Novel Signal Transduction Therapeutic Strategies
for Paralysis and Pain After Spinal Cord Injury (SCI)
Chen Guang Yu, Ph.D., M.D.
Assistant Professor
Spinal Cord and Brain Injury Research Center
Department of Anatomy and Neurobiology
University of Kentucky College of Medicine
Lexington, KY, USA
1
OUTLINE
(Therapeutic Strategies)
1
Targeting Individual ERK Isoform and Its Cross-talk with Calpain 1 for
Neuroprotection
2
FluBZ Therapy Targeting Multiple Signal Pathways for SCI
2
Traumatic Spinal Cord Injury (SCI)
•
Permanent locomotor disability
11,000 new cases occur each year in USA
Over 2.5 million SCI patients with paralysis in the world
•
SCI-pain (a common complication, 70-90%)
refractory to conventional analgesic treatment
•
Sustained activation of multiple signal pathways after SCI
Activation of the ERK1/2 signaling cascade by excitotoxic spinal cord
injury. Yu CG, Yezierski RP. 2005
Calpain in the CNS: from synaptic function to neurotoxicity.
Liu J, Liu MC, Wang KK. 2008
Cell cycle activation and spinal cord injury.
Wu J, Stoica BA, Faden AI. 2011
•
No effective treatments for Paralysis and Pain after SCI
Yu, et al., 2005, 2010; Crown et al., 2006; Zhao et al., 2007, We, et al. 2011
3
Part 1: Targeting Individual ERK and Its Cross-talk
with Calpain 1 for Treating paralysis and SCI-pain
Eur. J. Biochem. (Review) 271, 2050–2055 (2004)
Role of ERK 1 and 2 in neuronal survival
Michal Hetman1,2,3 and Agata Gozdz1
J Pharmacol Exp Ther. (Review) 2006 Dec;319(3):991-7.
A death-promoting role for ERK1/2.
Zhuang S, Schnellmann RG.
Hypothesis: reducing ERK2, while sparing ERK1, protects against SCI
ERK2 shRNA sequence
sense
loop
antisense
5’-CACCGCACCTCAGCAATGATCATCGAAATGATCATTGCTGAGGTGC-3’
BamHI
5’-LTR
RRE
U6
DNA Flap
XbaI
shRNA
CMV
EGFP
WPRE
3’LTR
Lentiviral ERK2 shRNA-GFP vector construction and production
4
The role of ERK2 in locomotor function after SCI
Locomotor function Test
BBB Score
Spinal ERK2 Knockdown
At 2 wks post-injury in rats
ERK1 44 kDa
ERK2 42 kDa
Viral titer:
5X107 to 1X108
22
20
18
16
14
12
10
8
6
4
2
0
-2
LV-Control
LV-ERK2 shRNA
*
*
*
*
pre
0
3
7
14
21
28
35
T10
230 kdyn
IH SCI Device
L-E Rats
n=7/group
42
Days Post-injury
Eriochrome cyanine (EC) stained spinal sections
II
6
3
Epicenter
3
Rostral
Distance from epicenter (mm)
I: LV-ERK2 shRNA;
II: LV-Control
6
Caudal
Total Tissue Sparing (mm
3
)
I
Tissue Sparing Assessment
*
4
3.5
3
2.5
2
1.5
1
.5
0
LV-Control
.
