Neurobiology of Disease 159 (2021) 105517
Available online 24 September 2021
0969-9961/© 2021 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
Amplification
of neurotoxic
HTTex1
assemblies
in human
neurons
Anjalika
Chongtham
a
, J. Mario
Isas
b
, Nitin
K. Pandey
b
, Anoop
Rawat
b
, Jung
Hyun
Yoo
a
,
Tara
Mastro
a
, Mary
B. Kennedy
a
, Ralf
Langen
b
, Ali
Khoshnan
a
,
*
a
Biology
and Bioengineering,
Caltech,
Pasadena,
CA 91125,
USA
b
Zilkha
Neurogenetic
Institute,
Keck School
of Medicine
of USC, Los Angeles,
CA 90089,
USA
ARTICLE
INFO
Keywords:
Huntington
’
s disease
Huntingtin
Huntingtin
exon1
Seeding
Neurotoxicity
ABSTRACT
Huntington
’
s disease
(HD)
is a genetically
inherited
neurodegenerative
disorder
caused
by
expansion
of a pol
-
yglutamine
(polyQ)
repeat
in the
exon-1
of huntingtin
protein
(HTT).
The
expanded
polyQ
enhances
the
amy
-
loidogenic
propensity
of HTT
exon
1 (HTTex1),
which
forms
a heterogeneous
mixture
of assemblies
with
a broad
neurotoxicity
spectrum.
While
predominantly
intracellular,
monomeric
and
aggregated
mutant
HTT
species
are
also
present
in the
cerebrospinal
fluids
of HD
patients,
however,
their
biological
properties
are
not
well
un
-
derstood.
To
explore
the
role
of extracellular
mutant
HTT
in aggregation
and
toxicity,
we
investigated
the
uptake
and
amplification
of recombinant
HTTex1
assemblies
in cell
culture
models.
We
find
that
small
HTTex1
fibrils
preferentially
enter
human
neurons
and
trigger
the
amplification
of
neurotoxic
assemblies;
astrocytes
or
epithelial
cells
are
not
permissive.
The
amplification
of HTTex1
in neurons
depletes
endogenous
HTT
protein
with
non-pathogenic
polyQ
repeat,
activates
apoptotic
caspase-3
pathway
and
induces
nuclear
fragmentation.
Using
a panel
of novel
monoclonal
antibodies
and
genetic
mutation,
we
identified
epitopes
within
the
N-terminal
17
amino
acids
and
proline-rich
domain
of HTTex1
to be
critical
in neural
uptake
and
amplification.
Synapto
-
some
preparations
from
the
brain
homogenates
of
HD
mice
also
contain
mutant
HTT
species,
which
enter
neurons
and
behave
similar
to
small
recombinant
HTTex1
fibrils.
These
studies
suggest
that
amyloidogenic
extracellular
mutant
HTTex1
assemblies
may
preferentially
enter
neurons,
propagate
and
promote
neurodegeneration.
1. Introduction
Huntington
’
s disease
(HD)
is an
autosomal
genetically
inherited
neurodegenerative
disorder
characterized
by
debilitating
motor,
psy
-
chiatric,
and
cognitive
symptoms
(Bates
et al.,
2015
; Ghosh
and
Tabrizi,
2018
). Expansion
of a CAG
repeat
(
>
35)
in the
exon
1 of huntingtin
HTT
gene,
which
translates
into
an
abnormal
polyglutamine
(polyQ)
tract,
is
the
underlying
cause
of Huntington
’
s Disease
(HD).
(The
Huntington
’
s
Disease
Collaborative
Research
Group,
1993
). Mutant
HTT
exon-1
(HTTex1)
released
by
the
enzymatic
cleavage
of
full-length
protein
and/or
by
aberrant
splicing
of
mutant
HTT
mRNA,
is
the
most
aggregation-prone
species,
accumulates
in the
brains
of HD
patients
and
is sufficient
to
induce
severe
HD-like
pathology
in
animal
models
(DiFiglia
et al.,
1997
; Davies
et al.,
1997
; Lunkes
et al.,
2002
; Sathasivam
et al.,
2010
; Bates
et al.,
2015
; Neueder
et al.,
2018
; Yang
et al.,
2020
).
