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Neuron, Volume 92
Supplemental Information
The Calcium Channel Subunit Alpha2delta2
Suppresses Axon Regeneration in the Adult CNS
Andrea Tedeschi, Sebastian Dupraz, Claudia J. Laskowski, Jia Xue, Thomas Ulas, MarcBeyer, Joachim L. Schultze, and Frank Bradke
DESeq normalization
16075 present genes over 8 groups
Maximum of group mean > 10
1560 variable genes over 8 groups
(FDR p-value < 3E-10)
1. Most variable genes
Selection of one cluster with higher expression
at E17.5 (n=141)
Hierarchical clustering
DE genes that fulfill the criteria (n=15):Embryonic E17.5 - E12.5 increase FC>2.5, FDR p<0.05Adult 24 hr - 6 hr decrease FC<-1.5, FDR p< 0.05Adult 36 hr - 24 hr no change 1.5< FC>1.5, FDR p>0.95Adult PNL - Sham decrease FC<-1.4, p<0.05
2. ANOVA model
Approaches1. and 2.
intersection
5 candidate genes
3 Paradigms, 8 groups, 24 RNA-seq
E12.5 lumbar DRGs E17.5 lumbar DRGs 6 hr cultured adult lumbar DRG 12 hr cultured adult lumbar DRG 24 hr cultured adult lumbar DRG 36 hr cultured adult lumbar DRG Sham L4,5 adult DRGs PNL L4,5 adult DRGs
Figure S1 A
Nor
mal
ized
exp
ress
ion
Dok4
E12.
5
E17.
5
6 hr
12 h
r
24 h
r
36 h
r
Sham
PNL0
50
100
150500
10001500200025003000 ***
****** ***
***n.s.
Magee2
E12.
5
E17.
5
6 hr
12 h
r
24 h
r
36 h
r
Sham
PNL0
100
200
300 ***
* **
n.s.n.s.n.s.
Tenm1
E12.
5
E17.
5
6 hr
12 h
r
24 h
r
36 h
r
Sham
PNL0
50
100
150
500
1000
1500***
*** ***
**n.s.
Cacna2d2
E12.
5
E17.
5
6 hr
12 h
r
24 h
r
36 h
r
Sham
PNL0
50
100
150500600700800900 ***
***** ***
***n.s.
Nor
mal
ized
exp
ress
ion
Scml4
E12.
5
E17.
5
6 hr
12 h
r
24 h
r
36 h
r
Sham
PNL0
50
100
150
400800
12001600 ***
*** *** *****
n.s.
B
D
6 hr 12 hr 24 hr 36 hrE12.5 E17.5 PNLShamA630075F10RikOTTMUSG00000016609Fam13aSgczNqo2Eepd12900041M22RikDok4Zdhhc22Camk1gCsmd3Magee2Cacna2d2Tenm1Scml4
0.00 2.92-2.92z-score
Paradigm 1 Paradigm 2 Paradigm 3
E
Cacna1fCacna1hCacna1eCacna1gCacna1dCacna2d2Cacna2d1Cacna2d3Cacna1iCacna1bCacna1aCacna1c
-3.60 3.600.00z-score
E12.5 E17.5 6hr 12hr 24hr 36hr Sham PNL
Paradigm 1 Paradigm 2 Paradigm 3
Nor
mal
ized
exp
ress
ion
Gene name Gene product Type
Cacna1c Cav1.2 L-type VGCCCacna1d Cav1.3 L-type VGCCCacna1f Cav1.4 L-type VGCC
Cacna1a Cav2.1 P/Q-type VGCCCacna1b Cav2.2 N-type VGCCCacna1e Cav2.3 R-type VGCC
Cacna1g Cav3.1 T-type VGCCCacna1h Cav3.2 T-type VGCCCacna1i Cav3.3 T-type VGCC
Cacna2d1 Alpha2delta1 accessory subunitCacna2d2 Alpha2delta2 accessory subunitCacna2d3 Alpha2delta3 accessory subunit
0123456789
10
E17.5 vs E12.5
6 hr vs 24 hr
Sham vs PNL
Fold
cha
nge
Effect size mean 51 47 43
111,777 99,010 52,115Effect size sum
C
Figure S2
A
B
2 2 Tuj1 merge
+ bl
ocki
ng p
eptid
e
C
GAPDH 35
15 30 15 30 µg
REIYKDNRNLFEVQENEP
S-S
VWA
2 (98-115)
E
2 2 Tuj1 merge
Wild
-type
C
acna
2d2
KO
Peptide sequence: Alpha2delta2
2 2
250
+ blocking peptide
2
Wild-type
Cacna2d2 KO
D
Tuj1
2 2
20 20 µg
F
G
Phalloidin
Tuj1
merge
12 hr
2 2
T12 GFP-M line
Adult spinal cord
GFP
merge
2 2
*
*
*
**
Eth
D-1
inte
nsity
(a
rbitr
ary
units
)
Figure S3
2 2
Tuj1
2 2
0
10
20
30
40
50
60
70
CTR Cacna2d2 CTR/Saponin
GFP
Tuj1
EthD-1
24 hr
DRG
vertebra
L6
Tweezer-like electrode
L4
L5
Cacna2d2 CTR
GFP 2 2
#1
#2
#3
#1
#2
#3
L5 D
RG
(ele
ctro
pora
ted
neur
ons)
A B C
D F G
H I J Day 1 In vivo L5 DRG electroporation Day 6 Sciatic nerve crush Day 10 Perfusion
Spinal cord
DRG DCL
PNL
E
L4, 5 DRG
2 2 NF-160 merge
Sham
PNL ***
Central Peripheral
Cac
na2d
2 no
rmal
ized
exp
ress
ion
0
20
40
60
80
100
120
Sham
DCL
Sham
PNL
0
100
200
300
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500 A
lpha
2del
ta2
inte
nsity
(n
orm
aliz
ed e
xpre
ssio
n) *
CTR
Cacna
2d2
CTR
Cacna
2d2
CTR/Sap
onin
PNL Sha
m
Cacna
2d2
CTR
GAPDH
GFP RFP merge
L5 D
RG
K L M
GFP
/RFP
col
ocal
izat
ion
(%)
0
20
40
60
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100
CTR
Cacna
2d2
0
5
10
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25
30
35
ns
No.
