1
Supp
orting
Information for
Chondroitin 4
-
O
-
sulfation regulates hippocampal perineuronal nets
and social memory
Huiqian Huang,
Améli
e
M. Joffrin, Yuan Zhao, Gregory M. Miller,
Grace
C.
Zhang
,
Yuki Oka,
Linda C. Hsieh
-
Wilson
*
*Correspondence: Linda C. Hsieh
-
Wilson
Email: lhw@caltech.edu
This PDF file
includes:
Figures
S1
to
S
13
Supplementary Materials and Methods
SI
References
2
Figure S1.
Disruption of t
he
4
-
O
-
sulfation pathway in
Chst11cKO mice.
(
A
)
HPLC disaccharide
analysis was used to quantify the a
mounts
of CS
-
C, CS
-
D, unsulfated CS and total CS in the cortex
of Ctrl (blue) and Chst11cKO (red) mice at postnatal day (P) 0, P7, P14, P28 and P60. The amounts
were calculated by div
iding the number of picomoles
(p
mol)
of CS by the total weight
(mg)
of the
dried homogenate. (
B
) Representative images
of brain slices
showing
the
visual cortex (VC)
of
Ctrl
and Chst11cKO mice
immuno
stained with a
n anti
-
CS
-
E monoclonal antibody. Scale
bars, 100
μ
m.
(
C
)
Representative images
of
brain slices
showing
the
hippocamp
us
(Hpc)
of
Ctrl and Chst11cKO
mice
immuno
stained with a CS
-
E monoclonal antibody. Scale bars, 500
μ
m.
3
Figure S2.
Increased PNN densities in the VC of
Chst11cKO mice.
(
A
)
Representative images
showing PNN
-
enwrapped (WFA
+
) and parvalbumin
-
expressing (PV
+
) neurons in the
VC
of Ctrl and
Chst11cKO mice. Brain sections were stained with WFA (green),
an anti
-
parvalbumin
antibody
(red), and DAPI (blue). Scale bar, 100
μ
m.
(
B
)
Quantification of the number of WFA
+
neurons in
the
VC
. **
P
< 0.01 vs. Ctrl, Student’s
t
-
test.
(
C
)
Percentage of WFA
+
PV
+
neurons amongst
the
PV
+
neurons. ***
P
< 0.001 vs. Ctrl, Student’s
t
-
test
.
(
D
)
Quantification of the number of PV
+
neurons in
the
VC
. Not significant.
(
E
)
Percentage of WFA
+
PV
+
neurons amongst
the
WFA
+
neurons. Not
significant.
n
= 14 brain slices each from 4 pairs of Ctrl and
Chst11cKO mice for
B
-
E
. All data are
shown as
the
mean ± SEM.
4
Figure S3.
Comparable aggrecan levels in the CA2 regions of Ctrl and Chst11cKO mice.
(
A
)
Representative images showing PNN and aggrecan staining in the hippocampus of Ctrl and
Chst11cKO mice. Brain sections were stained with WFA (green),
an anti
-
aggrecan
antibody
(red),
and DAPI (blue).
The
CA2 regions
a
re
marked with dotted white squares. Sca
le bar, 500
μ
m.
n
=
3 pairs each of
Ctrl and Chst11cKO mice
.
(
B
)
Magnified images showing the PNN
-
enriched CA2
regions of Ctrl and Chst11cKO mice, stained
for
aggrecan (red) and
PNNs (
WFA
,
green). Scale
bar, 50
μ
m.
5
Figure S4.
Low
,
comparable levels of Otx2 in the
CA2
regions
of Ctrl and Chst11cKO mice.
(
A
)
Representative images showing PNN and Otx2 staining in the hippocampus of Ctrl and
Chst11cKO mice. Brain sections were stained with WFA (green),
an anti
-
parvalbumin
antibody
(red)
, and
an anti
-
Otx2
antibody
(grey
in separate images; blue in merged images
). CA2 regions
a
re
marked with dotted white squares. Scale bar, 500
μ
m.
n
= 2 pairs each of
Ctrl and Chst11cKO
mice
.
(
B
)
Magnified images showing the PNN
-
enriched CA2 regions of Ctrl and Chst11cKO mice,
stained
for
Otx2 (grey scale),
PNNs (
WFA
,
green), and PV (red). Scale bar, 50
μ
m.
