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18
Figure Legends
Figure 1: Immunofluorescent imaging of intranuclear FUS and SFPQ
(A) The left panels show the hippocampal granule cells from representative cases. The
sections were immunostained with anti-FUS and anti-SFPQ antibodies combined with
DAPI-based (blue) nuclear staining. The FUS (red) and SFPQ (green) signals were
colocalized within the intranuclear matrixes in the control sample. In contrast, the signals
were dissociated within the nuclei of the FTLD-FUS, ALS-TDP, PSP, and CBD cases.
The Betz cells from the same cases are shown in the right panels. Signals for FUS and
SFPQ in Betz cell nuclei were also dissociated in FTLD-FUS, ALS-TDP, PSP, and CBD
cases. Scale bars = 5 μm for left, 10 μm for right panels. (B) Raw quantitative
colocalization indices for FUS and SFPQ from representative individual cases. The
colocalization indices (R2) for cases with ALS/FTLD-FUS (ALS/FTLD-FUS#1 and #2),
ALS/FTLD-TDP (ALS/FTLD-TDP#1), PSP (PSP#1 and #2), and CBD (CBD#1 and #2)
were lower than in cases with PiD (PiD#1), AD (AD#1 and #2), or the controls (Cont#1
and #2). Data shown are mean ± SEM.
Figure 2: Quantification of FUS and SFPQ intranuclear colocalization across
disease groups
The colocalization indices in the hippocampal granule cells were calculated for all
included cases and are displayed with mean ± SEM. The colocalization indices in the
hippocampal granule cells were calculated for each disease group. The average
colocalization indices were significantly lower in the ALS/FTLD-FUS, ALS/FTLDTDP, PSP, and CBD cases than in the controls. No significant differences in the R2 value
were observed between the AD cases and the controls, or between the PiD cases and the
19
controls. Statistical analysis was performed using a Kruskal-Wallis test with
significance levels set at p < 0.05 after Bonferroni/Dunn correction of the raw p-values
for 7 group comparisons: the control (n = 28) vs. ALS/FTLD-FUS (n = 14),
ALS/FTLD-TDP (n = 24), PSP (n = 25), CBD (n = 8), PiD (n = 5), or AD (n = 26).
Figure 3: Interactions between FUS and SFPQ are disrupted in the brain tissue of
ALS/FTLD and PSP cases.
Frozen tissues of frontal cortex (prefrontal area) were available for cases with
ALS/FTLD-TDP (n = 8), PSP (n = 5), or AD (n = 5), and controls (n = 13). The detailed
methods were described in Supplemental material (Supplemental Fig. 5). (A) Protein
extracts from the frontal lobe of ALS/FTLD-TDP cases and controls were
immunoprecipitated with an anti-FUS antibody (A300-293A) and blotted with antiSFPQ and anti-FUS antibodies (4H11). Immunoblots of the protein extracts (input)
using anti-SFPQ, anti-FUS, and anti-α-Tubulin antibodies are also shown. The signal
intensities for SFPQ in the FUS-immunoprecipitants were lower in ALS/FTLD-TDP
cases than in controls (right graph, n = 8 for each, student t- test). (B)
Immunoprecipitation of PSP cases also revealed lower FUS and SFPQ interactions than
controls (n = 5 for each, student t- test). (C) FUS-SFPQ interactions were not disrupted
in AD cases (n = 5 for each, student t- test). (D) The converse immunoprecipitation was
performed for validation. Protein extracts from ALS/FTLD, PSP, and AD cases as well
as controls were immunoprecipitated with an anti-SFPQ antibody (Bethyl Laboratories)
and blotted with anti-SFPQ (abcam) and anti-FUS antibodies (4H11). Immunoblots of
the protein extracts (input) using anti-SFPQ, anti-FUS, and anti-α-Tubulin antibodies
are also shown. The results revealed lower FUS and SFPQ interactions than controls in
20
ALS/FTLD and PSP, but not in AD. (E) RNA was simultaneously extracted from
samples shown in Fig. 3A from the frontal lobe of ALS/FTLD-TDP cases and controls.
Subsequent qPCR analysis revealed that the splicing ratio of MAPT exon 10+/exon 10(Ex10/Ex10-) was increased in ALS/FTLD-TDP cases relative to controls (n = 8 for
each, student t-test). (F) The qPCR analysis revealed that the splicing ratio of MAPT
exon 10+/exon 10- was increased in PSP cases relative to controls (n = 5 for each,
student t-test). (G) In contrast, the splicing ratio of MAPT exon 10+/exon 10- was not
elevated in AD cases (F) (n = 5 for each, student t-test). Data shown are mean ± SEM.