of e
lect
ropo
rate
d ne
uron
s pe
r gan
glio
n
N Sciatic nerve crush-site O DAPI
*** ***
A N
orm
aliz
ed e
xpre
ssio
n
C ns ns
ns ns
Cacna2d2
0
20
40
60
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20
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0
20
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60
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Nor
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ized
exp
ress
ion
Cacna2d1 Cacna2d3
siRNA CTR
siRNA #1
siRNA #2
GAPDH
Cacna2d1
Cacna2d2
Cacna2d3
B
H2O
Figure S4
2 1
Tuj1
CTRCacna2d1
2 3
Tuj1
CTRCacna2d3D E siR
NA CTR
siRNA #1
siRNA #2
siRNA CTR
siRNA #1
siRNA #2
Agarose gel
F
No.
of b
ranc
hes
/ 100
µm
Leng
th o
f the
long
est a
xon
(µm
)
** **
*** ***
0
100
200
300
400
500
600
ns
ns
0
0.3
0.6
0.9
1.2
1.5
1.8 *** **
siRNA CTR
siRNA #1
siRNA #2
siRNA #1 + Rescu
e
siRNA CTR
siRNA #1
siRNA #2
siRNA #1 + Rescu
e
G
2 2
Tuj1
siRNA CTR
siRNA #1
siRNA #2
siRNA #1 + Rescu
e
H
Tuj1
12 hr Vehicle 50 μM PGB 250 μM PGB
Lam
inin
I
0
50
100
150
200
250
300
350
0 0.2 0.4 0.6 0.8
1 1.2 1.4 1.6
******
******
Vehicle
50 μM PGB
250 μM PGB
No.
of b
ranc
hes
/ 100
µm
Leng
th o
f the
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est a
xon
(µm
)
Vehicle
50 μM PGB
250 μM PGB
J
0
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No.
of b
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/ 100
µm
Leng
th o
f the
long
est a
xon
(µm
)
LamininCSPGs
CSPGs / PGB
LamininCSPGs
CSPGs / PGB
******
******
***
ns
0
100
200
300
400
500
600
0
0.2
0.4
0.6
0.8
1
1.2
CTR
Cacna2d1
Cacna2d3 CTR
Cacna2d1
Cacna2d3
No.
of b
ranc
hes
/ 100
µm
Leng
th o
f the
long
est a
xon
(µm
)
ns ns
**
Figure S5
Pv
PGB Vehicle
A
E17
.5 s
pina
l cor
d
E17
.5 D
RG
2 2 Pv DAPI merge + blocking peptide
D
Neu
N+
neur
ons
/ sec
tion
% P
v+ n
euro
ns
0
50
100
150
200
250
Vehicle PGB 0
2
4
6
8
10
Vehicle PGB
ns ns
Bax
Bcl-xL
Tuj1
Vehicl
e
PGB B C
F
G
siRNA CTR 16 hr
siRNA #1
E17
.5 D
RG
Tuj1 GFP
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Leng
th o
f the
long
est a
xon
(µm
)
***
No.
of b
ranc
hes
/ 100
µm
siRNA CTR
siRNA #1
*
E H
0
20
40
60
80
100
120
siRNA CTR
siRNA #1
***
Nor
mal
ized
exp
ress
ion
Cacna2d2
0
100
200
300
400
500
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Wild-type
Cacna2d2 KO
Wild-type
Cacna2d2 KO
Leng
th o
f the
long
est
axon
(µm
)
No.
of b
ranc
hes
/ 100
µm
***
***
0
100
200
300
400
500
siRNA CTR
siRNA #1
Vehicle PGB0
2
4
6
8
10
ns
A
von Frey filament
L1
Sciatic nerve
Left (stimulated) Right
Figure S6
pERK
AAV-eGFP
D
V
B
E
Vehicle PGB0
1
2
3
4
5
Vehicle PGB0
10
20
30
40
50
60*** *** G F
1 2 3
4 5 6
I
H
2-Photon microscope
J R
C
GFP
C
***
AAV-eGFP
pERK
Vehicle PGB
Vehicle PGB0.0
0.5
1.0
1.5
D
Targ
et fo
rce
(gra
ms,
+/-
5%)
% S
upra
spin
al s
ensi
tizat
ion
Nor
mal
ized
pE
RK
exp
ress
ion
Syn
apto
phys
in 1
a.u
.(dor
sal h
orn)
Dorsal column axons
Spinal cord injury
WM GM
D
V
B Spinal cord injury
AAV-eGFP injection
Perfusion and imaging
Day 3 Day 14 Day 1
LS -200 -400 µm +200 +400
Rostral Caudal R C
AAV-eGFP
Horizontal scan of the injured spinal cord (M.I.P.) C
2-P
hoto
n m
icro
scop
e
A
Cau
dal
Ros
tral
#1 #2 #3 #4 #5
AAV-eGFP
Sagittal section of the injured spinal cord (M.I.P.)
AAV-eGFP GFAP DAPI
Coronal section of the injured spinal cord (M.I.P.) E D
HG
R C
Dorsal hemisection
-200 µm - 0 0
0 - 200 µm
200 - 400 µm
400-600 µm
600 - 800 µm
>800 µm0
20
40
60
80
100
Perc
ent o
f axo
ns Vehicle
Caudal LS Rostral
PGB***
***
*****
** *
AAV-eGFP
R CVehicle
PGB
Horizontal scan of the injured spinal cord (M.I.P.)
Vehicle or PGB administration (starting 1 hr after injury)
AAV-eGFP injection
SCI Perfusion Day 1 Day 28 Day 16
*
*
*
F
Hor
izon
tal s
can
of th
e in
jure
d sp
inal
cor
d (M
.I.P.