6
Figure S5. Dendritic spine morphology of hippocampal neurons
in
Ctrl and
Chst11cKO
mice
.
(
A
)
Representative images showing the hippocampal CA1, CA2, and
CA3
regions of Ctrl mice
injected with GFP
-
expressing lentivirus. Scale bar, 100
μ
m.
(
B
to
D
)
No significant difference was
observed
in the protrusion densities
in the CA1 (
B
), CA2 (
C
), and CA3 (
D
) regions
of
Chst11cKO
mice
compared to Ctrl mice. Not significant;
n
= 23, 22 dendrites in the CA1,
n
= 13, 16 dendrites
in the CA2, and
n
= 11, 15 dendrites in the CA3 regions of Ctrl and Chst11cKO mice, respectively.
Data are shown
as
the
mean ± SEM.
(
E
to
G
)
CA3 neurons
from Chst11cKO mice have larger
dendritic spine head widths
,
but
similar
mature spine density. Representative images
of
the
dendritic spines
from
GFP
-
expressing CA3 neurons
are shown in
E
. Scale bar, 5
μ
m.
Quantification
of the mature spine density
is
shown in
F
. Not significant;
n
= 11 and 15 dendrites from 3 pairs of
Ctrl and Chst11cKO mice, respectively. Data are shown as
the
mean ± SEM.
Cumulative
distribution curve
s
of spine head width
for
Ctrl and Chst11cKO CA3 neurons
are shown in
G
.
****
P
< 0.0001 vs. Ctrl, Kolmogorov
–
Smirnov test.
7
Figure S6.
Altered
PNN densities and synapse sizes in
cultured
hippocampal
Chst11cKO
neurons compared to Ctrl
neurons
.
(
A
) Schematic showing
the collection and culturing of
hippocam
pal neurons
from individual embryo
s
at embryonic
day
15 (E15). T
he t
ail of each embryo
was collected for genotyping.
(
B
)
Representative images showing hippocampal neuron cultures
from
Ctrl and Chst11cKO embryos stained with WFA (green) and
an anti
-
MAP2
antibody
(grey
scale).
The same
PNN
+
neurons were indicated with white or red arrowheads in separate channels
for WFA or MAP2, respectively.
Scale bar, 100 μm.
(
C
)
Quantification of the number of WFA
+
neurons per mm
2
. ****
P
< 0.0001 vs. Ctrl, Student’s
t
-
test;
n
= 22 and 21 regions of cultured neurons
from 3 pairs of Ctrl and Chst11cKO mice, respectively.
(
D
)
Quantification of PSD
-
95 puncta size.
****
P
< 0.0001 vs.
Ctrl, Student’s
t
-
test;
n
= 20 neurons each for Ctrl and Chst11cKO mice.
(
E
)
Quantification of gephyrin puncta size. ***
P
< 0.001 vs. Ctrl, Student’s
t
-
test;
n
= 20 neurons each
for Ctrl and Chst11cKO mice. All data are shown as
the
mean ± SEM.
8
Figure S7
.
ChABC treatment
of cultured
Chst11cKO neurons
remove
s
excess PNNs and
restores synapse sizes
to Ctrl neuron levels
.
(
A
)
Representative images showing wild
-
type
C57
hippocampal neurons treated with PBS (Control)
or
ChABC
for 2, 8, or 24 h, and stained
for PNNs
(
WFA
,
green) and MAP2 (grey
scale
). Scale bar, 100
μ
m.
(
B
)
Representative images of Ctrl,
Chst11cKO, and ChABC
-
treated Chst11cKO cultured hippocampal neurons st
ained
for PNNs
(
WFA
,
green)
and MAP2 (grey scale).
The same PNN
+
neurons were indicated with white or red
arrow heads in separate channels for WFA or MAP2, respectively
. Scale bar, 100
μ
m. (
C
)
Quantification of the number of WFA
+
neurons in each visual field, normalized to Ctrl. *
P
< 0.05,
***
P
< 0.001, ****
P
< 0.0001 vs. Ctrl, one
-
way ANOVA followed by Tukey’s multiple comparisons
test;
n
= 6, 6, and 8 regions of Ctrl, Chst11cKO, and ChABC
-
treated Chst11cKO cultured neurons,
resp
ectively.