Uncropped blots are available as Supplementary material.
21
Table.1 Clinical and pathological data
Pathological
phenotypes
(female/male)
Age at death
(year, SEM)
Average
Braak NFT
stage
(SEM)
Average Thal
Amyloid
phase
(SEM)
Used for IHC
(female/male)
FUS-SFPQ
colocalization
index (SEM)
ALS/FTL
D-FUS
14
(3/11)
60.0
(3.9)
0.58
(0.23)
ALS/FTL
D-TDP
24
(12/12)
69.7
(1.7)
1.09
(0.20)
PSP
CBD
PiD
AD
p Value
31
(15/16)
79.5
(1.6)
5.05
(0.17)
control
35
(10/25)
70.5
(1.3)
1.33
(0.16)
25
(8/17)
75.5
(1.2)
1.80
(0.19)
(4/4)
70.3
(1.6)
(4/1)
75.2
(4.4)
0.75
(0.43)
0.36
(0.14)
0.70
(0.24)
1.0
(0.82)
0.0
(0.0)
4.40
(0.13)
0.82
(0.20)
< 0.001*
15
(3/12)
24
(12/12)
25
(8/17)
(4/4)
(4/1)
26
(13/13)
28
(7/21)
0.21
(0.03)
0.24
(0.03)
0.21
(0.02)
0.21
(0.01)
0.50
(0.05)
0.41
(0.02)
0.38
(0.02)
= 0.001*
< 0.001*
= 0.004#
= 0.004##
< 0.001†
= 0.040††
FUS-SFPQ
0.05
0.04
0.07
0.07
013
0.14
0.20
= 0.038#
expression
(0.01)
(0.01)
(0.02) (0.03) (0.05) (0.02)
(0.03)
< 0.001##
variability
< 0.003†
index (SEM)
13
Used for IP
(3/5)
(2/3)
(2/3)
(6/7)
and WB
(female/male)
ALS, amyotrophic lateral sclerosis; FTLD, frontotemporal lobar degeneration; PSP, progressive
supranuclear palsy; CBD, cortico-basal degeneration; PiD, Pick’s disease; AD, Alzheimer’s disease
*AD versus controls, #ALS/FTLD-TDP versus controls, ##ALS/FTLD-TDP versus controls, †PSP versus
controls, ††CBD versus controls.
Kruskal-Wallis test, significance level was determined by Bonferroni/Dunn correction for seven groups.
22
Figure 2
FUS-SFPQ colocalization index
(R )
1.0
p=0.004
p=0.004
p<0.0001
p=0.040
N.S.
N.S.
CBD
PiD
AD
(n= 8)
(n= 5)
(n= 26)
0.8
0.6
0.4
0.2
0.0
Control
(n= 28)
ALS/FTLD ALS-FTLD
PSP
-FUS
-TDP
(n= 14)
(n= 24)
(n= 25)
SFPQ/FUS (IP:FUS)
ALS/FTLD
2.0
Signal intensity ratio
(ratio to Control)
SFPQ
FUS
SFPQ
FUS
αTubulin
0.5
0.0
on
SFPQ/FUS (IP:FUS)
SFPQ
FUS
input
SFPQ
FUS
αTubulin
Control ALS/FTLD
C9 C10 C11 F1
1.0
0.5
C9 C10 C11 C12 C13 A1
SFPQ
FUS
SFPQ
FUS
input
SFPQ
relative to control
relative to control
0.0
on
ol
LS
/F
TL
0.0
Control
AD
1.0
p<0.05 p<0.05
0.5
0.0
qRT-PCR
Ratio to Ex10+/ Ex10-
p<0.05
0.5
0.5
1.5
1.0
1.0
2.0
αTubulin
1.5
N.S.
