1. Buck
Institute
Technology
Summary:
Chronic
Disease
Therapy
with
Anti-­‐Aggregation
Compounds
Background
During
aging
there
is
a
disruption
to
normal
protein
processing,
leading
to
the
deposit
of
insoluble
misfolded
proteins,
or
aggregates.
In
particular,
protein
aggregates
are
a
hallmark
of
common
neurodegenerative
diseases,
for
example
α-­‐synuclein
aggregation
in
Parkinson’s
Disease
and
β-­‐amyloid
aggregation
in
Alzheimer’s
Disease.
For
decades,
compounds
have
been
used
to
image
these
aggregates
in
postmortem
brain
tissue
and
more
recently
in
patients,
using
modern
imaging
systems.
Scientists
at
the
Buck
Institute
hypothesized
that
these
compounds
may
prevent
aggregation
in
vivo
and
therefore
have
a
therapeutic
effect
beyond
their
current
diagnostic
use.
The
Technology
Dr.
Silvestre
Alavez
in
the
Lithgow
Lab
created
a
selective
screen
to
analyze
molecules
traditionally
employed
in
histopathology
to
stain
amyloids
in
tissues,
in
the
nematode
worm
C.
elegans.
This
screen
revealed
that
indeed
some
of
these
compounds
not
only
bind
protein
fibrils
but
slow
aggregation
and
increase
lifespan
in
C.
Elegans.
Promising
compounds
included
the
turmeric
component
curcumin,
the
antibiotic
rifampicin
and
other
compositions.
One
compound,
Thioflavin
T
(“ThT”),
was
found
to
extend
the
lifespan
of
worms
by
up
to
60%,
as
well
as
decreasing
the
physiological
aging
process
causing
morbdity,
in
particular
improving
frailty
by
decreasing
motor
dysfunction
(see
graphs).
These
results
are
reproducible,
occur
in
a
dose-­‐dependent
manner
and
were
published
in
the
journal
Nature
in
2011.
ThT
has
been
used
for
over
50
years
as
an
imaging
agent
to
understand
the
aggregation
of
soluble
amyloid
proteins
into
beta-­‐sheet
fibrils
and
has
been
explored
as
an
in
vivo
diagnostic
for
imaging
β-­‐amyloid
plaques.
This
is
the
first
time
that
ThT
and
related
compounds
have
been
identified
as
potential
therapeutics
to
reduce
aggregation
and
improve
healthy
aging.
The
Lithgow
lab
has
shown
that
this
novel
mechanism
of
action
likely
occurs
through
ramping
up
of
the
endogenous
stress-­‐response
and
protein
degradation
pathways.
While
this
finding
was
made
in
nematode
worms,
preliminary
results
in
collaboration
with
researchers
at
the
Barshop
Institute
suggest
that
HBX,
a
ThT
structurally
related
compound
that
crosses
the
blood
brain
barrier,
improves
the
prognosis
in
a
mouse
model
of
neurodegenerative
disease.
In
addition,
the
Andersen
lab
at
the
Buck
Institute
has
shown
that
HBX
effectively
suppresses
neuronal
inflammation
associated
with
neurological

2. David J. S. Zucker1,2, Ida M. Klang1,3 & Gordon J. Lithgow1
homeostasis is a major contri-butor
disease.
These
findings
indicate
a
key
role
for
the
maintenance
of
protein
homeostasis
during
aging.
protein homeostasis
accumulation of inso-luble
amyloids2. A group of
A B
the bioavailability and/or pharmacological properties of
histopathology to stain amy-loid
aggregationin vitro and
animalswith such com-pounds
compounds influence lifespan.