The
aggregation
of mutant
HTTex1
produces
a heterogeneous
mixture
of
assemblies
including
oligomers,
fibrils
and
inclusion
bodies,
which
may
have
different
biological
properties
(Arrasate
et al.,
2004
; Kim
et al.,
2016
; Sahoo
et al.,
2016
; B
̈
auerlein
et al.,
2017
). For
example,
transient
expression
of
mutant
HTTex1
in
culture
models
revealed
that
the
accumulation
of soluble
oligomeric
species
coincides
with
neurotoxicity
(Arrasate
et al.,
2004
; Nucifora
et al.,
2012
). On
the
other
hand,
fibrils
of
HTTex1
may
interact
with
cellular
membranes
and
disrupt
their
archi
-
tectures
and
vital
functions
(B
̈
auerlein
et al.,
2017
). The
promiscuous
interaction
of misfolded
mutant
HTTex1
with
cellular
proteins
may
also
contribute
to the
heterogeneity
of assemblies
and
toxicity
spectrum
(Kim
et al.,
2016
; Wanker
et al.,
2019
).
The
oligomerization
of mutant
HTTex1
in vitro
occurs
by
a stepwise
mechanism
and
is influenced
by
several
factors
such
as interaction
with
biological
membranes
and
the
formation
of an oligomeric
seed
structure,
Abbreviations:
HD,
Huntington
’
s disease;
HTT,
huntingtin;
HTTex1,
huntingtin
exon1;
polyQ,
polyglutamine;
PRD,
proline-rich
domain;
CSF,
cerebrospinal
fluids;
IPSC,
induced
pluripotent
stem
cells;
NPCs,
neuronal
progenitor
cells;
WB,
Western
blots;
SDD-AGE,
semi-denaturing
detergent
agarose
gel
electrophoresis.
* Corresponding
author.
E-mail
address:
Khoshnan@caltech.edu
(A.
Khoshnan).
Contents
lists
available
at ScienceDirect
Neurobiology
of Disease
journal
homepage:
www.elsevi
er.com/loc
ate/ynbdi
https://doi.org/10.1016/j.nbd.2021.105517
Received
20
July
2021;
Received
in revised
form
24
August
2021;
Accepted
21
September
2021
Neurobiology of Disease 159 (2021) 105517
2
which
acts
as a nucleating
agent
and
accelerates
aggregation
(Pandey
et al.,
2018
; Tao
et al.,
2019
). Seed
structures
may
also
assemble
in vivo,
however,
little
is known
about
their
structures
and
roles
in aggregation
and
toxicity.
Seeding-competent
mutant
HTT
assemblies
capable
of
promoting
the
aggregation
of monomeric
HTTex1
in vitro
and
in cell
models
have
been
isolated
from
the
brains
of HD
animal
models
and
postmortem
brain
homogenates
and
cerebrospinal
fluids
(CSF)
of HD
patients.
In
these
studies,
the
levels
of
mutant
HTT
seeds
positively
correlated
with
disease
severity
in the
HD
patients
(Morozova
et al.,
2015
; Ast
et al.,
2018
; Lee
et al.,
2020
). Interestingly,
similar
to other
amyloidogenic
proteins
such
as
α
-synuclein
and
Tau,
mutant
HTTex1
may
have
the
propensity
to
propagate
by
a prion-like
mechanism
(Jucker
and
Walker,
2018
; Pearce
and
Kopito,
2018
; Masnata
et al.,
2019
). For
example,
mutant
HTTex1
assemblies
spread
between
neurons
in
the
brains
of
Drosophila
models
of
HD
and
form
aggregates
by
recruiting
monomeric
HTTex1
(Pearce
et al.,
2015
). Moreover,
cere
-
brospinal
fluids
(CSF)
of HD
patients
seed
mammalian
cells
engineered
to express
mutant
HTTex1-EGFP
and
induce
protein
aggregation
(Tan
et al.,
2015
; Lee
et al.,
2020
). These
encouraging
findings
provide
a new
direction
in HD
research
and
may
have
implications
for
the
spread
and
propagation
of neurotoxic
mutant
HTT
assemblies
in HD.
The
neurodegenerative
aspect
of
HD
may
release
various
mutant
HTT
assemblies
in the
circulation
and
CSF.
