Cell, Volume 138 Supplemental Data Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling Thorsten Trimbuch, Prateep Beed, Johannes Vogt, Sebastian Schuchmann, Nikolaus Maier, Michael Kintscher, Jörg Breustedt, Markus Schuelke, Nora Streu, Olga Kieselmann, Irene Brunk, Gregor Laube, Ulf Strauss, Arne Battefeld, Hagen Wende, Carmen Birchmeier, Stefan Wiese, Michael Sendtner, Hiroshi Kawabe, Mika Kishimoto-Suga, Nils Brose, Jan Baumgart, Beate Geist, Junken Aoki, Nic E. Savaskan, Anja U. Bräuer, Jerold Chun, Olaf Ninnemann, Dietmar Schmitz, and Robert Nitsch
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Cell, Volume 138
Supplemental Data
Synaptic PRG-1 Modulates
Excitatory Transmission
via Lipid Phosphate-Mediated Signaling Thorsten Trimbuch, Prateep Beed, Johannes Vogt, Sebastian Schuchmann, Nikolaus Maier, Michael Kintscher, Jörg Breustedt, Markus Schuelke, Nora Streu, Olga Kieselmann, Irene Brunk, Gregor Laube, Ulf Strauss, Arne Battefeld, Hagen Wende, Carmen Birchmeier, Stefan Wiese, Michael Sendtner, Hiroshi Kawabe, Mika Kishimoto-Suga, Nils Brose, Jan Baumgart, Beate Geist, Junken Aoki, Nic E. Savaskan, Anja U. Bräuer, Jerold Chun, Olaf Ninnemann, Dietmar Schmitz, and Robert Nitsch
2
A
B-/-+/-+/+ -/-+/-+/+
~4.1 kb
~6 kb~9.8 kb
~6.7 kb
5‘Probe 3‘Probe
-/-+/-+/+
305 bp
706bp
C
Targeting construct
5‘ arm (4.6kb) 3‘ arm (1.3kb)
Wild type locus
1 2 4 5 6 7
3
Not I BamH I BamH I Sal I
Targeted locus
3
1 2 73
Xba I Xba I~9.8kb
Xba I BamH I~6.7kb
5‘ProbeEcoR I EcoR I
EcoR I EcoR I
~6kb
~4.1kb
3‘Probe
7MC1-neoIRES LacZ3xstop MC1-neoIRES LacZ3xstop
MC1-neoIRES LacZ3xstop MC1-neoIRES LacZ3xstop
11 2
33 2
D-/-+/+
175
25
47.5
6283
kDa
Anti-PRG1
Anti-ß-Actin
E-/-+/+
Anti-PRG1
CA1CA1
Figure S1. Generation of constitutive PRG-1 KO mice.
(A) Diagram of the targeting strategy. Numbered exons of the Prg-1 gene are represented as grey
boxes and their relative positions are indicated. The black box represents the first 77 bp of exon 4 that
are present in the targeting vector. Red boxes indicate the relative position of the putative
phosphatase domains on exons 4 to 6. The 5’ (4.6 kb) and 3’ (1.3 kb) homology arms were inserted
via Not I / BamH I and BamH I / Sal I restriction digest from PCR products in the targeting construct.
For homologous recombination, an IRES-LacZ-MC1-neo cassette was inserted, leading to a
translational stop of PRG-1 in exon 4 due to the 3 stop codons in each frame, which preceded the
IRES sequence. Assessment of correct recombination was performed by southern blot analysis. The
corresponding restriction enzymes, fragment lengths and relative position of the external radioactive-
3
labeled probes (hatched boxes) are indicated. Relative position of oligonucleotides for genotyping of
tail tip DNA is shown as black arrows numbered from 1 to 3. 1: 5’-GGG CTG ACC GCT TCC TCG
TGC TTT AC-3’, 2: 5’- CGG GGA TGT GCA CCA ATT GGG AAG AG-3’, 3: 5’-CCG TGA TTG CTT
GTT CCT TCT AGT GTG-3’. (B) Southern Blot analysis from wildtype (WT) (+/+), heterozygous (+/-),
and homozygous mutant (-/-) mouse tail DNA hybridized with the 32P-labeled 5’ and 3’ probes. WT
and mutant-specific bands are indicated. (C) Genotyping analysis of WT, heterozygous and PRG-1
KO mice using primers, shown in (A), results in the amplification of a 706 bp fragment for the WT allele
and 305 bp fragment for the Prg-1 KO allele. (D) Western Blots of brain homogenate of WT and PRG-
1 KO mice using a PRG-1-specific antibody revealed absence of the PRG-1 protein in PRG-1-deficient
animals. ß-Actin antibody serves as loading control. (E) Immunohistochemistry of WT and PRG-1 KO
hippocampal CA1 pyramidal cells revealed absence of PRG-1 signal in PRG-1-deficient animals
(Scale bar 100 μm).