effects of ThT on protein homeostasis we exploited two
models of human proteotoxic disease: the strain CL4176
3::Ab3–42 let 39UTR(pAF29); pRF4 (rol-6(su1006))])12,
in vivo and increase
adult Caenorhabditis
T (ThT) resulted in
ageing. ThT also sup-pressed
metastable proteins and
expresses an aggregating amyloid-b(3–42) peptide (Ab(3–42))
tissue13 and AM140 (rmIs132[P(unc-54) Q35::YFP]), which
polyglutamine beneficial effects (polyQ) of
protein. Amyloid-b aggregates are
with lesions in Alzheimer’s disease, and polyQaggregationis a
several neurological conditions14.Whenraisedat 25uC, nema-todes
network regulator heat
longevity transcrip-tion
expressing these proteins in muscle accumulate protein aggregates
autophagy and proteoso-mal
that pharmacological
network has a profound
paralysed. We found that 50 mM ThT and 100mM curcu-min
decreased the proportion of paralysed worms (Fig. 2a, b). By
immunohistochemistry we found that ThT reduced Ab(3–42) aggrega-tion
development of novel thera-peutic
a b
100 Control
80
60
40
20
in vivo. a, b, The paralysis phenotype associated aggregation is suppressed by 25 mM ThT, 50mM ThT, 100 CL4176 (*P,0.001, **P,0.0001) expressing Ab(3–42) (*P,0.05, **P,0.01) expressing polyQ (b) after 1 and respectively. Error bars represent the mean6s.e.m. of four experiments. c, Temperature-sensitive strain HE250 [unc-after 36 h at 25 uC showing the typical paralysis phenotype LETTER 100
75
50
25
0
–25
–50
–75
c d
2
0
–2
–4
–6
100
80
60
40
100
80
60
40
20
Control 25 μM ThT 50 μM ThT 100 μM curcumin Control 25 0
50 Control
40
30
20
10
30
25
20
100
80
Opportunity
Research
conducted
in
the
Lithgow
Lab
at
the
Buck
Institute
represents
the
first
example
of
amyloid-­‐binding
dyes
for
use
in
extending
lifespan
and
reducing
age-­‐related
frailty.
Furthermore,
these
studies
suggest
a
novel
and
critical
role
for
protein
aggregation
in
controlling
physiological
aging.
ThT
and
related
compounds
have
potential
as
therapeutic
agents,
improving
healthspan
by
controlling
protein
aggregation
in
chronic
human
disease.
Work
continues
at
the
Buck
regarding
this
new
approach.
The
Buck
seeks
developmental
partners
who
can
collaborate
and
further
develop
these
agents
to
treat
the
myriad
diseases
caused
or
exacerbated
by
aggregation.
The
Buck
Institute
has
filed
patents
on
these
compounds
and
derivatives
thereof
for
treatment
of
multiple
age-­‐related
processes,
such
as
frailty,
and
chronic
diseases,
such
as
arthritis,
macular
degeneration
and
cardiovascular
disease.
and preservedmuscle integrity in CL4176 (Fig. 2e).We also
ThT rescued Ab(3–42) aggregation-induced paralysis even
nematodes were treated 18 h after the induction of aggregate
e f
indicating that ThT can ameliorate detrimental effects dur-ing
development of aggregate-100 related pathology (Supplemen-tary
Control
80
60
amyloid-binding compounds 40
extended lifespan through
proteinhomeostasis, then we 20
expected that they would influ-ence
15
10
5
CL4176 CL4176 + ThT N2 HE250 60
40
20
100 Control
80
60
40
20
heterologous disease-related models but also nematode
tested ThT and curcumin on mutant worms that express
proteins previously exploited as indicators of the protein
network capacity15. Strains carrying mutations in the
100 Control
(HE250 [unc-52(e669su250)II]) and unc-54 80
(CB1157
e1157)I]) produce temperature-sensitive muscle proteins
perlecan) and UNC-54 (myosin class II heavy chain),
50
50
The
Buck
Institute
is
the
only
free
standing
60
institute
dedicated
to
aging
and
age-­‐related
research
in
the
United
States.
We
actively
partner
with
industry
40
to
develop
therapeutics,
diagnostics
or
tools
that
make
a
difference.
The
Buck
Institute
welcomes
interested
parties
20
to
inquire
regarding
licensure
or
collaboration
of
this
technology.
For
more
information
on
this
or
another
technology
or
opportunity,
please
contact:
that exhibit altered structure and cause paralysis at
0
We found that ThT suppressed paralysis of these mutants
prevented the disruption of the muscle sarcomeres (Sup-plementary
Fig. 8) and restored perlecan organization (Fig. 2d). We
these observations to other temperature-sensitive missense
folding mutations expressed in the neuromuscular junction
nervous system18. We found that ThT suppressed ethanol
Carlotta
Duncan,
Ph.D
.