Moreover,
HTT
is actively
and
passively
secreted
from
cultured
cells
and
neurons
in the
HD
animal
models
(Trajkovic
et al.,
2017
; Caron
et al.,
2021
). Indeed,
the
levels
of
mutant
HTT
in plasma
and
CSF
of HD
patient
are
being
used
as bio
-
markers
evaluating
the
efficacy
of therapeutics
in HD
patients
(Tabrizi
et al.,
2019
). While
few
recent
studies
demonstrate
that
extracellular
amyloidogenic
HTTex1
fibrils
have
the
propensity
to enter
cell
lines
and
neurons
in mouse
models
(Masnata
et al.,
2019
; Lee
et al.,
2020
), the
biological
properties
of extracellular
mutant
HTT
seeds
including
the
mechanism
for
cell
entry,
amplification,
and
neurotoxicity
remain
to be
investigated.
Here,
we
performed
a structural
and
biological
charac
-
terization
of small
HTTex1
fibrils.
We
report
that
small
HTTex1
fibrils
preferentially
enter
human
neurons,
accumulate
in the
nucleus,
amplify
and
produce
neurotoxic
assemblies.
We
further
demonstrate
that
con
-
formations
within
the
N-terminal
17
amino
acids
and
proline-rich
do
-
mains
(PRD)
of HTT
are
critical
for
the
neural
entry
and
amplification.
We
propose
that
extracellular
assemblies
of HTTex1
may
contribute
to
amplification
of neurotoxic
assemblies.
2. Materials
and methods
2.1. Antibodies
PHP1-PHP3
mouse
monoclonal
antibodies
(mAbs)
were
reported
previously
(Ko
et al.,
2018
). The
new
PHP5
and
PHP6
mAbs
were
iso
-
lated
from
a mAb
library
made
to the
N-17
peptide
with
7 glutamines
at
the
C-terminus,
and
PHP7-PHP10
were
produced
to sonicated
mutant
HTTex1
fibrils
(Khoshnan
et al.,
in preparation).
Clones
were
selected
from
each
hybridoma
library
for
binding
to
HTT
species
by
ELISA,
Western
blots
and
dot
blots
as previously
described
(Ko
et al.,
2018
,
Supplementary
fig.
2A
and
B).
Antibody
to beta
III
Tubulin
was
from
Abcam
(Cat#
ab18207,
1:1000).
The
secondary
antibodies
were
goat
anti-rabbit
Alexa
Fluor
488
Cat#
A32731,
goat
anti-mouse
Alexa
Fluor
488,
Cat#
A28175,
goat
anti-mouse
Alexa
Fluor
594
Cat#
A32742,
Life
Technologies,
1:1000
and
goat
anti-mouse,
Horseradish
Peroxidase
(HRP)
Cat#
A16072,
Invitrogen,
1:10000.
2.2. Dot blot assay
A strip
of PVDF
membrane
was
pre-wet
in 100%
methanol
for
15
s,
soaked
in water
for
2 min
and
equilibrated
for
5 min
in TBS-T
(0.05%
Tween,
pH
7.4).
A sheet
of Whatman
filter
paper
was
then
soaked
in
TBS-T
and
placed
on
a dry
sheet
of Whatman
filter
paper
on
top
of some
dry
paper
towels.
The
PVDF
membrane
was
placed
on
top
of filter
stack
and
2
μ
L of each
protein
was
spotted
on
a pre-marked
grid.
The
mem
-
brane
was
dried
to fix
proteins
to it for
1.5
h at room
temperature.
The
membrane
was
then
blocked,
probed
with
each
indicated
primary
antibody
followed
by
treatment
with
goat-anti-mouse
HRP
secondary
antibody,
and
detected
by
the
addition
of
chemiluminescent
agent
Clarity
™
Western
ECL
Substrate
(Cat#
1705060,
Bio-rad).
2.3. Engineering
of recombinant
PHP2
antibody
and lentivirus
production
The
DNA
fragments
encoding
the
antigen
binding
domain
of VH
and
VL
of PHP2
were
amplified
from
a cDNA
library
made
to
mRNA
of
parental
hybridoma
by
standard
PCR
methods
using
mixture
of com
-
mercial
primers
and
sequenced
(Khoshnan
et al.,
2002
). The
amplified
cDNAs
were
assembled
into
an
IgG2
backbone
(provided
by
Dr.
Ale
-
jandro
Balazs
at the
Ragon
Institute
of
MGH,
MIT
and
Harvard)
by
Gibson
assembly
(New
England
biolabs)
and
subsequently
cloned
into
a
lentiviral
vector
(FUGW).