LV-ERK2 shRNA
Yu et al., J Neurochemistry 2010
5
Contributions of ERK1/2 to pain behaviors after excitotoxic SCI
QUIS: Quisqualic acid
an AMPA receptor agonist
ERK1/2 activation after Excitotoxic SCI
Saline
________
QUIS
_____________
Phospho ERK
44 Kda (ERK1)
42 Kda (ERK2)
Total ERK
44 Kda (ERK1)
42 Kda (ERK2)
_________________________________________
Group
n
Grooming Behavior Incidence
____________________________________
PD98059 4
0
0%
Vehicle 6
5
83%
_________________________________________
Self-injurious over-grooming
of the affected dermatome
(PD98059 1 μg/15μl delivered to surface of the cord
For 1 h pre and immediately post-injection of QUIS)
pERK2, but not pERK1, contributes to the complete Freund’s Adjvant-induced
and formalin-induced pain (Xu Q, et al, 2008; Alter BJ et al., 2010)
6
Determination of targets of ERK1/2 activation after SCI
ERK1/2 inhibition (U0126) down-regulates
calpain 1 expression 4h after SCI in rats
.03
**
Calpain 1
Calpain 1
76 KDa
GAPDH
76 KDa
36 KDa 36 KDa
GAPDH
SCI+
SCI+
U0126 iv U0126 ip
SCI+
Sham
Vehicle
Calpain-1 Protein level
(Calpain-1/GAPDH)
.025
SCI+U0126-IV (n=4)
.02
SCI+U0126-IP (n=4)
#
SCI+Vehicle (n=4)
.015
#
sham (n=4)
.01
.005
0
.
U0126 iv (2mg/kg) and ip (10mg/kg) pretreatment
7
Role of calpain 1 in SCI
Locomotor Function Test
Spinal CAPN1 knockdown
At 2 wks post-injury in rats
T10
180 kdyn
IH SCI Device
L-E Rats
Viral titer:
5X107 to 1X108
Tissue Sparing Assessment
I: LV-Control shRNA
II: LV-CAPN1 shRNA (Scale bar: 100 μm)
b
c
e
f
g
I
h
i
j
l
k
m
n
II
4 mm
2 mm
1 mm
Lesion epicenter
Rostral
2 mm
1 mm
Caudal
Eriochrome cyanine (EC) staining
Yu et al., Journal Neurotrauma, 2012
4 mm
Total Tissue Sparing (mm3 )
a
6
d
*
LV-calpain 1 shRNA
LV-control
5
4
3
2
1
0
.
n=10 per group, ANOVA followed by the Bonferroni post hoc test
8
Determination of CAPN1 targets
Using cocktail antibodies
Phospho-SHP2 (72 kDa)
Phospho-AKT (60 kDa)
Phospho-p44/p42 ERK1/2
(44 kDa and 42 kDa)
eIF4E (25 kDa,
loading control protein)
WT-Sham
WT-SCI 6h
CAPN1 KO SCI 6h
CAPN1 deletion increased ERK1/2
activation after acute SCI in mice
CAPN1 deletion exacerbated SCI-pain after acute SCI in mice
CAPN1 deletion worsened
SCI-pain after SCI in mice
CAPN1 deletion showed
earlier onset of grooming
WT
CAPN1 KO
WT
CAPN1 KO
12
4
Severity of Grooming
Onset of Grooming (Days)
14
10
**
8
6
4
2
3.5
3
2.5
2
1.5
1
.5
0
0
.
.
CAPN1 deletion exacerbated
SCI-pain after SCI in mice
——————————————————————————
Group
n
Grooming Behavior
Incidence
Onset
____________________________________________________
CAPN-KO
5
4
80%
6
WT
4
3
75%
12.5
____________________________________________________
10
Summary for Part 1: Targeting calpain 1 and ERK2
Cross-talk may provide synergistic effects after SCI
CAPN1reduction
or deletion
ERK1
Activation
ERK2
Activation
ERK2
Inhibition
Spared
ERK1
Neuroprotective
Improves
Locomotion
Neuroprotective
Worsens
SCI-Pain
Improves locomotion
Reduces pain
ERK1-/- mice exhibit exacerbated paralysis in EAE.
(Agrawal A, 2006)
ERK1 plays a critical protective role against NMDA injury.