4
0
5
10
15
20
Bod
y w
eigh
t [g]
*** ***
******
***
P5-6
P9-11
P10-
14
P20-22
P30-3
5P4
2-56
P25-
27
A
n = 155, status at the third week of life
Het WT KO dead KO0
10
20
30
40
50
Perc
enta
geof
tota
l num
bero
f mic
eC
WTP40-60
KoP40-60
0
200
400
Bra
inw
eigh
t[m
g]
*B
WT-littersPRG-1-KO
1
34
2
6
78
5
9
Bod
y le
ngth
[cm
]
WT PRG-1-KO
P21
Figure S2. General observations concerning the PRG-1 KO mouse line.
(A) Body weight measurement of the offspring revealed that the PRG-1 KO mice had a significantly
lower body weight up to day P35 (ANOVA with post hoc Bonferroni for selected groups, p < 0.001 (WT
n = 43, KO n = 44)). The image shows a WT and a PRG-1 KO mouse at P21. (B) Brain weight of WT
and PRG-1 KO mice revealed a significantly lower brain weight in the PRG-1 KO mice (unpaired t test,
p < 0.05, WT n = 21, KO n = 22). The image illustrates the morphologic difference between the brains
from a WT and a PRG-1 KO mouse. (C) Mating of PRG-1-heterozygous mice results in genotypes,
which correspond to mendelian expectations. While all genotypes were viable at birth, approximately
50% of the PRG-1 KO mice died around the third week of life. Data are represented as mean +/- SEM.
5
P21 WT P21 PRG-1 -/-
Synaptoporin Synaptoporin
CA3CA3
CA3
CA1
CA3
CA1
VGlut1 punctae/50 µm
WT PRG1-KO0
25
50
75
100
VG
lut1
pun
ctae
/50
µm
150 µm
50 µm
50 µm
A
E
DC
B
F
P21 WT
P21 PRG-1 -/-
VGlut1 5 µm
VGlut1
H
G
I
Figure S3. No changes in synaptic innervation at P21.
Synaptoporin staining revealed similar mossy fiber (MF) innervation pattern of the CA3 pyramids in the
PRG-1 KO mice at P21 (A,B). Higher magnification of the synaptoporin expression in the
infrapyramidal MF (C,D) and in the terminal part of the suprapyramidal MF (E,F) at the CA3/CA1-
border displayed similar innervation pattern. VGlut1 staining reveals the density of the glutamatergic
innervation of the apical CA1 dendrites in the stratum radiatum in the WT and PRG-1 KO animals
(G,H). Note the negative contours of the unstained apical CA1-dendrites. (I) Quantitative analysis of
VGlut1-punctae revealed no significant changes (t-test) in the glutamatergic innervation (n = 5 animals
per group; 4 slices per animal). Data are represented as mean +/- SEM.
6
5‘ probe 3‘ probe 3‘ probe (dNeo)
~18.5kb
~11kb
~18.5kb
~9.4kb
~18.5kb
~7.4kb
+/+ +/+ +/fl +/fl +/fl(dNeo)
~9.4kb
A
B
C
+/fl
1kb
0.8kb
0.6kb
0.4kb
0.2kb
D
175
83
47.5
25
Anti-PRG-1+/+ +/fl fl/fl +/- -/-
Anti-ß-Actin
+/+ +/fl fl/fl +/-
kDa
Targetingconstruct
5‘ arm (10kb) 3‘ arm (3.5kb)
Wild typelocus
1 2 4 5 6
Not I
Targetedlocus
3
Spe I~18.5kb
5‘Probe 3‘Probe
2 4 5 6 73
Neo
Spe I Not I
Spe I
1 2 4 5 63
Spe I Spe ISpe I
Neo
~11kb ~9.4kb (~7.4kb after Neo deletion)
7
7
1 2 43
Figure S4. Generation of conditional PRG-1 KO mice.
(A) Diagram of targeting strategy. Numbered exons of the Prg-1 gene are represented as grey boxes
and their relative positions are indicated. The red boxes indicate the relative position of the putative
phosphatase domains on exons 4 to 6. In the targeting vector two green triangles flanking exons 4 to 6
represent LoxP sites, while the yellow triangles flanking the neomycin selection cassette (Neo)
represent frt sites. Correct homologous recombination and deletion of Neo by FLP recombinase
expression within the ES cells was detected by southern blot analysis utilizing Spe I as restriction
enzyme and two external 32P-labeled DNA probes (hatched boxes). Relative locations of
oligonucleotides for genotyping by PCR analysis are indicated as black arrows numbered 1 to 4. 1: 5’-
of the recombination events are shown. Corresponding fragments sizes of WT allele (+/+, 18.5 kb),
heterozygous floxed allele (+/fl, 11 kb 5’ probe, 9.4 kb 3’ probe) and heterozygous floxed allele after
Neo deletion (+/fl(ΔNeo), 7.4 kb 3’ probe) are indicated. (C) Genotyping by PCR analysis of mouse tail
DNA. By using the primer 1 and 2 (shown in (A)), PCR of WT DNA produced a DNA product of 698 bp
(Lane 2); heterozygous floxed DNA, two products (698 bp and 771 bp (Lane 3)); homozygous floxed
DNA, a PCR product of 771 bp (Lane 4). For functional tests we bred homozygous floxed mice with
Cre-deleter mice, expressing Cre recombinase within the germ line, which resulted in deletion of
exons 4 to 6. Lane 4 shows the PCR product of a heterozygous PRG-1 KO mouse using primers 3
and 4 (180bp). (D) Western blot of WT, heterozygous and homozygous floxed, as well as
heterozygous and homozygous PRG-1-deficient brain homogenates, using PRG-1 antibody and anti-
ß-Actin as loading control are shown.