Control
and
PHP2
recombinant
viral
particles
were
produced
in HEK-293
cells
as described
previously
(Khoshnan
and
Patterson,
2012
). Viral
titers
were
determined
using
a GFP
lentivirus
as a
reference.
Subsequently,
MESC2.10
neural
progenitor
cells
(NPCs)
were
transduced
at multiplicity
of
(2:1).
Supernatant
of
neurons
form
the
transduced
NPCs
were
tested
for
antibody
secretion
and
PHP2
antibody
binding
to HTT
fibrils
was
confirmed
by
Western
blots.
2.4. Purification
of thioredoxin
(TRX)-HTTex1
fusion
protein
Expression
and
purification
of wild-type
(Q25)
and
mutant
(Q46)
HTTex1
fusion
proteins
has
been
described
previously
(Bugg
et
al.,
2012
; Isas
et al.,
2015
; Isas
et al.,
2017
). Briefly,
bacterial
cell
pellet
containing
the
expressed
fusion
protein
(6XHis-TRXA-HTTex1/or
HTTex1-111C
variant
for
labeling)
were
lysed
and
cell
debris
were
removed
by
centrifugation.
The
clarified
lysates
containing
the
fusion
protein
were
loaded
onto
Ni-column
(NiHis60
super
flow
resin)
and
washed
with
a low
concentration
of imidazole
saline
buffer.
The
fusion
protein
was
eluted
with
high
concentration
of imidazole
saline
buffer.
The
HTTex1-111C
variant
was
first
labelled
with
AlexaFlour
555
(1 to 3
M ratios
for
3 h),
diluted
1 to 10
with
low
salt
buffer,
loaded
on
to
anionic
exchange
resin
(Mono
Q),
and
further
fractionated
with
a linear
gradient
(25
mM
to 1 M salt)
to remove
free
label
and
elute
the
labelled
Alexa
sample.
2.5. Preparation
and purification
of HTTex1
fibrils
Monomer
HTTex1
(Q46)
((HTTex1)
(Q46_111Alexa555))
was
pro
-
duced,
by
the
removal
of the
TRX
fusion
tag
enzymatically
(EKMax),
followed
by
separation
on
reverse
phase
column
(C4)
with
an
acetoni
-
trile
gradient
as previously
described
(Pandey
et al.,
2018
). HTTex1
fi
-
brils
or
(Alexa555
labelled
fibrils)
were
made
by
first
solubilizing
lyophilized
protein
powder
from
previous
step
in 0.5%
TFA
(
v
/v)
in
methanol,
determined
the
concentration,
and
removed
the
organic
sol
-
vent
by
gentle
N
2
gas
stream,
resulting
in a thin
clear
protein
film.
The
protein
film
was
resuspended
in ice
cold
buffer
(20
mM
Tris
pH
7.4,
150
mM
NaCl)
and
adjusted
to between
20
and
25
μ
M concentration.
Fibrils
reaction
started
by
adding
1%
(molar
ratio)
of sonicated
HTTex1
seeds
as previously
described
(Isas
et al.,
2017
) and
incubated
at 4
◦
C over
-
night.
In a separate
reaction,
20%
Alexa
labelled
fibrils
were
made
by
adding
4 to 5
μ
M of HTTex1_111alexa555
solubilized
monomer
to the
Httex1
unlabeled
sample.
After
overnight
incubation
fibrils
were
har
-
vested
by ultra-centrifugation,
and
the
resulting
translucent
pellets
were
resuspended
in TFA:H20
(1:4000)
at a concentration
of 1 mg/ml
and
fragmented
using
sonication.
Mutant
HTTex1
in which
each
residue
in
PRD
domain
was
replaced
with
a Pro
residue
(HTTex1mL17)
was
generated
for
probing
the
binding
site
of PHP1
and
PHP2
as recently
reported
(Ko
et al.,
2018
). HTTex1mL17
Fibrils
were
made
as described
for
the
HTTex1
in the
section
above.
A. Chongtham
et al.
Neurobiology of Disease 159 (2021) 105517
3
2.6. Treatment
of cells with HTTex1
seeds
Neurons
were
derived
from
embryonic
human
MESC2.10,
IPSC-
derived
neuronal
progenitor
cells
(NPCs)
(provided
by
Dr.
