
a
debilitating
disease
characterized
by
the
degeneration
of
motor
neurons
leading
to
a
complete
loss
of
voluntary
muscle
control.
TDP-‐43
has
been
identified
as
a
major
component
of
cellular,
protein
aggregates
characteristically
found
in
the
cytoplasm
of
affected
neurons
in
a
variety
of
neurodegenerative
diseases,
including
ALS.
TDP-‐43
controls
the
structure
and
stability
of
mRNA’s,
preventing
them
from
forming
double
stranded
RNA’s
(dsRNA).
In
the
absence
of
nuclear
TDP-‐43,
dsRNA
accumulates
and
aggregates.
In
recent
years,
researchers
have
identified
two
genes,
sut-‐1
and
sut-‐2,
that
when
mutated
suppress
the
toxic
effects
of
tau
expression,
causing
symptoms
of
Alzheimer’s
disease.
Both
Alzheimer’s
disease
and
ALS
share
a
similar
pattern
of
protein
aggregation
within
neurons
leading
to
cell
death.
This
similarity
suggests
that
a
single
mechanism
involving
failure
of
protein
degradation
pathways
plays
a
role
in
both
diseases.
If
sut-‐2
mutation
can
suppress
the
toxic
effects
of
tau
containing
protein
aggregates,
it
may
also
suppress
the
toxic
affects
of
TDP-‐43
containing
protein
aggregates
in
ALS.
Transgenic
C.
elegans
worms
expressing
human
TDP-‐
43
exhibit
an
uncoordinated
phenotype,
which
I
used
to
indicate
the
presence
of
motor
neuron
degeneration.
I
created
a
C.
elegans
strain
expressing
both
transgenic,
human
TDP-‐
43
and
non-‐functional,
mutated
sut-‐2;
in
doing
so,
I
found
that
this
single
point
mutation
lead
to
a
partial
amelioration
of
the
uncoordinated
phenotype
seen
with
the
expression
of
human
TDP-‐43
alone.
BACKGROUND
Amyotrophic
Lateral
Sclerosis
Amyotrophic
Lateral
Sclerosis
(ALS)
is
a
progressive
adult-‐onset
neurodegenerative
disorder
causing
degeneration
of
upper
and
lower
motor
neurons.
In
almost
all
cases
of
ALS,
affected
motor
neurons
show
ubiquitin-‐positive,
tau-‐negative
aggregates
in
the
cytoplasm,
suggesting
that
ALS
is
a
protein
aggregate
disorder.
Motor
neurons
extend
from
the
central
nervous
system
to
all
muscles
of
the
body,
controlling
their
voluntary
movement.
When
these
neurons
die
a
person
loses
the
ability
to
control
their
muscles,
leading
to
paralysis
and
loss
of
muscle
function.
Consequentially,
ALS
is
a
debilitating
disease
causing
severe
disability
and
eventually
death.
2.
TDP-‐43’s
role
in
ALS
TDP-‐43
has
been
identified
as
a
major
component
of
aggregates
characteristically
found
in
the
cytoplasm
of
affected
neurons
in
a
variety
of
neurodegenerative
diseases,
including
ALS,
Alzheimer’s
disease,
hippocampal
sclerosis,
Lewy
body
disease,
parkinsonism-‐
dementia
complex
of
Guam,
corticobasal
degeneration,
Pick’s
disease,
Perry
syndrome
and
frontotemporal
lobar
dementia
(FTLD).
The
normal
nuclear
localization
of
this
protein
is
disrupted
and
TDP-‐43
forms
large,
insoluble
aggregates
in
the
cytoplasm
along
with
other
misfolded
proteins.
TDP-‐43
within
these
aggregates
is
hyper-‐phosphorylated,
ubiquitinated
and
cleaved.
Phosphorylated
TDP-‐43
is
not
seen
in
healthy
tissues
(Inukai
et
al.,
2013).
TDP-‐43
is
normally
localized
within
the
nucleus
of
the
cell,
and
cytoplasmic
localization
is
concurrent
with
nuclear
clearance.
The
toxicity
caused
by
these
TDP-‐43
aggregates
is
likely
caused
by
both
the
cytoplasmic
aggregation
and
the
nuclear
loss
of
function
(Tan
et
al.,
2015).