)
AAV-eGFP
Vehicle
PGB
*
*
R C
Figure S7
Supplemental Figure Legends
Figure S1. Narrowing Down Critical Regulators of Axon Growth and Regeneration, Related to Figure 2 (A) Schematic describing the workflow used to determine significantly differentially expressed genes in the RNA-Seq data set. (B) Mean fold change rate, mean effect size and sum effect size were calculated across the three experimental paradigms to determine the cut-off values for calling differentially expressed genes. (C) Heatmap of hierarchical clustering (z-transformed, blue to red) to visualize expression changes of the 15 genes resulting from the filtering strategy in (A). Each box represents data from a single array, each column a single sample, each row a single gene (n=3 independent biological replicates per condition). (D) For each differentially expressed candidate gene, expression values were validated by qRT-PCR. Mean and SEM (*p<0.05, **p<0.01, ***p<0.001; ns: not significant; triplicate experiments). (E) Heatmap of hierarchical clustering (z-transformed, blue to red) to visualize expression changes of the VGCCs subunits across the 3 paradigms. Each box represents data from a single array, each column a single sample, each row a single gene (n=3 independent biological replicates per condition). Figure S2. Validation of a Rabbit Polyclonal Antibody Directed Against the N-terminal Sequence of Murine Alpha2delta2, Related to Figure 2 (A) Sequence of the synthetic peptide used for immunization. Highlighted in different colors are specific protein domains of Alpha2delta2, including the VWA domain (yellow). Red dots indicate potential glycosylation sites. (B) Adult DRG neurons were cultured for 12 hr and stained with Alpha2delta2 and Tuj1 antibodies (upper panels). To determine its specificity, Alpha2delta2 antibody was also pre-incubated with its immunizing peptide (lower panels). Scale bar, 50 µm. The antibody specificity was also validated by immunoblot using DRG extracts from (C) adult mice and (D) 3 weeks old Cacna2d2 KO and control littermates. The upper band could represent the glycosylated form of the protein. GAPDH and Tuj1 are shown as a loading control (n=3 independent replicates per condition). Quantity of loaded protein extracted is indicated below the blot. (E) DRG neurons from Cacna2d2 KO and control littermates were cultured for 12 hr, and stained with Alpha2delta2 and Tuj1 antibodies. Scale bar, 50 µm. (F) Cultured adult DRG neurons stained with Alpha2delta2, Phalloidin and Tuj1 antibodies. Alpha2delta2 is expressed along the neurites and at the growth cone. Scale bar, 10 µm. Inset, higher magnification view of a region in the main panel. The arrows indicate Alpha2delta2 punctate staining at the growth cone. Images were processed with Huygens deconvolution software. Scale bar, 4 µm. (G) Horizontal sections of the spinal cord with GFP-positive DC axons were stained with Alpha2delta2 antibody. Inset, higher magnification view of the region indicated by the asterisk. The arrows indicate Alpha2delta2 clusters along DC axons. Scale bar, 50 µm.
Figure S3. Injury- and Electroporation-mediated Changes in Cacna2d2 Expression, Related to Figure 3 (A) Schematic representation of a central or peripheral branch axotomy (DCL or PNL respectively). (B) Cacna2d2 expression was measured by qRT-PCR in L4-5 DRGs 7 days after injury. Mean and SEM (***p<0.001; triplicate experiments). (C) Cryosections of L4-5 DRGs immunostained with Alpha2delta2 and NF-160 antibodies (Sham n=3 and PNL n=3 animals; 4 DRGs/animal). Inset, higher magnification view of a region in the main panel. DAPI stained nuclei. Scale bar, 50 µm. Immunoblots show Alpha2delta2 expression in (D) L4-6 DRGs after PNL or in (E) adult DRG neurons cultured for 24 hr after electroporation with CTR or Cacna2d2-expressing plasmid DNA. GAPDH or Tuj1 are shown as loading controls (n=3 independent replicates per condition). (F) Representative fluorescence images of adult dissociated DRG neurons electroporated as in (E). Before fixing and staining for Tuj1, DRG neurons were stained with the viability indicator ethidium homodimer-1 (EthD-1) 24 hr after plating. The white dashed line indicates the border of the nucleus. Scale bar, 20 µm. (G) Quantification of (F). Mean and SEM (**p<0.01; triplicate experiments; 50-60 nuclei per condition). (H) Schematic of L5 DRG electroporation in vivo.
(I) Representative fluorescence images of L5 electroporated neurons. Cryosections were stained using the generated Alpha2delta2 antibody (GFP n=3 and Cacna2d2 n=3 animals). Scale bar, 20 µm. (J) Quantification of (I). Mean and SEM (*p<0.05; 25-30 neurons per condition). (K) Representative fluorescence images of L5 DRG electroporated in vivo with a mixture of GFP and RFP-expressing plasmid DNA. Scale bar, 100 µm. (L) Quantification of (K). Mean and SEM (274 electroporated neurons analyzed in n=8 animals). (M) Electroporation efficiency (ns: not significant; CTR n=8 and Cacna2d2 n=7 animals). (N) Photomicrograph of the sciatic nerve 4 days after crush injury. The arrow indicates the lesion site. Scale bar, 500 µm. (O) Longitudinal section of the sciatic nerve was counterstained with DAPI to visualize accumulation of inflammatory cells within the site of injury. The asterisk indicates the crush-site. Scale bar, 500 µm.