(Nakazawa T, 2008)
11
Part 2: FBZ/FluBZ Therapy Targeting Multiple Signal Pathways for SCI
Fenbendazole (FBZ)
• A benzimidazole anthelminth for animal use only
• FBZ inhibits microtubule formations, and thereby blocking mitosis in
nematodes
• FBZ has greater sensitivity for nematodes as compared to mammalian
tubulin
• Its influence on the outcomes of ongoing experiments are a concern
Villar et al., 2007; Friedman et al., 1978
12
FBZ improves locomotor function and
tissue sparing after moderate SCI in mice
T9
50 kdyn
IH SCI Device
C57BL/6 mice
Locomotor
Function
Tissue Sparing
Eriochrome cyanine
(EC) staining for myelin
Yu CG et al., 2014
FBZ-medicated feed (8 mg/kg/day) for 4 wks prior to SCI, n=7/group
13
FluBZ improved locomotor function
And tissue sparing after SCI in rats
•
•
Approved for human use,
Long term treatment without
adverse effects
Locomotor Function
T10
180 kdyn
IH SCI Device
SD rats
Total Tissue Sparing
SCI+Vehicle SCIFluBZ+
***
35
30
25
20
15
10
5
0
.
Total White Matter Sparing (mm3 )
Total Tissue Sparing (mm3 )
50
45
40
SCI-FluBZ
SCI-Vehicle
35
SCI-FluBZ
SCI-Vehicle
***
30
25
20
15
10
5
0
Gray Matter Sparing
Total Gray Matter Sparing (mm 3)
White Matter Sparing
Total Tissue Sparing
14
SCI-FluBZ
SCI-Vehicle
*
12
10
.
FluBZ IP treatment, 3 hrs post-injury for 2 wks (10 mg/kg/day, n=10 per group)
8
6
4
2
0
.
J Clin Invest. 2009 Oct;119(10):2990-9.
B cells produce pathogenic antibodies and impair recovery after
spinal cord injury in mice.
Ankeny DP, Guan Z, Popovich PG.
J Am Assoc Lab Anim Sci. 2009 May;48(3):251-7.
Effects of fenbendazole on the murine humoral immune system.
Landin AM, Frasca D, Zaias J, Van der Put E, Riley RL, Altman NH,
Blomberg BB.
FBZ suppresses B cell proliferation and production of antibodies.
Microtubule inhibitors (Colchicine, vinblastine):
Cell cycle inhibition, antiinflammation, antiproliferation, and
inhibition of pERK1/2 (Gaba, 2014, Spagnuolo, 2010)
15
FBZ reduced IgG levels and CD45R-positive B cells
at lesion site six weeks after SCI in mice
Immunohistochemistry for IgG
Immunofluorescence staining for CD45R-B cells
CD45R DAPI
Sham
SCI-Control
SCI+FBZ
SCI-Control
Yu CG et al., 2014
SCI-FBZ
Sham
16
FLuBZ improves recovery of B cell population 4 weeks after SCI
a
Sham
SCI+Vehicle
b
R3: 35.39%
R3: 30.27%
c
SCI+FluBZ
d
SCI+FluBZ 10mg/kg
Sham
R3: 26.56%
B-Cell Population (%)
SCI+Vehicle
40
35
*
#
30
25
20
15
10
5
0
.
Flow cytometry analysis for CD45RA-positive B cell population
17
FluBZ inhibited ERK1/2, cyclin B1, and
astroglial activation 4 wks after SCI
pERK1 44 KDa
pERK2 42 KDa
ERK1/2 Activation
Sham
SCI+Vehicle
eIF4E (25 KDa)
(loading control)
SCI+FluBZ
Sham
SCI+Vehicle
Cyclin B1
upregulation
Cyclin B1
(58 kDa)
GAPDH
(36 kDa)
Sham
SCI+Vehicle SCI+FluBZ
Cyclin B1 Upregulation
(Cyclin B1/GAPDH)
.9
SCI+FluBZ
.8
***
.7
##
.6
.5
.4
.3
.2
.1
0
.
Astroglial Activation
B
GFAP
(43-45 kDa)
GAPDH
(36 kDa)
Sham
SCI+Vehicle
SCI+FluBZ
GFAP Expression (GFAP/GAPDH)
A
Sham
SCI+Vehicle
SCI+FluBZ
60
***
50
40
30
20
##
10
0
.
18
Brain Res Rev. 2009 Apr;60(1):135-48. doi:
10.1016/j.brainresrev.2008.12.011. Epub 2008 Dec 25.
MAP kinase and pain.
Ji RR1, Gereau RW 4th, Malcangio M, Strichartz GR.