Alysson
Muotri,
UCSD)
according
to standard
protocols
(Khoshnan
and
Patter
-
son,
2012
). Astrocytes
were
derived
from
IPSCs
by culturing
progenitors
in DMEM/F12
supplemented
with
N2
growth
factor
and
0.5%
FBS
(Cat#
ES-009-B,
Millipore)
for
14
–
20
days.
Caco-2
and
Neuro2A
were
obtained
from
ATCC
and
cultured
according
to instructions
provided.
Each
line
was
treated
with
1.25
nM,
5 nM
or 10
nM
of sonicated
HTTx1
fibrils
for
the
indicated
time
points
in
the
figure
legends
and
processed
for
immunocytochemistry
and
confocal
microscopy
as described
previously
(Khoshnan
and
Patterson,
2012
). For
antibody
inhibition
assay,
10
nM
of sonicated
fibrils
were
pre-incubated
with
1
μ
g of each
antibody
clone
indicated
in the
figures
for
2 h at RT
and
subsequently
added
to growth
medium
and
processed
as above.
2.7. In vitro seeding
assay with HTT species
Sonicated
HTTx1
seeds
(10
ng)
were
incubated
with
recombinant
WT
HTTex1
monomers
or 100
μ
g of total
protein
from
human
neural
lysates
for
4 h at 25
◦
C with
continuous
agitation.
For
examining
the
seeding
capability
of amplified
products,
5%
(
v
/v)
of seeded
neuronal
lysate
was
incubated
with
100
μ
g of fresh
lysate
for
4 h at 25
◦
C. Semi-
denaturing
detergent
agarose
gel
electrophoresis
(SDD-AGE)
was
per
-
formed
to examine
the
amplified
products
and
the
corresponding
seeds
as
described
previously
(Halfmann
and
Lindquist,
2008
) with
some
modifications.
Briefly,
seeded
lysates
were
loaded
in a 1.5%
agarose
gel
in 1
×
Tris-acetate-EDTA
(TAE)
(Tris
base,
glacial
acetic
acid
and
EDTA)
containing
a final
concentration
of 0.1%
SDS.
Using
1
×
Tris-buffered
saline
(TBS),
downward
capillary
action
was
used
to transfer
protein
to immune-blot
PVDF
membrane
(Merck
cat#
IPVH00010).
Membranes
were
blocked
with
5%
milk
in wash
buffer
(0.05%
Tween
in PBS)
and
incubated
with
the
indicated
primary
anti-HTT
antibodies
overnight
at
4
◦
C. HRP-conjugated
goat
anti-mouse
secondary
antibody
(1:10000)
in
blocking
buffer
was
then
applied
for
2 h and
developed
with
ECL
sub
-
strate
and
X-ray
film.
2.8. Western
blot analysis
of seeded
neurons
MESC2.10
derived
neurons
grown
in
10
cm
culture
plates
were
treated
with
10
nM
of sonicated
HTTex1
fibrils
for
24
h. The
seeded
neurons
were
harvested
and
lysed
in RIPA
buffer
(50
mM
Tris-HCl
(pH
7.4),
150
mM
NaCl,
1%
NP-40,
0.1%
SDS
and
protease
inhibitor).
50
μ
g
of neuronal
lysate
and
corresponding
mHTTex1
seed
were
boiled
for
5
min
with
sample
loading
buffer
and
loaded
into
a 4
–
20%
poly
-
acrylamide
gel
(SDS-PAGE,
Criterion
Bio-Rad)
to
detect
monomeric
endogenous
HTT
or
by
semi-denaturing
detergent
agarose
(1.5%)
gel
electrophoresis
(SDD-AGE)
to probe
for
the
seeded
aggregated
products.
Different
antibodies
indicated
in the
figures
and
figure
legends
were
used
for
detecting
different
species
of HTT.
For
proteinase
K resistance
assay,
0.1
μ
g of mHTTex1
seed
or 50
μ
g of
seeded
neuronal
lysate
was
incubated
with
varying
doses
of proteinase
K
(0 to 0.5
μ
g/mL,
Qiagen)
for
30
min
at 25
◦
C and
heat
inactivated
at 75
◦
C for
10
min.
The
digested
products
were
analyzed
by
SDD-AGE
and
western
blot.
PHP2
was
used
as the
detection
antibody.