Figure S4. Cacna2d2 Gene Knockdown Promotes Axon Growth in vitro, Related to Figure 3 (A) Real-time qPCR shows Cacna2d2 expression in adult DRG neurons after electroporation with CTR or Cacna2d2 siRNA (***p<0.001; triplicate experiments). (B) Agarose gel electrophoresis of RT-PCR products shows Cacna2d1, Cacna2d2 and Cacna2d3 are expressed in adult DRG neurons. (C) Real-time qPCR shows Cacna2d1 and Cacna2d3 expression following knockdown of Cacna2d2 (ns: not significant; triplicate experiments). (D) Immunoblots show Alpha2delta1 and Alpha2delta3 expression in adult DRG neurons cultured for 24 hr after electroporation with CTR or Cacna2d1 or Cacna2d3-expressing plasmid DNA. Tuj1 is shown as loading control (n=3 independent replicates per condition). (E) Quantification of maximal axon length and branching of adult DRG neurons cultured for 24 hr after electroporation as in (D) (*p<0.05; ns: not significant; triplicate experiments; 97-124 neurons per condition). (F) Quantification of maximal axon length and branching of adult DRG neurons cultured for 16 hr after electroporation with CTR or Cacna2d2 siRNAs with a GFP-expressing plasmid DNA. Cacna2d2 expression was rescued using a siRNA-resistant plasmid DNA expressing a mutant Alpha2delta2. Mean and SEM (**p<0.01 and ***p<0.001; ns: not significant; triplicate experiments; 120-140 neurons per condition). (G) Immunoblot for Alpha2delta2 shows knockdown and rescue of Alpha2delta2 protein expression. Tuj1 is shown as a loading control (n=3 independent replicates per condition). (H) Representative fluorescence images of adult DRG neurons cultured for 12 hr in the presence of vehicle or PGB (50 or 250 µM). Scale bar, 200 µm. (I) Quantification of (H). Mean and SEM (***p<0.001; triplicate experiments; 107-178 neurons per condition). (J) Quantification of adult DRG neurons cultured for 24 hr on laminin or CSPGs in the presence of vehicle or PGB (250 µM). Mean and SEM (***p<0.001; triplicate experiments; 122-212 neurons per condition). Figure S5. Parvalbumin Positive E17.5 DRG Neurons Express Alpha2delta2, Related to Figure 5 (A) Cryosections of E17.5 DRGs immunostained with Alpha2delta2 and Parvalbumin antibodies. Scale bar, 20 µm. To determine specificity of Alpha2delta2 staining, the antibody was pre-incubated with a blocking peptide. (B) Quantification of E17.5 DRG neuronal survival and differentiation. Mean and SEM (ns: not significant; vehicle n=6 and PGB n=7 embryos; 3-4 DRGs/embryo). (C) Immunoblots show Bax and Bcl-xL expression in E17.5 DRG neurons after vehicle or PGB chronic administration. Tuj1 is shown as loading control (n=3 independent replicates per condition). (D) AMIRA reconstruction of Parvalbumin-positive primary afferents in E17.5 spinal cords after vehicle or PGB chronic administration. Scale bar, 100 µm. (E) Quantification of axon length and branching in dissociated E17.5 DRG neurons from Cacna2d2 KO and control littermates cultured for 16 hr (***p<0.001; CTR n=4 and Cacna2d2 KO n=5; 175-176 neurons per condition). (F) Representative fluorescence images of dissociated E17.5 DRG electroporated with CTR or Cacna2d2 siRNA and cultured for 16 hr. Neurons were stained with a Tuj1 antibody. Scale bar, 100 µm. (G) Quantification of (F). Mean and SEM (*p<0.05 and ***p<0.001; triplicate experiments; 136-176 neurons per condition).
(H) Real-time qPCR shows Cacna2d2 expression in dissociated E17.5 DRG neurons after electroporation with CTR or Cacna2d2 siRNA as in (F) (***p<0.001; triplicate experiments). Figure S6. Chronic Pregabalin Weakens Synaptic Transmission in vivo, Related to Figure 6 (A) Schematic of the stimulation paradigm. (B) Primary sensory afferents were labeled by injecting AAV-eGFP into the left sciatic nerve 10 days prior to stimulation. After electrical stimulation, coronal sections of the adult spinal cord were stained using a pERK antibody. White arrows indicate stimulation-induced pERK activation in the ipsilateral spinal dorsal horn. Scale bar, 200 µm. (C) Coronal sections of adult spinal cords immunostained with a Phospho-ERK (pERK) antibody. Sensory primary afferents were labeled by injecting AAV-eGFP into the left sciatic nerve. Scale bar, 200 µm. (D) pERK expression in (C), dashed box, was quantified. Scatter plot with mean (***p<0.001; vehicle n=5 and PGB n=5 animals; 3-4 regularly spaced lumbar sections/animal). (E) Synaptophysin 1 expression in the dorsal horn was quantified. Scatter plot with mean (ns: not significant; vehicle n=5 and PGB n=5 animals; 3 regularly spaced lumbar sections/animal). (F) Schematic of the experimental procedure. (G) Von Frey hair threshold of the plantar surface of the mouse hindpaw was examined 7 days after vehicle or PGB chronic administration (***p<0.001; Vehicle n=14 and PGB n=14 animals). (H) Top and side view photograph of the spinal window. Scale bar, 10 mm. (I) Photographs showing the series of steps during window implantation (described in Extended Experimental Procedures). (J) Photograph showing a multiphoton excitation fluorescence imaging session after window implantation on a GFP-M mouse. Z-stack projection of DC axons expressing GFP. Scale bar, 200 µm (R: rostral, C: caudal). Figure S7. A Spinal Cord Injury Model that Completely Transects Ascending DC Axons, Related to Figure 7 (A) Schematic drawing of longitudinal ascending DC axons and the spinal cord injury model we used to completely transect them (D: dorsal, V: ventral, WM: white matter, GM: grey matter). (B) Experimental procedure and schematic representation of multiphoton imaging data acquisition (LS: lesion site). (C) Multiphoton scan of the unsectioned spinal cord two weeks after injury. Asterisk indicates the lesion epicenter (M.I.P.: max intensity projection). (D) Coronal sections of the spinal cord rostral to the lesion confirmed completeness of lesions (n=5 animals). (E) Sagittal sections of the injured spinal cord stained using a GFAP antibody. DAPI stained nuclei. The white dashed line indicates the border of the lesion. (F) Wider view of the same multiphoton scans shown in Figure 7B was generated by automated tile scanning. Asterisk indicates the lesion epicenter (R: rostral, C: caudal). (G) Multiphoton scan of the unsectioned adult spinal cord 4 weeks after dorsal hemisection was generated by automated tile scanning. DC axons were labeled by injecting AAV-eGFP into the left sciatic nerve. Asterisk indicates the lesion epicenter (R: rostral, C: caudal). (H) Quantification of regenerating axons (*p<0.05, **p<0.01, ***p<0.001; LS: lesion site; vehicle n=9, PGB n=8). Scale bars, 200 µm.