8
Figure S5. Subcellular fractionation of mouse cerebral cortex from PRG-1 KO and WT litter
mice.
Western blot analysis was performed on subcellular fractionations of the PRG-1 KO and WT mouse
cerebral cortices using anti-PSD-95 and anti-RabGDI antibodies (#1, #2, #5, #6, and #7, Prg-1 KO; #3,
#4, #8, #9, and #10, WT) to control the PSD preparations. Note that PSD-95 is enriched in the PSD
fraction while RabGDI is abundant in the SC+CSV fraction, but not in the PSD fraction.
9
A
B
C
H253KGFP
YZ XY
XZ
XYYZ
XZ
PRG-1GFP
PRG-1 GFP merge
YZ
H253K GFP
YZ
merge
PRG-1H253K/Homer1
PRG-1/Homer1
Figure S6. 3D reconstruction of in utero electroporated Prg-1 constructs.
(A) Prg-1 was electroporated at E14 and analyzed at postnatal day (P) 21. Images of the PRG-1
staining (red) and the co-electroporated GFP signal (green) are shown in the xy-, xz- and yz-planes in
the section view overlay mode. Xz- and yz-planes are located as displayed by the white crosshair.
Note the membrane localization of the reconstituted PRG-1 in the yz-plane (arrows). PRG-1 staining in
the xz-plane at the intersection with the yz-plane, which is located at the dendritic membrane, confirms
membrane localization. Sections were scanned with a Leica confocal laser-scanning microscope
equipped with Argon and HeNe lasers and the excitation lines 488 nm and 543 nm. Imaging of serial
stacks was performed with a 63x oil objective, 4.8x zoom and a z-separation of 0.1 μm. 3D
reconstructions were performed with Volocity software (Improvision, Tubingen, Germany). Further
immune stainings showed colocalization of PRG-1 with the postsynaptic marker Homer1 (indicated by
the purple staining, arrows) confirming localization of the reconstituted PRG-1 at the postsynaptic
membrane. (B) Prg-1-H253K was electroporated and analyzed under the same conditions as
described above. PRG-1 staining revealed similar distribution and membrane localization (arrows) of
the mutated PRG-1 molecule. Immune staining confirmed colocalization of the H253K mutated PRG-1
with Homer1 pointing to localization of the mutated PRG-1 at the postsynaptic membrane (purple
staining, arrowheads). (C) Higher magnification of a cross section of a dendrite in the yz-plane
revealed similar distribution and membrane localization of PRG-1 (left), as well as of the mutated
PRG-1 (H253K) protein (right).
10
PRG-1-KO mEPSCs
50 pA0.5 s
mIPSCsWT
+ LPA
mIP
SC
freq
uenc
yra
tio
0
1
0-5-10 5 10
LPA
LPA
0
1
0-5-10 5 10
mE
PS
Cfr
eque
ncy
ratio
+ LPA
30 pA1 s
0
1
PRG-1-KO
mE
PSC
freq
uenc
yra
tio[L
PA
/ co
ntro
l]
**
0
1
WTmIP
SCfr
eque
ncy
ratio
[LP
A / c
ontr
ol]
A
B
Figure S7. Effect of exogenously applied LPA on mIPSCs in C57/B6 and on mEPSCs in PRG-1 -/- mice. (A) Application of 10 µM LPA did not alter the mIPSCs in C57/B6 (wild-type) hippocampal CA1
neurons. For the analysis the mIPSC frequency of each analyzed neuron was averaged for the last 4
minutes under control conditions and in steady state after LPA application, and these values then
compared. This revealed a mean ratio of 1.02 ± 0.04 (n = 7, p = 0.61, paired t-test). (B) In PRG-1 -/-
mice the mEPSC frequency increased after the application of 10 µM LPA. The analysis was performed
as in (A) and disclosed a mean ratio of 1.34 ± 0.08 (n = 7, p < 0.01). Note that this increase was
smaller compared to wild-type animals (p < 0.001, independent t-test). Data are represented as mean