(
D
)
ChABC treatment of Chst11cKO neurons
restored the
PSD
-
95 puncta size
to Ctrl
9
neuron levels
. ****
P
< 0.0001 vs. Ctrl, one
-
way ANOVA followed by Tukey's multiple comparisons
test;
n
= 10, 11, and 10 Ctrl, Chst11cKO, and ChABC
-
treated Chst11cKO
cultured neurons,
respectively.
(
E
)
ChABC treatment of Chst11cKO neurons
res
tor
ed
the gephyrin puncta size
to Ctrl
neuron levels
. ****
P
< 0.0001 vs. Ctrl, one
-
way ANOVA followed by Tukey's multiple comparisons
test;
n
= 10 neurons for each condition. All d
ata are shown as
the
mean ± SEM.
10
Figure S
8.
Chemical manipulation of CS 4
-
O
-
sulfation
levels
mod
ulates PNN densit
ies
and
synapses
.
(
A
and
B
)
Treatment of Chst11cKO neurons
(
13
–
15 DIV
)
with
natural
CS
polysaccharides (20
μ
g/ml) enriched in the 4
-
O
-
sulfated CS
-
A or CS
-
E motifs, but not the 6
-
O
-
sulfated CS
-
C motif, restored PNN densities to Ctrl
neuron
levels. Representative images
of
hippocampal neuron
al
cultures for the conditions indicated
are shown in
A
. Scale bar, 100
μ
m.
Quantification of
the
n
umber of WFA
+
neurons per mm
2
is shown in
B
.
**
P
< 0.01, ***
P
< 0.001 vs.
Chst11cKO, one
-
way ANOVA followed by Tukey's multiple comparisons test;
n
= 9, 8, 9, 9, and 9
regions of Ctrl, Chst11cKO, and CS
-
A, CS
-
E or CS
-
C
-
treated Chst11cKO cultured neurons,
11
r
espectively.
(
C
to
E
)
Addition of natural CS polysaccharides to
cultured Chst11cKO hippocampal
neurons
did not rescue the increased density or size of gephyrin puncta
.
Representative images of
gephyrin puncta (red) distributed along the dendrites of neurons
(MAP2, blue)
cultured under the
indicated conditions
are shown in
C
. Scale bar, 10
μ
m.
Quantification of gephyrin puncta number
per 10
μ
m
(
D
)
and size
(
E
).
**
P
< 0.01
, ***
P
< 0.001
vs. Chst11cKO, one
-
way ANOVA followed by
Tukey's multiple comparisons test;
n
= 10, 12, 10, 10, and 10 for Ctrl, Chst11cKO, and CS
-
A, CS
-
E or CS
-
C
-
treated Chst11cKO neurons, respectively.
(
F
)
Addition of natural
CS polysaccharides
enriched
in
the CS
-
A or CS
-
E motifs, but not the CS
-
C motif
, rescued the decreased
size of PSD
-
95 punc
ta in Chst11cKO neurons
. Quantification shows PSD
-
95 puncta size
.
**
P
< 0.01, ***
P
<
0.001 vs. Chst11cKO, one
-
way ANOVA followed by Tukey's multiple comparisons test;
n
= 15, 13,
14, 10, and 12 neurons for Ctrl, Chst11cKO, and Chst11cKO neurons treated wit
h CS
-
A, CS
-
E or
CS
-
C, respectively.
(
G
)
Treatment with a sulfotransferase inhibitor (
Inhib
,
10
μ
M, 24
h) reduced
the
PSD
-
95 puncta size. Quantification of PSD
-
95 puncta size
.
***
P
< 0.001 vs. Con, Student’s
t
-
test;
n
=
10
and
9
neurons treated with
Inhib
in DMSO
or DMSO alone (Con)
, respectively. (
H
)
Inhib
treatment (10
μ
M
, 24
h) did not change
the g
ephyrin
puncta size. Quantification of
g
ephyrin
puncta
size
.
n
=
9
and
8
neurons treated with
Inhib
in DMSO or DMSO alone (
Con
)
, respectively. All data
are shown as
the
mean ± SEM.
12
Figure S9
.
mIPSC and mEPSC recordings of CA2 neurons from Ctrl and Chst11cKO mice.
(
A and
B
)
Raw trace
s
of mIPSC (
A
) and mEPSC (
B
) of CA2 neurons from Ctrl and Chst11cKO
mice.