Table S2 Gene Associated GO term
Cacna2d2 BIOLOGICAL REGULATION Cacna2d2 REGULATION OF SYNAPTIC TRANSMISSION Cacna2d2 REGULATION OF CELLULAR PROCESS Cacna2d2 CELLULAR PROCESS Cacna2d2 REGULATION OF TRANSMISSION OF NERVE IMPULSE Cacna2d2 REGULATION OF NEUROLOGICAL SYSTEM PROCESS Cacna2d2 REGULATION OF BIOLOGICAL PROCESS Cacna2d2 REGULATION OF SYSTEM PROCESS Cacna2d2 REGULATION OF MULTICELLULAR ORGANISMAL PROCESS Cacna2d2 REGULATION OF LOCALIZATION Cacna2d2 REGULATION OF CELL COMMUNICATION Cacna2d2 REGULATION OF ION TRANSPORT Cacna2d2 CATION TRANSPORT Cacna2d2 MULTICELLULAR ORGANISMAL DEVELOPMENT Cacna2d2 ANATOMICAL STRUCTURE DEVELOPMENT Cacna2d2 REGULATION OF ION TRANSMEMBRANE TRANSPORT Cacna2d2 REGULATION OF TRANSMEMBRANE TRANSPORT Cacna2d2 SYSTEM DEVELOPMENT Cacna2d2 DEVELOPMENTAL PROCESS Cacna2d2 POSITIVE REGULATION OF BIOLOGICAL PROCESS Cacna2d2 ION TRANSPORT Cacna2d2 REGULATION OF TRANSPORT Cacna2d2 METAL ION TRANSPORT Cacna2d2 CELL DEVELOPMENT Cacna2d2 TRANSMEMBRANE TRANSPORT Cacna2d2 MULTICELLULAR ORGANISMAL PROCESS Cacna2d2 LOCALIZATION Cacna2d2 TRANSPORT Cacna2d2 ESTABLISHMENT OF LOCALIZATION Cacna2d2 CELL DIFFERENTIATION Cacna2d2 ION TRANSMEMBRANE TRANSPORT
Dok4 BIOLOGICAL REGULATION Dok4 REGULATION OF CELLULAR PROCESS Dok4 REGULATION OF BIOLOGICAL PROCESS Dok4 NERVOUS SYSTEM DEVELOPMENT Dok4 SIGNALING Dok4 SIGNALING PROCESS Dok4 SIGNAL TRANSMISSION
Dok4 MULTICELLULAR ORGANISMAL DEVELOPMENT Dok4 ANATOMICAL STRUCTURE DEVELOPMENT Dok4 SYSTEM DEVELOPMENT Dok4 DEVELOPMENTAL PROCESS Dok4 MULTICELLULAR ORGANISMAL PROCESS Dok4 SIGNAL TRANSDUCTION Dok4 SIGNALING PATHWAY
Scml4 BIOLOGICAL REGULATION Scml4 REGULATION OF CELLULAR PROCESS Scml4 CELLULAR PROCESS Scml4 REGULATION OF BIOLOGICAL PROCESS Scml4 REGULATION OF CELLULAR METABOLIC PROCESS Scml4 REGULATION OF NITROGEN COMPOUND METABOLIC PROCESS Scml4 REGULATION OF CELLULAR BIOSYNTHETIC PROCESS Scml4 REGULATION OF METABOLIC PROCESS Scml4 REGULATION OF BIOSYNTHETIC PROCESS Scml4 REGULATION OF PRIMARY METABOLIC PROCESS
Tenm1 BIOLOGICAL REGULATION
Supplemental Table Legends Table S1. GO enrichment analysis of gene cluster 1, Related to Figure 2. Table S2. GO enrichment analysis for the four differentially expressed candidate genes, Related to Figure 2.
Supplemental Video Legend Video S1, Related to Figure 7 Multidimensional rotation of the injured spinal cords shown in Figure 7B. Asterisk indicates the lesion epicenter (R: rostral, C: caudal).
Supplemental Experimental Procedures Morphometric Analysis DRG neurons were fixed with 4% paraformaldehyde and 4% sucrose, and stained for Tuj1 (Covance). Images were randomly taken with an Axiovert microscope (Zeiss) and analyzed using Image J software (NIH). This was carried out for three independent experiments. The number of neurons quantified for each condition is indicated in the corresponding figure legend. Peripheral Nerve Lesion (PNL) Mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). The left sciatic nerve was exposed at the mid-thigh level, ligated and cut distally to the ligation site. In one set of experiments, the left sciatic nerve was exposed at the mid-thigh level and crushed for 20 seconds using #2 forceps (FST) allowing to accurately and persistently marking the crush-site. Mice were allowed to recover for seven days before isolating L4-5 DRGs. For the PNS regeneration study, mice were perfused 4 days after sciatic nerve crush. Regeneration of labeled DRG axons was assessed in longitudinal sections of the left sciatic nerve using standard fluorescence microscopy. Sections were counterstained with DAPI to visualize accumulation of inflammatory cells within the site of injury and confirm the exact location of the crush-site before further analysis. RNA-Seq Data Analysis After mapping of the reads to the genome using TopHat2, we imported the data into Partek Genomics Suite V6.6 (PGS) to calculate the number of reads mapped to each transcript against the RefSeq mm10 annotation download on January 2014. These raw read counts were used as input to DESeq2 for calculation of the normalized signal for each transcript using the default parameters. After DESeq2 normalization, the normalized read counts were imported back into PGS and floored by setting all read counts to at least a read count of 1. Subsequent to flooring, all transcripts having a maximum over all group means lower than 10 were removed. After dismissing the low expressed transcripts, the data comprised of 16.075 transcripts to be expressed in at least one sample. To visualize the structure within the data we performed hierarchical clustering (HC) on genes with the highest variance (the most variable genes) defined by a an FDR-corrected p-value<3E-10 (n=1,560) according to the expression values of the samples across all conditions (dismissing class labels) using default settings in PGS. To study the biological functions of cluster 1 (Figure 2A, n=141 genes) identified by hierarchical clustering, we applied GO enrichment analysis on respective gene sets using the Cytoscape (http://www.cytoscape.org/) plug-in BiNGO (v2.44) (Maere et al., 2005). To include only significant results, the FDR threshold was set to 0.05. The Cytoscape plugins Enrichment Map (v1.1) (Merico et al., 2010) and Word Cloud (Oesper et al., 2011) were used to visualize the GO networks. The cutoff for the Jaccard coefficient was set to 0.25 and the FDR q-value to 0.1. Enriched GO-terms were visualized by red nodes, where color and size represent the corresponding FDR-adjusted enrichment p-value (q-value). To reduce the number of possible candidate genes for further analysis from the cluster identified by HC, we used an ANOVA model followed by a defined filtering strategy. For the three experimental paradigms we determined the overall mean fold change rate, the mean effect size and the sum effect size as parameters