1.5
FUS/SFPQ(IP:SFPQ)
FUS
2.0
A2 A3 A4 A5
FUS
AD
2.0
SFPQ
F2 F3 P1 P2 P3 A1 A2 A3
qRT-PCR
Ratio to Ex10+/ Ex10-
SFPQ/FUS (IP:FUS)
AD
Control PSP
IP:anti-SFPQ
LD
FT
LS
αTubulin
0.0
PSP
ol
Control
Signal intensity ratio
(ratio to Control)
p<0.05
tr
input
C1 C2 C3 C4 C5 P1 P2 P3 P4 P5
Signal intensity ratio
(ratio to Control)
1.5
IP:anti-FUS
PSP
p<0.01
1.0
Control
IP:anti-FUS
1.5
Signal intensity ratio
(ratio to Control)
C1 C2 C3 C4 C5 C6 C7 C8 F1 F2 F3 F4 F5 F6 F7 F8
input
IP:anti-FUS
Control
p<0.05
Control PSP
on
S-
TL
PS
qRT-PCR
Ratio to Ex10+/ Ex10N.S.
relative to control
Figure 3
1.5
1.0
0.5
0.0
Control
AD
Supplementary Information
Supplementary Figure 1: Quantification of FUS and SFPQ intranuclear
colocalization
(A) We compared the immunostaining of different anti-FUS and anti-SFPQ antibody
clones on a control brain sample using double immunofluorescence. A mousemonoclonal anti-FUS antibody from Santa Cruz (4H11) was combined with a rabbitpolyclonal anti-FUS antibody from either Bethyl Laboratories (293A) or SigmaAldrich. Convergence of the fluorescent antibody signals within the nuclei of
hippocampal granule cells was considered evidence for reliable immunolabeling of the
FUS protein (data not shown). The immunostaining of two additional anti-SFPQ
antibodies, a mouse-monoclonal antibody from Sigma-Aldrich and Abcam (B92), and a
rabbit-polyclonal antibody from Bethyl Laboratories, were also compared. Signals from
these two clones likewise indicated their sufficiency for use. Secondary fluorescent
antibodies we used were conjugated Alexa-488 and 546 (1:1000, Thermo Fisher
Scientific). (B) Hippocampal granule cells that were immunostained with anti-SFPQ,
FUS, and NeuN antibodies and counter-stained with DAPI are shown at 40x
magnification (left) and at 630x magnification (right). Fluorescent signals for
intranuclear FUS and SFPQ were obtained at 630x magnification. Scale bars, 200 μm
for left, 10 μm for right images. (C) Fluorescent signals for intranuclear FUS and SFPQ
were obtained at 630x magnification. Wavelengths of 488 and 546 nm were set as the
fluorescent unit, and signal intensities from each intranuclear matrix were automatically
measured along the largest nucleus diameter by ZEN2012 software (left images in the
upper image-graph sets). The middle graphs represent FUS (red) and SFPQ (green)
signal intensities on the y-axis given for each pixel on the x-axis along the diameter of
the nucleus. As shown in the right panels, correlation indices (R2) were high when the
FUS and SFPQ signals were coincident with each pixel. In contrast, the R2 values were
low when the proteins were spatially dissociated in the nucleus. For instance,
hippocampal granule cells in the control sample had FUS-SFPQ colocalization indices
of 0.45, 0.51, 0.78, and 0.48, whereas neurons from the disease group had values of
0.11, 0.025, 0.0082, and 0.029 (lower panels). Scale bars, 5 μm.
Supplementary Figure 2: Immunofluorescent imaging of intranuclear FUS and
SFPQ
Images for the cases depicted in Fig. 1A are shown in lower magnification. The sections
were immunostained with anti-FUS and anti-SFPQ antibodies and counter-stained with
DAPI-based (blue) nuclear staining. Scale bars = 10 μm for upper images, 20 μm for
lower images.
Supplementary Figure 3: Comparison of FUS-SFPQ interactions between neurons
with and without FUS-positive cytoplasmic inclusions.
Triple immunostaining was performed using anti-SFPQ (monoclonal-mouse, SigmaAldrich), anti-FUS (polyclonal rabbit, Bethyl Laboratories, A300-293A), and anti-FUS
(polyclonal rabbit, Sigma-Aldrich) antibodies. (A) Three of the included ALS/FTLDFUS cases (case #3, #4, and #5) had FUS-immunopositive cytoplasmic inclusions in the
hippocampal granule cells. The interaction of FUS and SFPQ was similarly impaired in
neurons with and without cytoplasmic inclusions. Arrows indicate FUS-positive
cytoplasmic inclusions. Opal 4-Color Kit (Parkin Elmer) was used for triple
immunofluorescent study. Scale bars = 5 μm. (B) Quantification data also revealed no
significant differences in FUS-SFPQ interactions between neurons with and without the
cytoplasmic FUS-positive inclusions. Data shown are mean ± SEM.