2.9. Immunodepletion
of HTT in neural
lysates
Immunodepletion
of
HTT
was
performed
by
incubating100
μ
g of
neuronal
lysates
with
2
μ
g of each
anti-HTTex1
or
control
antibodies
overnight
at
4
◦
C with
rotation.
The
antibody-HTT
complexes
were
precipitated
with
protein
G covalently
conjugated
to magnetic
beads
(Thermo
Scientific,
P188803).
The
HTT-depleted
lysates
were
seeded
with
10
ng
of sonicated
HTTex1
fibrils
for
4 h at 25
◦
C with
rotation.
SDD-AGE
was
performed
to detect
the
amplified
products.
The
primary
antibody
used
for
western
blot
analysis
was
PHP1,
which
is reactive
to
mutant
HTT
aggregates
in HD
animal
models
(Ko
et al.,
2018
).
2.10.
Sucrose
fractionation
of HD brains
Synaptosome
and
ER/Golgi
fractions
were
prepared
from
individual
mouse
forebrains
according
to
a recently
published
method
(Mastro
et al.,
2020
). Briefly,
forebrains
of 9-month
old
ZQ175
HD
mice
were
dissected
from
each
animal,
rinsed
in Buffer
A (0.32
M sucrose,
1 mM
NaHCO
3
, 1 mM
MgCl
2
, 0.5
mM
CaCl
2
, 0.1
mM
phenylmethylsulphonyl
chloride
(PMSF,
Sigma
Millipore,
St.
Louis,
MO).
Each
individual
fore
-
brain
was
homogenized
in Buffer
A (10%
w
/
v
, 4.5
ml
for
mice)
with
12
up
and
down
strokes
of a Teflon/glass
homogenizer
at 900
rpm.
Ho
-
mogenates
were
subjected
to centrifugation
at 1400
g
for
10
min.
The
pellet
was
resuspended
in Buffer
A to 10%
w/v
(3.8
ml),
homogenized
(three
strokes
at 900
rpm)
and
subjected
to centrifugation
at 710
g
for
10
min.
The
final
resultant
pellet
(P1)
was
harvested
as a crude
fraction
containing
the
nuclei.
The
two
supernatants
(S1)
were
combined
and
subjected
to centrifugation
at 13,800
g
for
10
min.
The
resulting
pellet
(P2)
was
resuspended
in Buffer
B (0.32
M sucrose,
1 mM
NaHCO
3
; 2 ml
for
mice),
homogenized
with
6 strokes
at 900
rpm
in a Teflon/glass
homogenizer,
and
layered
onto
a discontinuous
sucrose
gradient
(equal
parts
0.32
M,
0.85
M,
1.0
M,
and
1.2
M sucrose
in 1 mM
NaH
2
CO
3
buffer
(10.5
ml).
Gradients
were
subjected
to centrifugation
for
2 h at 82,500
g
in a swinging
bucket
rotor.
The
bands
between
0.32
M and
0.85
M su
-
crose
(light
membranes,
Myelin),
0.85
M
and
1.0
M
sucrose
(light
membranes,
Endoplasmic
Reticulum,
Golgi),
1.0
M and
1.2
M sucrose
sections
(Synaptosomes)
were
collected.
2.11.
Assessment
of seeding
activity
of protease-resistant
brain fractions
Synaptosome
and
ER/Golgi
fractions
isolated
from
ZQ175
mouse
brain
by
sucrose
fractionation
were
dialyzed
using
Slide-A-Lyzer
Dial
-
ysis
Cassette
(Thermo
Fisher
Scientific,
66,203).
The
dialyzed
mouse
brain
fractions
isolated
by
sucrose
fractionation
were
incubated
with
0.01
μ
g/mL
of Proteinase
K at 37
◦
C for
1 h and
heat
inactivated
at 75
◦
C
for
10
min.
MESC2.10
neurons
in 12-well
plate
were
then
incubated
with
50
μ
g of each
fraction
at 37
◦
C for
24
h. This
was
followed
by
immunocytochemistry
to
examine
for
the
presence
of
seeding-
competent
fibrils
and
assembly
formation.
2.12.
Immunocytochemistry
and nuclear
damage
quantifications
Cells
were
fixed
with
4%
formaldehyde
in PBS
at room
temperature
for
30
min.
After
permeabilization
and
blocking
(70%
methanol
in PBS,
at least
1 h at