Daryl
A.


Bosco

Alzforum
Webinar

Stress
Granules
and
Neurodegenera<ve
Disease‐

What's
the
Scoop?

 Background
of
FUS
in
ALS


•  Muta<ons
in
FUS
account
for
5%

of
familial
ALS
(Kwiatkowski,

Science,
2009;
Vance,
Science,

2009)


•  Most
muta<ons
are
in
the
nuclear

localiza<on
signal
(different
from

TDP‐43),
leading
to
cytoplasmic

reten<on
(Gal,
Neuro.
Aging,

2010)


•  Unknown
mechanism:
gain
of

toxic,
cytoplasmic
func<on
and/or
 Lagier-Tourenne, Human Molecular Genetics, 2010

loss
of
normal
nuclear
func<on?

 ALS‐FUS
mutants
mislocalize
to
the
cytoplasm


•  Cells:
inducible
HEK‐293
cells
stably

expressing
GFP‐FUS
constructs


•  Exogenous
FUS
expressed
at
levels

similar
to
endogenous
FUS
 less

FUS
aggrega<on
and
mislocaliza<on

compared
to
transient
transfec<ons


•  Trunca<on
mutants
(R495X
and

G515X)
exhibit
the
most
pronounced

mislocaliza<on

 ALS‐FUS
mutants
assemble
into
stress
granules


•  ALS‐FUS
mutants
assemble
into
stress

granules
in
response
to
oxida<ve

stress
(NaAS,
TG)
and
heat
shock


•  Most
striking
result
is
for
trunca<on

mutants;
the
extent
of
stress
granule

associa<on
correlates
with

cytoplasmic
mislocaliza<on


•  WT‐FUS
and
endogenous
FUS
(data

not
shown)
do
not
assemble
into

stress
granules

–  In
contrast
to
WT
and
endogenous
TDP‐43

–  Appears
specific,
untagged‐GFP
is

cytoplasmic
but
does
not
assemble
into

stress
granules

The
assembly
of
FUS
into
SG
is
fast
in
response
to

stress,
and
reversible

 ALS‐FUS
does
not
assemble
into
P‐bodies


•  P‐bodies
are
associated
with

stress
granules,
as
previously

reported


•  P‐bodies
func<on
in
the

degrada<on
of
mRNA


•  GFP‐FUS
does
not
co‐localize
with

P‐body
markers

 All
results
in
cells
are
recapitulated
in
vivo
in
zebrafish


•  Zebrafish
eggs
were
injected
with

mRNA
encoding
GFP‐FUS
variants


•  In
the
absence
of
stress,
cytoplasmic

mislocaliza<on
is
most
striking
for

trunca<on
mutants


•  Autosomal
recessive
H517Q
variant

exhibits
greater
mislocaliza<on
in

response
to
heat
shock


•  ALS‐FUS
mutants
assemble
into
stress

granules
in
response
to
heat
shock

 Unanswered
ques<ons


•  Does
the
associa<on
of
FUS
into
stress
granules
contribute
to
the

pathogenesis
of
ALS
and
other
neurodegenera<ve
diseases?


•  Do
FUS
and
TDP‐43
play
a
normal
role
in
stress
granule
assembly?


•  Does
the
incorpora<on
of
ALS‐FUS
(or
TDP‐43)
into
stress
granules
alter

the
func<on
of
these
structures?


•  Are
the
effects/roles
of
FUS
and
TDP‐43
with
respect
to
stress
granules

similar?

Or
are
there
differences?


•  Do
stress
granules
represent
precursors
to
end‐stage,
pathological

aggregates?

If
so,
do
they
contribute
to
aggrega<on,
or
are
they
simply

sequestered
into
the
aggregates?