Supplementary Figure 4: Comparison of FUS-SFPQ interactions between neurons
with and without inclusions positive for phosphorylated-TDP-43 or phosphorylatedtau
(A) The FUS-SFPQ intranuclear colocalization was compared between neurons with
and without phosphorylated TDP-43 (p-TDP-43) inclusions in a representative
ALS/FTLD-TDP case (left images). A comparison between neurons with or without
phosphorylated-tau (p-tau) inclusions was also undertaken in PSP and CBD cases
(middle and right images). The interaction of FUS and SFPQ was similarly impaired in
neurons with and without the cytoplasmic inclusions. Scale bars = 10 μm. Arrows
indicate neurons harboring p-TDP-43 or p-tau inclusions. Scale bars = 5 μm. (B)
Quantification data also revealed no significant differences in FUS-SFPQ interactions
between neurons with and without cytoplasmic FUS-positive inclusions. Data shown
are mean ± SEM.
Supplementary Figure 5: Interactions between FUS and SFPQ are disrupted in the
brain tissue of ALS/FTLD and PSP cases
Frozen frontal lobe tissues, which contained the cortex and subcortical white matter,
from autopsied brains were suspended in 3 ml of cold TNE buffer with protease
inhibitors (Roche), and homogenized in a dounce tissue grinder with 15 strokes of a
loose pestle. The homogenate was centrifuged at 3000 x g for 10 min to remove debris
and was centrifuged again at 14000 x g for 12 min. The antibodies used in
immunoprecipitation and immunoblotting are listed in Supplementary Table 1. The
band intensities were measured using Multi Gauge software (Fujifilm).
(A) The control immunoprecipitation experiment using anti-FUS (A300-293A) and
rabbit IgG with two control samples (C1 and C2) is shown. (B) Tau protein profiles were
shown by a fractionation assay using sarcosyl. Protein extracts from the cases in Fig. 3
were fractionated into TBS-soluble and sarcosyl-insoluble fractions. The TBS-soluble
fractions were immunoblotted with anti-4R-tau (4R-T) and anti-3R-tau (3R-T) antibodies.
(C) The ratio of signal intensities for 4R-T/3R-T in Supplementary Fig. 5A are shown.
Although the 4R-T/3R-T ratio was not significantly altered, it trended towards 4R-T
dominance in ALS/FTLD cases relative to controls (left graph, n = 8 for each, student ttest). In contrast, the 4R-T/3R-T ratio was significantly increased in PSP cases relative to
controls (middle graph, n = 5 for each), whereas there was no difference in AD cases and
controls (right graph, n = 5 for each, student t-test). Data shown are mean ± SEM. (D)
The sarcosyl-insoluble fractions were immunoblotted with anti-total tau (Tau-5) and antiphosphorylated tau (HT7) antibodies. Phosphorylated tau was present in sarcosylinsoluble fractions from the PSP and AD samples but not from ALS/FTLD cases.
Supplementary Figure 6: Variability of FUS and SFPQ expression in the
hippocampal granule cells
To evaluate the expression levels of FUS or SFPQ in the individual neuronal nuclei,
the signal intensities of the two proteins were acquired from 400 nuclei in the
hippocampal granule cell nuclei. Sample images were acquired and analyzed using a
BZ-X700 microscope (Keyence). We determined the degree of correlation between the
two proteins with a regression analysis to evaluate the proportion of FUS and SFPQ
expression in the hippocampal granule cells for each sample. We defined the R2
correlation coefficient as an FUS-SFPQ expression variability value. When the values
are large, the expression of FUS and SFPQ in individual neurons is less variable. The
FUS-SFPQ expression variability values were plotted and compared among the disease
states.