RNA
processing
depends
of
TDP-‐43
TDP-‐43
controls
the
structure
and
stability
of
RNA’s,
preventing
them
from
forming
double
stranded
RNA’s
(dsRNA)
and
ensuring
proper
translation.
In
the
absence
of
nuclear
TDP-‐
43,
dsRNA
accumulates
(Saldi
et
al.,2014).
Wild-‐type
TDP-‐43
is
implicated
in
multiple
steps
of
mRNA
processing,
including
the
binding
of
TDP-‐43
to
the
3’
UTR
of
an
mRNA,
influenced
mRNA
stability.
This
increases
the
abundance
of
certain
transcripts
and
reduces
the
abundance
of
others
in
post-‐transcriptional
regulation
(Saldi
et
al.,
2014).
TDP-‐43
maintains
proper
splicing
patterns
of
many
transcripts
by
exon
skipping
and
exon
inclusions
(Hazelette
et
al.,
2012).
A
large
proportion
of
altered
transcripts
have
the
potential
to
form
dsRNA.
This
suggests
that
TDP-‐43
has
a
fundamental
function
in
the
control
and
metabolism
of
dsRNA,
and
that
without
its
proper
functioning,
dsRNA’s
accumulate
within
the
cell.
dsRNA
buildup
triggers
an
innate
immune
response
It
is
common
in
proteinopathies
for
cells
to
aggregate
and
expel
extra
material
built
up
in
the
cytoplasm;because
the
misfolded-‐protein
response
is
overwhelmed
by
the
massive
synthesis
of
misfolded
proteins
and
transcripts.
An
accumulation
of
faulty,
damaged
and
misfolded
proteins
within
the
cell
leads
to
aggregation
of
these
proteins,
causing
inclusion
body
formation
and
cell
death
(Brehm
and
Kruger,
2015).
Under
normal
circumstances,
a
cell
responds
to
misfolded
proteins
with
degradation
by
autophagy.
When
the
production
of
misfolded
proteins
outpaces
autophagy,
the
cell
begins
expelling
these
aggregated
proteins,
allowing
them
to
activate
toll-‐like
receptors
(TLR’s),
which
trigger
an
innate
immune
response
(Murdock
et
al.,
2015).
TLR3
(in
mammals)
can
facilitate
an
excessive
and
unregulated
immune
response,
which
can
be
harmful
to
the
host
and
contribute
to
the
disease
pathology
(Lester
and
Li,
2014).
Thus,
the
pathology
of
ALS
and
other
neurodegenerative
diseases
may
not
be
based
on
the
nature
of
the
damage,
but
on
the
body’s
reaction
to
the
damage,
specifically,
an
innate
immune
response
leading
to
cell
death.
3. sut-‐2
as
a
potential
modulator
of
proteinopathy
In
200
7,
Kraemer
and
Schellenberg,
(2007)
preformed
forward
genetic
screens
for
mutations
that
prevent
tau-‐induced
pathology
(one
of
the
major
components
of
protein
aggregates
involved
in
Alzheimer’s
disease).
They
discovered
that
a
recessive
mutation
in
a
single,
well
conserved
gene,
which
the
named
suppresser
of
tau-‐1
(sut-‐1),
partially
suppresses
the
Alzheimer’s
phenotype
of
tau
aggregation
and
neurodegenerative
changes
caused
by
tau.
They
identified
the
sut-‐1
gene
and
found
it
encodes
a
novel
protein.
Further
study
lead
to
the
discovery
of
a
second
sut
protein
(sut-‐2)
that
also
suppresses
the
tau
pathology
when
mutated
(Guthrie
et
al.,
2009).
Both
Alzheimer’s
disease
and
ALS
share
a
similar
pattern
of
protein
aggregation
within
neurons
leading
to
cell
death.
This
similarity
suggests
that
a
single
mechanism
involving
failure
of
protein
degradation
pathways
plays
a
role
in
both
diseases.
If
sut-‐2
mutation
can
suppress
the
toxic
affects
of
tau
containing
protein
aggregates,
it
may
also
suppress
the
toxic
affects
of
TDP-‐43
containing
protein
aggregates
in
ALS.