(
C
and
D
)
Quantification revealed that mIPSC frequency (
C
) for Chst11cKO CA2 neurons
showed an increasing trend compared to Ctrl CA2 neurons.
P
= 0.07,
Wilcoxon rank
-
sum one
-
sided test. The mIPSC amplitude (
D
)
was not significantly different between Ctrl an
d Chst11cKO
neurons;
n
= 14 and 15 neurons from
3
Ctrl and
5
Chst11cKO mice, respectively.
(
E
and
F
)
mEPSC
frequency (
E
) and amplitude (
F
) were comparable for Ctrl and Chst11cKO CA2 neurons. Not
significant;
n
= 14 neurons each from
4
Ctrl and
6
Chst11cKO mice. All data are shown as
the
mean ± SEM.
13
Figure S10. PNN densities modulate
CREB
phosphorylation
levels,
but not total
CREB
levels
,
in the hippocampus.
(
A
)
Representative images showing the CA1, CA2, and CA3 regions
of the hippocampus stained with
a p
-
CREB antibody (red) and WFA (green). Each region was
cropped with a rectangular box, as indicated, for analysis of p
-
CREB levels at the cellular level.
Scale b
ar, 500
μ
m.
(
B
)
Representative images showing the CA2 and CA3 hippocampal regions of
Ctrl and Chst11cKO mice immunostained for CREB (red). Scale bar, 200
μ
m.
(
C
)
Representative
images of cropped CA1, CA2, and CA3 regions of total CREB. Scale bar, 50
μ
m. (
D
and
E
)
Quantification of the average p
-
CREB fluorescence intensity per neuron (normalized to Ctrl Pen)
in the hippocampal CA1
(
D
) and CA3 (
E
) regions of Ctrl and Chst11cKO mice, injected with ChABC
or penicillinase (Pen). *
P
< 0.05, two
-
way ANOVA, F(1,33)
=
0.7723,
P
=
0.3859 for CA3;
n
= 10,
9, 9, and 9 slices from 3 mice each for Ctrl Con, Ctrl ChABC, Chst11cKO Con, and Chst11cKO
ChABC conditions, respectively. All data are shown as
the
mean ± SEM.
14
Figure S11.
Modulation of
PNN density
and
CS 4
-
O sulfation
levels affects
p
-
CREB
levels
in
hippocampal neurons.
(
A
) Representative images of
cultured
hippocampal neurons stained with
WFA (green) and
anti
-
p
-
CREB or
anti
-
CREB antibodies (red) as
indicated. WFA
+
neurons are
marked with
a
yellow arrowhead. Scale bar, 25
μ
m. (
B
) Quantification of p
-
CREB and CREB
intensities, normalized to WFA
−
as a control. *
P
< 0.05 vs. WFA
−
, Student’s
t
-
test;
n
= 11 and 8
individual WFA
−
and WFA
+
neurons stained with p
-
CREB, respectively;
n
= 8 and 5 individual WFA
−
and WFA
+
neurons stained for CREB, respectively.
(
C
)
Quantification of p
-
CREB and CREB
intensities in neurons treated with ChABC for 2, 8 and 24
h. **
P
<
0.01, ****
P
< 0.0001 vs. Con,
one
-
way ANOVA followed by Tukey's multiple comparisons test;
n
= 100
–
150 neurons for each
condition.
(
D
)
Representative images showing Ctrl and Chst11cKO hippocampal neurons treated
15
with ChABC or PBS as a control and stained with a
n
anti
-
p
-
CREB antibody (u
pper panels, grey
scale; lower panels, red) and AF488 phalloidin (green). Scale bar, 50
μ
m.
(
E
)
Quantification of total
CREB levels for Ctrl, Chst11cKO, and ChABC
-
treated Chst11cKO neurons (normalized to Ctrl).
**
P
< 0.01 vs. Chst11cKO, one
-
way ANOVA follo
wed by Tukey's multiple comparisons test;
n
=
100
–
150 neurons for each condition.
(
F
)
Quantification of total CREB levels for Ctrl, Chst11cKO,
and CS
-
A
-
,
CS
-
E
-
and CS
-
C
-
treated (20
μ
g/ml, 24
h) Chst11cKO neurons, respectively. **
P
< 0.01
vs. Chst11cKO, one
-
way ANOVA followed by Tukey's multiple comparisons test;
n
= ~100
–
150
neurons for each condition. All data are shown as
the
mean ± SEM.