Business
Development
&
Licensing
Officer
Technology
Transfer,
Buck
Institute
for
Research
on
Aging.
Phone
-­‐
415-­‐209-­‐2000;
cduncan@buckinstitute.org
related diseases.
elegans is influenced
signalling pathway (ILS)
factors FOXO-like
Together with the stress
also regulates protein
that chemical modu-lation
We reasoned that
protein-aggregate-binding
protein homeostasis and
lifespan effects. We
nematodes to the fibril-binding
benzothiazol-3-ium-2-
100 mMthroughout
and maximal lifespan
Supplementary Table 1).
all ages (P,0.001,
spontaneous movement
adulthood. At higher
lifespan (Fig. 1a, b). Other
properties, including cur-cumin
lesser extent (up to 45%)
curcumin treatments were
lifespan (Supplemen-tary
structural features to
properties: 2-(2-hydroxyphe-nyl)-
benzothiazole(HBT)and
0 10 20 30 40
0
1 μM ThT
10 μM ThT
50 μM ThT
100 μM ThT
500 μM ThT
Time (days)
Per cent survival
–100 0 1 10 50 100 500
ThT
Curcumin
Compound (μM)
Median lifespan gain (%)
5 10 15 20 25 30 35 40
–8
N2
ThT
Time (days)
In (mortality)
0 2 4 6 8 10 12 14 16
0
50 μM ThT
100 μM curcumin
Days of treatment
No. bends min–1
0 10 20 30 40
0
1 nM BM
10 nM BM
100 nM BM
1 μM BM
Time (days)
Per cent survival
Control 50 μM ThT 100 μM curcumin
0
* *
No. bends min–1
40
30
20
0 10 20 30 40
1 nM HBX
10 nM HBX
100 nM HBX
1 μM HBX
Time (days)
Per cent survival
0 10 20 30 40
0
1 nM HBT
10 nM HBT
100 nM HBT
1 μM HBT
Time (days)
Per cent survival
g
10
40
30
20
10
Figure 1 | Amyloid-binding compounds extend C. elegans lifespan.
a, Dose–response Kaplan–Meier survival curves of synchronously ageing
hermaphrodite wild-type (N2) populations exposed to 0 mM (control) to
500 mMThT at 20 uC. b, Per cent change in median lifespan of N2 populations
cultured on 0–500 mM ThT and curcumin. c, ln-linear plot of age-specific
mortality rate with age for control and 50 mMThT-treated C. elegans. d, Effect
of 50 mMThT and 100 mMcurcumin on motility of N2 worms evaluated as the
mean number of body bends in a 20-s period in 15 individual worms
throughout life (upper panel) and after 12 days of treatment (lower panel) with
ThT and curcumin. Data are presented as bends min21 and represent the
average of three independent experiments. P,0.0001. e–g, Dose–response
strain carrying the gas-1(fc21) mutation in a subunit of
complex I and levamisole resistance in a strain carrying
an a-subunit of the nicotinic acetylcholine receptor
Supplementary Fig. 9), indicating that ThT could act in a variety of
a b
c d
e
f
25 °C HE250 15 N2 + HS
20
Paralysis (%)
0
Paralysis (%)
Control 50 μM ThT 100 μM Cur 100 μM Rif
0
Paralysis (%)
HE250
CB1157
0
Aβ aggregates per sarcomere
0
No. of aggregates per 2,000 μM2
Control
ThT
Figure 2 | ThT and curcumin rescue a paralysis phenotype aggregation A.
Dose-­‐dependent
survival
increase
for
worms
with
ThT
treatment.
B.
Decreased
paralysis
of
worms
with
ThT
and
curcumin
treatment.
Paralysis
is
detected
by
a
halo-­‐effect
of
bacteria
around
the
control
stressed
worms
unable
to
feed
(asterisks)
compared
to
the
treated
stressed
worms
with
decreased
frailty.
Contr
50
uM
*
*