C.
elegans
as
a
model
for
ALS
C.
elegans
is
a
small,
translucent,
round
worm
with
a
rapid
reproductive
cycle
and
a
short
lifespan
(http://wormbook.org/).
These
features
make
C.
elegans
a
valuable
model
organism.
Additionally,
human
transgenes
show
a
remarkable
ability
to
function
within
C.
elegans
and
genetic
mutations
that
cause
dysfunction
and
disease
in
humans
often
exhibit
similar
phenotypes
in
C.
elegans
(Wolozin
et
al.,
2011).
Transgenic
worms
expressing
human
TDP-‐43
exhibit
an
uncoordinated
phenotype,
which
can
be
measured
by
a
variety
of
movement
assays.
Using
the
uncoordinated
phenotype
as
an
indicator
of
TDP-‐43
induced
neurotoxicity
has
allowed
researchers
to
investigate
the
wild
type
function
of
TDP-‐43
and
it’s
role
in
disease
pathology
(http://www.mayo.edu/research/labs/neurodegenerative-‐
diseases/cell-‐animal-‐models-‐tdp-‐43-‐proteinopathies#).
METHODS
C.
elegans
maintenence
Maintenance
and
growth
of
C.
elegans
were
performed
as
described
in
(Brenner,
1974)
and
all
strains
were
raised
at
20°C.
The
two
transgenic
strains
used
in
this
study
were
created
by
gonad
injection
and
integration
of
DNA
array.
Strains
N2
–
wild
type
C.
elegans
strain
used
as
a
control
TDP-‐43
–
C.
elegans
strain
expressing
human
TDP-‐43
and
marked
with
intestinal
GFP.
These
C.
elegans
exhibit
an
uncoordinated
phenotype
and
are
used
to
model
ALS.
sut-‐2(bk741)
–
C.
elegans
strain
with
sut-‐2
knocked
down
by
point
mutation
bk741
Cl6049;sut-‐2(bk741)
–
C.
elegans
strain
expressing
human
TDP-‐43
and
the
sut-‐2
point
mutation
bk741
Crosses
I
preformed
a
synchronized
egg
lay
on
sut-‐2(bk741)
and
then
moved
L3
C.
elegans
to
34°
for
8
hours
to
induce
the
generation
of
males.
These
males
were
then
placed
on
a
plate
with
L3
TDP-‐43
C.
elegans.
Progeny
from
these
plates
were
chosen
for
the
presence
of
intestinal
4. GFP
indicating
that
they
carried
the
TDP-‐43
transgene.
Each
of
these
C.
elegans
was
placed
on
an
individual
plate
and
left
overnight
at
15°.
C.elegans
expressing
the
sut-‐2
point
mutation
bk741
do
not
lay
many
eggs
at
this
temperature,
so
those
without
eggs
were
assumed
to
be
expressing
both
the
TDP-‐43
trans
gene
and
the
sut-‐2
mutation.
I
ran
genotyped
these
C.
elegan
to
ensure
that
these
C.
elegans
did
indeed
carry
the
sut-‐2
point
mutation.
Motility
assay
I
preformed
a
synchronized
egg
lay
and
let
C.
elegans
grow
up
for
3
days
at
20°.
I
washed
the
C.
elegans
off
the
plates
using
S-‐basal
solution
and
spun
them
down
in
a
centrifuge
for
1
minute
at
13000rpm.
I
removed
the
supernatant,
containing
food
from
the
plates,
and
re-‐
suspended
the
C.
elegans
in
S-‐basal
solution.
C.
elegans
were
placed
on
an
unspotted
plate
and
allowed
to
sit
for
3
minutes.
I
then
used
the
ultrascope
to
take
a
2-‐minute
video
of
each
plate.
I
analyzed
the
videos
with
worm
tracker
for
imageJ
to
calculate
the
body-‐lengths
per
second
moved
by
each
worm
on
each
plate.
RESULTS
I
used
body-‐lengths-‐per-‐second
as
a
measure
of
coordination
in
each
of
the
four
C.
elegans
strains.
This
measure
corrects
for
differences
in
average
C.
elegans
size
between
strains,
which
is
not
taken
into
account
by
analyzing
speed
alone.