(A) Anti-SFPQ and anti-FUS immunofluorescence images of hippocampal granule
cells from representative cases are shown. The SFPQ (green) and FUS (red) signals
were equivalent in a control sample but exhibited a variable mosaic expression pattern
in cases with ALS-FUS, FTLD-TDP, PSP, and CBD. Scale bars, 10 μm. (B) Schematic
diagram depicting our method for quantitatively determining the degree of FUS-SFPQ
expression variability in representative cases. The left box denotes a control case,
whereas the right box is for an ALS/FTLD-FUS case. Wavelengths at 488 and 546 nm
were set as the fluorescent unit, and fluorescent signals were automatically acquired
from the individual hippocampal granule cells using ZEN2012 software. After plotting
the FUS and SFPQ signals for each hippocampal granule cell, a correlation index (R2)
was calculated. In the neurons of a control, the proportion of FUS and SFPQ was
equivalent hence well correlated with a correlation index of 0.474. In a case with FTLDFUS, the expressions of FUS and SFPQ were disproportional with a correlation index of
0.006. Scale bars, 10 μm. (C) In controls, the average FUS-SFPQ expression variability
value was 0.20 ± 0.18. By contrast, the values were significantly lower in the cases with
ALS/FTLD-FUS (0.053 ± 0.013, p = 0.038), ALS/FTLD-TDP (0.043 ± 0.008, p <
0.0001), and PSP (0.071 ± 0.158, p = 0.003) than that of controls (0.14 ± 0.12). The
cases with AD (0.14 ± 0.12), CBD (0.070 ± 0.029), and PiD (0.13 ± 0.05) did not show
significant differences when compared with the control. Kruskal-Wallis test was under
taken. Significance level was set at p < 0.05 after Bonferroni/Dunn correction of the raw
p-values for seven group comparisons: the control (n = 28) vs. ALS/FTLD-FUS (n =
14), ALS/FTLD-TDP (n = 24), PSP (n = 25), CBD (n = 8), PiD (n = 5), or AD (n = 26).
Data shown are mean ± SEM. (D) Scatter plots for all included individuals showed a
significant correlation between the FUS-SFPQ expression variability indices and the
FUS-SFPQ colocalization indices (linear least square method, R2 = 0.128, p < 0.001).
Supplementary Table 1
Antibodies used in the study.
Figure
primary antibody
vender
#clone or product
source
experiment
dilution
Figure 1
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IP
5 μg/ mg lysate
anti-SFPQ
abcam
B92
mouse-IgG
WB
1:1000
anti-FUS
Santa Cruz
4H11
mouse-IgG
WB
1:500
anti-SFPQ
Bethyl Laboratories
A301-321A
rabbit-IgG
IP
5 μg/ mg lysate
anti-αTubulin
Santa Cruz
10D8
mouse-IgG
WB
1:500
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-FUS
Santa Cruz
4H11
mouse-IgG
IF
1:300
anti-FUS
Sigma-Aldrich
HPA008784
rabbit-IgG
IF
1:200
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-SFPQ
Bethyl Laboratories
A301-321A
rabbit-IgG
IF
1:1000
anti-NeuN
abcam
EPR12763
rabbit-IgG
IF
1:1000
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-FUS
Sigma-Aldrich
HPA008784
rabbit-IgG
IHC
1:200
Figure 3
Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure 4
Supplementary Figure 5
Supplementary Figure 6
Pathological validation
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-phosphorylated TDP-43
Cosmo Bio
S409/410
rabbit-IgG
IF
1:1000
anti-phosphorylated tau
Thermo Fisher
AT8
mouse-IgG
IF
1:200
anti-3R-T
Millipore
RD3
mouse-IgG
WB
1:2000
anti-4R-T
Millipore
RD4
mouse-IgG
WB
1:1000
anti-phosphorylated tau
Thermo Fisher
HT7
mouse-IgG
WB
1:1000
anti-total-tau
abcam
TAU-5
mouse-IgG
WB
1:2500
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IF
1:1000
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IF
1:300
anti-TDP-43
Proteintech
10782-2-AP
rabbit-IgG
IHC
1:200
anti-phosphorylated TDP-43
Cosmo Bio
S409/410
rabbit-IgG
IHC
1:1000
anti-phosphorylated tau
Thermo Fisher
AT8
mouse-IgG
IHC
1:200
anti-β-amyloid
Dako
6F3D
mouse-IgG
IHC
1:300
anti-FUS
Bethyl Laboratories
A300-293A
rabbit-IgG
IHC
1:1000
anti-FUS
Sigma-Aldrich
HPA008784
rabbit-IgG
IHC
1:200
anti-SFPQ
Sigma-Aldrich
WH0006421M2
mouse-IgG
IHC
1:300
Supplementary Table 2
Primers used for qPCR.
Gene
Forward
Reverse
Internal probe
human MAPT exon10- (3R-T)
ACCTGAAGAATGTCAAGTC
GATGGATGTTGCCTAATGA
AGACTATTTGCACCTTCCCGCCTC
human MAPT exon10+ (4R-T)
GTGCAGATAATTAATAAGAAGC
GATGGATGTTGCCTAATG
...
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