to adapt the cut-off values for calling differentially expressed genes. All three parameters were highest in Paradigm 1, followed by Paradigm 2 and 3. Accordingly, the highest cutoff values were chosen for Paradigm 1. Next, we searched for genes that would fulfill the following filtering criteria: being 1) at least 2.5-fold upregulated in E17.5 compared to E12.5 DRGs, 2) at least 1.5-fold downregulated in cultured adult neurons 24 hr after plating, 3) not changed at later time points (36 hr vs 24 hr), and 4) 1.4-fold downregulated in PNL compared to Sham. To narrow down the list of candidate genes for further analysis, the 141 genes derived from the analysis of the most variable genes were intersected with the 15 genes derived from the ANOVA model. Hierarchical clustering, 1-way ANOVA models, determination of variance were all performed using PGS. Quantitative Real-time PCR Total RNA was extracted from mouse DRG neurons using RNeasy kit (Qiagen) and cDNA was synthesized from 1 µg of RNA using random hexamers from the SuperScript® VILO™ cDNA synthesis kit (Applied Biosystem). The subsequent cDNA (1:10 dilution) was used in a real time PCR (StepOne Plus, Applied Biosystem) using Fast SYBR® Green Master Mix (Applied Biosystem). Melting curve reactions
were run with each primer set. The GAPDH gene was used for normalization. The sequences of the primers used were: Dok4_s 5’-CGGCTGGAGAAGTATCCTGA-3’ Dok4_as 5’-TGATGTCATTGAGCCGTGAT-3’ Magee2_s 5’-CGACAGTCAGAAATGCTGGA-3’ Magee2_as 5’-AGGAGACCCGCTTTTGGTAT-3’ Tenm1_s 5’-TCACAGTGACTTTGGCCTTG-3’ Tenm1_as 5’-TGTCAACTTCCCCAGTGTCA-3’ Cacna2d1_s 5’- GACCTTGTCACACTGGCAAA-3’ Cacna2d1_as 5’- GGCTGCTTGGACTTTCTCTG-3’ Cacna2d2_s 5’- ACAAGGACAATCGGAACCTG-3’ Cacna2d2_as 5’- TGGGCTTTCTGGAAATTCTCT-3’ Cacna2d3_s 5’- TGGTGGGGAGATAAAATCCA-3’ Cacna2d3_as 5’- CCTCGGACTTCTTGTGGAAC-3’ Scml4_s 5’-CCGTTTCCTGACCAAACTGT-3’ Scml4_as 5’-TGAGGAAGAGTGGGTTACGG-3’ GAPDH_s 5’-GGTGCTGAGTATGTCGTGGA-3’ GAPDH_as 5’-CCTTCCACAATGCCAAAGTT-3’. Normalized expression was calculated as dCt (gene norm) = Ct (gene of interest) – Ct GAPDH
Normalized expression = 2^-dCt (gene norm)
Generation of a Rabbit Polyclonal Antibody Directed Against Murine Alpha2delta2 A custom made polyclonal antibody recognizing murine Alpha2delta2 was generated using the Antibody Service at Charles River Germany (BMS-PAK-0003). The sequence of the synthetic peptide used for immunization was: REIYKDNRNLFEVQENEP. The antibody was purified using an immunoaffinity purification column. Immunohistochemistry Neuronal tissues were fixed at 4°C in 4% paraformaldehyde overnight, and subsequently transferred to 30% sucrose. Tissues were embedded in optimum cutting temperature (OCT) compound (Tissue-Tek), frozen and sectioned (CM3050 S, Leica). Cryosections were warmed at 37°C for 30 min and OCT was washed away with PBS. They were then blocked at room temperature (RT) with 10% normal goat serum (NGS), 0.2% triton X100 in PBS (or 2.5% bovine serum albumin in PBS with 0.1% triton X100 for Parvalbumin staining) for 1 hr and then incubated at 4°C with the primary antibody overnight. After washing three times with PBS, cryosections were incubated with Alexa Fluor conjugated secondary antibodies (1:500, Invitrogen). When necessary, sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (1:10000, Molecular Probes). Images were taken using a LSM700 confocal microscope (Zeiss). Linear fluorescence intensity was calculated using Photoshop (version 12.0.1, Adobe). Colocalization was measured using Image J software. The imaging software Amira (Version 5.4.5, FEI Visualization Sciences Group) was used for three-dimensional (3D) rendering. An isosurface was applied to the Parvalbumin signal (Figure S5D). E17.5 DRG neuronal survival and differentiation was measured by counting the number of NeuN positive neurons and calculating the percentage of Parvalbumin-positive neurons in 3-4 DRGs per embryo. At least three independent replicates for each condition were analyzed. Immunocytochemistry DRG neurons were grown on laminin-coated coverslips and fixed with 4% paraformaldehyde in 4% sucrose. Coverslips were blocked at RT with 10% NGS and 0.2% Triton-X100 in PBS for 30 min. Coverslips were subsequently incubated at 4°C overnight with the appropriate primary antibodies. After several rinses in PBS, the coverslips were incubated with Alexa Fluor conjugated secondary antibodies (1:500, Invitrogen) and washed in PBS before mounting onto microscope slides. Images were taken using a LSM700 confocal microscope (Zeiss). When necessary, linear fluorescence intensity was calculated using Photoshop (Mean grey value). Alpha2delta2 expression along the longest neurite was normalized to the background. In a set of experiments, images were processed with Huygens Deconvolution software
(HuCore version 14.6.1, Scientific Volume Imaging, Netherlands) and Huygens Remote Manager (version 3.2) in confocal mode with theoretical point spread function. At least three independent replicates for each condition were analyzed. Immunoblotting For immunoblotting, cultured and dissected DRG from embryonic and adult mice were lysed on ice in RIPA buffer (500 mM Tris-HCl pH 4.8, 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing phosphatase (PhosSTOP, Roche) and protease inhibitors (Complete, Roche), centrifuged and the supernatant collected. The protein concentration of the lysate was determined using the Bradford Reagent (Bio-Rad). A portion of the lysate (5-30 µg total protein) was then fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and the separated proteins were transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane was stained with Ponceau S (Sigma) to confirm equal loading and transfer of the samples. After blocking at RT with 5% milk or 5% bovine serum albumin (BSA) in TBST for 1 hr, the membrane was then probed