Table
1:
Average
Body-‐Lengths-‐per-‐Second
of
C.
elegans
compared
between
three
transgenic
strains
and
the
N2
wild
type
control
strain.
Strain
Average
Body
Lengths
per
Second
Standard
Error
Sample
Size
N2
0.2520
0.0054
2333
sut-‐2(bk741)
0.2360
0.0033
3430
TDP-‐43
0.1290
0.0012
17986
TDP-‐43;sut-‐2(bk741)
0.2002
0.0017
8689
Table
2:
P-‐Values
calculated
by
independent
T-‐tests
on
average
Body-‐Lengths-‐per-‐
Second
of
C.
elegans
compared
between
three
transgenic
strains
and
the
N2
wild
type
control
strain.
N2
N2
sut-‐2(bk741)
0.01179
sut-‐2(bk741)
TDP-‐43
9.19E-‐182
1.171E-‐1000
TDP-‐43
TDP-‐43;sut-‐2(bk741)
2.60E-‐21
1.07E-‐19
4.38E-‐239
Wild
type
C.
elegans
moved
an
average
of
0.2520(±0.0054)
body
lengths
per
second.
sut-‐
2(bk741)
C.
elegans
moved
an
average
of
0.2360(±0.0033)
body
lengths
per
second.
TDP-‐
5. 43
C.
elegans
moved
an
average
of
0.1290(±0.0012)
body
lengths
per
second.
sut-‐
2(bk741);TDP-‐43
C.
elegans
moved
an
average
of
0.2002(±0.0017)
body
lengths
per
second.
A
multi-‐way
ANOVA
showed
significance
and
a
Tukey-‐Kramer
post-‐hoc
analysis
showed
significant
differences
between
all
four
strains.
Figure
1:
Average
body-‐lengths-‐per-‐second
moved
by
C.
elegans
as
a
measure
of
coordination
compared
between
four
strains.
All
strain
comparisons
show
significance
at
an
adjusted
alpha
of
0.0833,
n=32438.
DISCUSSION
The
data
show
that
C.
elegans
expressing
a
TDP-‐43
phenotype
have
significantly
decreased
motility
compared
to
both
the
wild-‐type
strain
(N2)
(p=0.0118)
and
the
strain
expressing
mutated
sut-‐2
(sut-‐2(bk741))
(p<0.001).
This
is
expected
because
these
worms
have
dysfunctional
motor
neurons
as
part
of
an
ALS
phenotype.
What
is
not
expected
is
that
C.
elegans
expressing
the
sut-‐2
mutation
also
move
significantly
fewer
body-‐lengths-‐per-‐
second
than
wild-‐type
C.
elegans.
This
difference
is
much
smaller
than
between
the
wild
type
strain
and
either
of
the
strains
expressing
TDP-‐43;
both
of
which
movedramatically
slower
than
either
the
N2
or
SUT-‐2(BK741)
strains,
due
to
the
uncoordinated
phenotype
induced
by
expression
of
human
TDP-‐43.
sut-‐2(bk741);TDP-‐43
C.
elegans
display
a
movement
phenotype
closer
to
that
of
the
wild-‐
type
than
the
uncoordinated
TDP-‐43.
This
demonstrates
a
partial
rescue
of
the
pathogenic
phenotype
by
inhibiting
sut-‐2
function.
Further
exploration
of
the
relationship
between
the
sut-‐2
protein
and
TDP-‐43
will
provide
useful
clues
as
to
the
role
of
TDP-‐43
in
ALS.
sut-‐2
likely
plays
a
critical
role
in
the
mechanism
by
which
the
cell
responds
to
the
abundance
of
0.0000
0.0500
0.1000
0.1500
0.2000
0.2500
0.3000
Average
body-‐lengths
per
second
6. dsRNA
which
accumulate
in
the
absence
of
nuclear
TDP-‐43.
Further
exploration
of
these
findings
may
lead
to
a
better
understanding
of
what
causes
neurodegeneration
and
provide
possible
new
neuroprotective
strategies
for
the
treatment
of
ALS
and
other
neurodegenerative
diseases.
7. REFERENCES
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S.
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Genetics
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C.
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and
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R.,
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D.
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and
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C.
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