Life Sci. 2004 Apr 9;74(21):2643-53.
MAPK activation in nociceptive neurons and pain
hypersensitivity.
Obata K1, Noguchi K.
Rev Med Suisse. 2013 Jun 26;9(392):1342-5.
[Glial cells and chronic pain: from the laboratory to clinical hope].
[Article in French]
Clarke CB1, Suter MR, Gosselin RD.
Eur J Pharmacol. 2013 Sep 15;716(1-3):120-8. doi:
10.1016/j.ejphar.2013.03.023. Epub 2013 Mar 22.
Astrocytes--multitaskers in chronic pain.
Hansen RR1, Malcangio M.
19
FluBZ attenuates pain behaviors
after excitotoxic SCI in rats
Grooming Onset
Grooming Area (cm )
.9
20
17.5
15
12.5
10
7.5
5
2.5
0
.8
Grooming Severity
QUIS+FluBZ
QUIS+Vehicle
QUIS+FluBZ
QUIS+Vehicle
4.5
.7
Grooming Severity
QUIS+FluBZ
QUIS+Vehicle
22.5
Grooming Onset (Days)
Grooming Area
.6
.5
.4
.3
.2
4
3.5
3
2.5
2
1.5
.1
1
0
.5
.
.
0
.
Vehicle-treated
FluBZ-treated
Grooming incidence
75%
40%
Onset grooming (days)
11
19
20
Summary for Part 2
(FBZ/FluBZ therapy)
• Fenbendazole and flubendazole Improve experimental outcomes
following SCI
• Flubendazole inhibits
pERK1/2
cyclin B1
B-cell response
astroglial activation
• Flubendazole is clinically approved and could easily be translated to
human clinical trials.
21
Future Direction
SCI primary injury
FluBZ
G1
G1
G1
M
M
inhibit
microtubule
formation
SS
pERK1/2
Molecular
mechanisms
G2
G2
Cyclin B1-CDK1
cell cycle progression at G2/M
B cells
Autoimmune
Astrocytes
astroglial scar
Microglia
Cell nucleus
Inflammation, Nociception
Neuronal/axonal Failed axonal
damage
regeneration
Locomotor Chronic
deficits
Pain
Cellular
mechanisms
Pathological
mechanisms
Dysfunction
22
ACKNOWLEDGEMENTS
Dr. James W. Geddes, Ph.D.
University of Kentucky
Drs. Yu/James W. Geddes’s lab
Vimala Bondada
Carolyn Crowdus
Colin Rogers, Ph.D.
Kashif Raza
Sarbani Ghoshal, Ph.D.
Ranjana Singh, Ph.D.
Brantley Graham
Charles Mashburn, Ph.D.
Lauren Thompson
Allie Zeller
Lauren Power
Mackenzie Jones
Jessica Jones
Dr. Hall’s lab
Dr. Rabchevsky’s lab
Dr. Saatman’s lab
Dr. Springer’s lab
Dr. Sullivan’s lab
Dr. Snow’s lab
Dr. Smith’s lab
Dr. Bondada’s lab
Dr. Gensel’s lab
Ms. Zel Frye,
Ms. Liz Jones,
Ms. Julie Combs
Dr. Jeanie F. Kincer
Funding Sources
•
•
Dr. Robert P. Yezierski, Ph.D. •
University of Florida
•
•
University of Florida Seed Grant:
“Effects of ERK1/ERK2 siRNA on Spinal Injury Pain”
PI: Chen Guang Yu, 2004-2005
Paralysis Project of America Grant:
“Inhibition of ERK1/2 for treatment for spinal cord injury
PI: Chen Guang Yu/James Geddes, 2006-2007
KSCHIRT Grant 7-6A:
“Inhibition of ERK2 with lentiviral ERK2-shRNA for SCI”
PI: James Geddes/Chen Guang Yu, 2008-2014
KSCHIRT Grant 11-19A:
“Calpain knockdown minimize damage and deficits after SCI”
PI: Chen Guang Yu/James Geddes, 2012-2015
NIH CTSA Grant: ULTR000117
PI: Chen Guang Yu, 2014-2016
23