with a custom-made rabbit polyclonal anti-Alpha2delta2 (1:1000), rabbit anti-Cav2.1 (1:200), rabbit anti-Alpha2delta1 (1:200), rabbit anti-Alpha2delta3 (1:200), rabbit anti-Bax (1:500) or rabbit anti-Bcl-xL (1:1000). As a negative control, 1 µg of Alpha2delta2 antibody was preincubated with 1 µg of the blocking peptide for 1 hr at RT. Monoclonal anti-GAPDH (1:20000) or polyclonal anti-Tuj1 (1:10000) antibodies were used as protein loading controls. For quantitative analysis, Alpha2delta2 expression was normalized to tubulin expression levels. Dorsal Column Lesion (DCL) Mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) and a T11-T12 laminectomy was performed. The dorsal half of the spinal cord was cut using a microknife (10316-14, Fine Science Tools (FST)). For the control surgery, a laminectomy was performed without any direct manipulation of the spinal cord. Mice were allowed to recover for seven days before isolating L4-5 DRGs. Cell Viability and Cytotoxicity Analysis Cell viability and cytotoxicity was measured using CytoSelectTM Assay Kit (CBA-240, Cell Biolabs Inc.). Briefly, adult dissociated DRG neurons were electroporated with GFP (2.5 µg) plus either Cacna2d2 (3 µg) or empty (3 µg) expressing plasmid DNA. Medium was gently removed from the wells 24 hr after plating and 1X Saponin solution (240 µl/well in a 4-well plate) was added to control wells to initiate cell death. After washing, EthD-1 solution (384 µl/well in a 4-well plate cells) was added and wells were incubated at 36.5°C and 5% CO2 for 30 min. After removing the EthD-1 solution, wells were washed twice with medium and, after fixation, neurons were imaged for the presence of red EthD-1 (Excitation: 525 nm, Emission: 590 nm) fluorescence. Nuclear fluorescence intensity was calculated using Photoshop. At least three independent replicates for each condition were analyzed. DRG Electroporation in vivo Surgeries were performed as previously reported (Saijilafu et al., 2011). Briefly, mice were anesthetized and the L5 DRGs were exposed. Concentrated plasmid DNA (4-5 µg/µl) was injected using a pulled-glass pipette connected to a Picospritzer (30 psi, 8 ms). Immediately after injection, L5 DRGs were electroporated using a custom-made tweezer-like electrode connected to an Electro Square Porator (five 15 ms pulses at 35 V with 950 ms interval, ECM 830, BTX Harvard Apparatus). Finally, the skin was closed with suture clips and mice were allowed to recover for 5 days before a conditioning lesion or sciatic nerve crush was performed. L5 DRGs were isolated 7 days after PNL. For PNS regeneration study, animals were perfused 4 days after crush. To determine the percentage of co-expression of two proteins, L5 DRGs were electroporated in vivo with a mixture of RFP and GFP plasmid DNA. DNA Constructs Cacna2d2 was purchased from Origene (pCMV6-Entry-Cacna2d2, Cat Nr. MC223740). Cacna2d1 was a gift from Diane Lipscombe (Addgene plasmid #26575) (Lin et al., 2004). Cacna2d3 was a gift from Annette Dolphin (Addgene plasmid #58727) (Davies et al., 2010). Cacna2d3 coding sequence was PCR amplified with the following primers: 5’-AGCTTGCATGCCTGCAGGTCGACGCCACCATGGCCGGGCCGGGCTCG-3’ and 5’-
GTCACAGGGATGCCACGGAATTCTCACCTTGAGAAGAGACT-3’. The resulting PCR product containing flanking sequences matching a homemade pCMV6 vector was used to clone Cacna2d3 by T4 polymerase-based ligase independent reaction. Cacna2d2-RNAi resistant and Cacna2d2 R282A carrying constructs were both generated by overlapping PCR mutagenesis using pCMV6-Entry flanking VP1.5 forward primer and XL39 reverse primer (Origene) together with internal primers containing the desired mutations (underlined text): Cacna2d2-RNAi resistant forward, 5'-CTGGTGCAGCGGCCTCGGTACCGAAGAGGTCC-3'; Cacna2d2-RNAi resistant reverse, 5'-TCTTCGGTACCGAGGCCGCTGCACCAGCTC-3'. Cacna2d2-R282A forward, 5’-GTACGATGTCAGAAGAGCACCCTGGTATATACAG-3’; Cacna2d2-R282A reverse, 5’-CTGTATATACCAGGGTGCTCTTCTGACATCGTAC -3’. Following amplification, the mutated cDNAs were gel-purified and cloned in the original pCMV6-Entry vector (Origene) in Sgf I and Mlu I sites. GFP expressing plasmid (pmaxGFP) was purchased as part of the Amaxa Nucleofector Kit. All constructs were sequence verified. Sciatic Nerve Stimulation Adult mice were injected with either vehicle (0.9% saline) or PGB (46 mg/kg body weight) 3 times/day. Six days before, 1.5-2 µl of AAV-eGFP was injected into the left sciatic nerve to label primary afferents. Seven days after the first administration, mice were deeply anesthetized with a mixture of ketamine and xylazine. The left sciatic nerve was exposed at the mid-thigh level and electrically stimulated at 5 Hz for 5 min using a square pulse stimulator (SD9, Grass Technologies) followed by a 5 min pause. Mice were transcardially perfused with 1% paraformaldehyde followed by 4% paraformaldehyde. Spinal cords were dissected, and transferred to 30% sucrose in PBS. Coronal sections or the spinal cord were stained according to standard protocols (see also immunohistochemistry section). Von Frey Testing Adult mice were injected with either vehicle (0.9% saline) or PGB (46 mg/kg body weight) 3 times/day. Seven days after the first administration, mice were placed in a testing chamber and plantar threshold was measured using retractable monofilaments (Ugo Basile) calibrated to deliver a target force spanning from 0.4 (normal) to 4 gr (diminished light touch). A quick withdrawal of the hind paw was considered as a positive response. Signs of supraspinal sensitization (i.e. sustained paw withdrawal and/or licking the paw) were also annotated. A pause of 20-30 sec was given to allow sensory receptors to reach baseline levels between each monofilament application. After 20 stimuli presentations, the lowest force, which produced a retraction at least 50% of the time, determined the response threshold. During the entire session (no longer than 15 min/each animal), pieces of fruit cereal were placed for acclimation. Spinal Cord Window Implantation and 2-Photon Chronic Imaging Window implantation was performed as previously reported (Farrar et al., 2012). Briefly, adult female transgenic mice expressing GFP under the control of Thy1 promoter (Feng et al., 2000) were injected with either vehicle (0.9% saline) or PGB (46 mg/kg body weight) 3 times/day until the end of the study. Seven days after the first injection, mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). A small incision in the skin was made at the T11-T13 level, and the T12 vertebra was clamped using stainless steel bars with a notched groove. After fixing the bars with dental acrylic and cyanoacrylate, a T12 laminectomy was performed without damaging the dura. A top plate was screwed to the top of the bars, and a bilateral dorsal column crush was performed using #5 forceps (FST). The space between the spinal cord and the top plate was filled with Kwik-Sil silicone elastomer (World Precision Instruments) and a 4 mm diameter coverslip was positioned on top. Finally, the implant was sealed at the rostral and caudal vertebrae and skin attached to the edge of the window using cyanoacrylate glue and dental acrylic. Intravital multiphoton imaging (LSM 7MP, Zeiss) of the spinal cord was performed daily for 3 days. At least 6 axons/animal were quantified for each condition. Spinal Cord Injury (SCI)
Mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). A T11-T12 laminectomy was performed and spinal cord was crushed with #5 modified forceps (Dumont, FST). The forceps were deliberately positioned to sever dorsal column axons completely (Figure S7). In a set of experiment, a PNL was performed seven days prior to a SCI. Additional experiments included a dorsal hemisection model using a microknife (10316-14, FST) (Figure S7). Beginning 1 hr or 14 days after injury, PGB (46 mg/kg body weight) or an equal amount of vehicle (0.9% saline) was administered (intraperitoneal injections, 3 times/day for the first week, 2 times/day until the end of the study). DC axons were traced by injecting 1.5-2 µl of AAV-eGFP (AAV1.CMV.PI.EGFP.WPRE.bGH, 1-2e13 GC/ml, Penn Vector Core Facility) into the left sciatic nerve. Mice were terminated after two or four weeks following SCI. Horizontal imaging of the unsectioned spinal cord was performed using a 2-photon microscope (LSM 7MP, Zeiss) and images were used for quantification of dorsal column regeneration. Serial cryosections (60 um thick) of the spinal cord were cut in the sagittal plane. Transverse sections of the spinal cord (3 mm caudal and 7-8 mm rostral to the lesion) were taken to confirm the completeness of the lesion and tracing efficiency among experimental groups (Figure S7D). Mice with incomplete lesions were excluded from the analysis. The multidimensional rotation video of the injured spinal cord was made using Imaris software (Version 7.7.1, Bitplane). Staining for GFAP (1:500, Dako) was performed following standard protocols (see also immunohistochemistry section). Lesion area was quantified based on GFAP staining using Image J software on three consecutive AAV-eGFP labeled sections (c.a. 180 µm of tissue; n=4 per condition). Standardized randomization and blinding strategies were adopted. Quantification of Dorsal Column Regeneration after SCI For each multiphoton microscopy image of the unsectioned injured spinal cord, the number of regenerating axons at different distances from the lesion epicenter was normalized to the number of labeled axons caudal (200-400 µm) to the lesion. The lesion epicenter was identified based on axon morphology and the presence of infiltrating macrophages containing autofluorescent phagocytic material (Greenhalgh and David, 2014). Imaging of the unsectioned spinal cord excluded the possibility that regenerating axons with tortuous trajectory were counted multiple times, as is often the case with sagittal sections of the injured spinal cord. Samples with poor tracing for each specific experiment or altered by clear experimental flaw (i.e. incomplete lesion) were excluded from the analysis. Blinding strategies were adopted in an independent replication study.
Supplemental References Davies A., Kadurin I., Alvarez-Laviada A., Douglas L., Nieto-Rostro M., Bauer C.S., Pratt W.S., Dolphin A.C. (2010). The alpha2delta subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function. Proc Natl Acad Sci. 107(4):1654-9. Farrar M.J., Bernstein I.M., Schlafer D.H., Cleland T.A., Fetcho J.R., Schaffer C.B. (2012). Chronic in vivo imaging in the mouse spinal cord using an implanted chamber. Nature methods 9, 297-302. Feng G., Mellor R.H., Bernstein M., Keller-Peck C., Nguyen Q.T., Wallace M., Nerbonne J.M., Lichtman J.W., Sanes J.R. (2000). Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000 Oct;28(1):41-51 Greenhalgh A.D., David S. (2014). Differences in the phagocytic response of microglia and peripheral macrophages after spinal cord injury and its effects on cell death. J Neurosci. 34, 6316-6322 Lin Y., McDonough S.I., Lipscombe D. (2004). Alternative splicing in the voltage-sensing region of N-Type CaV2.2 channels modulates channel kinetics. J Neurophysiol. 92(5):2820-30. Maere S., Heymans K., Kuiper M. (2005). BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks, Bioinformatics 21, 3448-3449. Merico D., Isserlin R., Stueker O., Emili A., Bader G.D. (2010). Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PloS One 5, e13984. Oesper L., Merico D., Isserlin R., Bader G.D. (2011). WordCloud: a Cytoscape plugin to create a visual semantic summary of networks. Source Code Biol. Med. 6:7. Saijilafu, Hur E.M., Zhou F.Q. (2011). Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons. Nat Commun. 2, 543.
