Novel Signal Transduction Therapeutic Strategies

for Paralysis and Pain After Spinal Cord Injury (SCI)

Chen Guang Yu, Ph.D., M.D. 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 SCI2 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

• 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

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

Eur. J. Biochem. (Review) 271, 2050–2055 (2004)

Role of ERK 1 and 2 in neuronal survivalMichal 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.

Part 1: Targeting Individual ERK and Its Cross-talk

with Calpain 1 for Treating paralysis and SCI-pain

4

Hypothesis: reducing ERK2, while sparing ERK1, protects against SCI

ERK2 shRNA sequencesense loop antisense

5’-CACCGCACCTCAGCAATGATCATCGAAATGATCATTGCTGAGGTGC-3’

RRE U6 shRNADNA Flap EGFPCMV 3’LTRWPRE

BamHI XbaI

5’-LTR

Lentiviral ERK2 shRNA-GFP vector construction and production

0

2

4

6

8

10

12

14

16

18

20

22

BB

B S

co

re

LV-ERK2 shRNA

LV-Control

*

* *

*

ERK1 44 kDaERK2 42 kDa

Spinal ERK2 Knockdown

At 2 wks post-injury in rats

Locomotor function Test

T10230 kdynIH SCI DeviceL-E Ratsn=7/group

The role of ERK2 in locomotor function after SCI

Viral titer:Viral titer:Viral titer:Viral titer:5X105X105X105X107777 to 1X10to 1X10to 1X10to 1X108888

-2

0

pre 0 3 7 14 21 28 35 42

Days Post-injury

5Yu et al., J Neurochemistry 2010

6 3 Epicenter 3 6

Rostral Distance from epicenter (mm) Caudal

I: LV-ERK2 shRNA; II: LV-Control

I

II

Eriochrome cyanine (EC) stained spinal sections

0

.5

1

1.5

2

2.5

3

3.5

4

To

tal

Tis

su

e S

pari

ng

(m

m

)

.

*

LV-Control LV-ERK2 shRNA

33 33

Tissue Sparing Assessment

5X105X105X105X107777 to 1X10to 1X10to 1X10to 1X108888

QUIS: QUIS: QUIS: QUIS: Quisqualic acid an AMPA receptor agonist

Contributions of ERK1/2 to pain behaviors after excitotoxic SCI

_________________________________________

←44 Kda (ERK1)

←42 Kda (ERK2)

Phospho ERK

Total ERK

←44 Kda (ERK1)

←42 Kda (ERK2)

Saline QUIS________ _____________

ERK1/2 activation after Excitotoxic SCI

6

Self-injurious over-grooming of the affected dermatome

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)

_________________________________________

Group n Grooming Behavior Incidence

____________________________________

PD98059 4 0 0%

Vehicle 6 5 83%

_________________________________________

(PD98059 1 µg/15µl delivered to surface of the cord For 1 h pre and immediately post-injection of QUIS)

ERK1/2 inhibition (U0126) down-regulates

calpain 1 expression 4h after SCI in rats

76 KDa

36 KDa

Calpain 1

GAPDH.015

.02

.025

.03

Ca

lpa

in-1

Pro

tein

le

ve

l

(Ca

lpa

in-1

/GA

PD

H)

sham (n=4)

SCI+Vehicle (n=4)

SCI+U0126-IP (n=4)

SCI+U0126-IV (n=4)

**

#

#

Calpain 1 76 KDa

36 KDaGAPDH

Determination of targets of ERK1/2 activation after SCI

7

ShamSCI+

Vehicle

SCI+

U0126 ip

SCI+

U0126 iv

0

.005

.01

Ca

lpa

in

(Ca

lpa

in

.

U0126 iv (2mg/kg) and ip (10mg/kg) pretreatment

Spinal CAPN1 knockdownAt 2 wks post-injury in rats

Locomotor Function Test

T10180 kdynIH SCI DeviceL-E Rats

Role of calpain 1 in SCI

Viral titer:Viral titer:Viral titer:Viral titer:

8Yu et al., Journal Neurotrauma, 2012 n=10 per group, ANOVA followed by the Bonferroni post hoc test

Lesion epicenter 1 mm 2 mm1 mm2 mm4 mm 4 mm

Rostral Caudal

I: LV-Control shRNAII: LV-CAPN1 shRNA

I

II

a b c d e f g

h i j kl m n

(Scale bar: 100 μm)

Eriochrome cyanine (EC) staining 0

1

2

3

4

5

6

To

tal T

issu

e S

pari

ng

(m

m )

.

LV-control

LV-calpain 1 shRNA*

3

Tissue Sparing Assessment

Viral titer:Viral titer:Viral titer:Viral titer:5X105X105X105X107777 to 1X10to 1X10to 1X10to 1X108888

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 CAPN1 deletion increased ERK1/2 CAPN1 deletion increased ERK1/2 CAPN1 deletion increased ERK1/2 activation after acute SCI in miceactivation after acute SCI in miceactivation after acute SCI in miceactivation after acute SCI in mice

CAPN1 deletion exacerbated SCICAPN1 deletion exacerbated SCICAPN1 deletion exacerbated SCICAPN1 deletion exacerbated SCI----pain after acute SCI in micepain after acute SCI in micepain after acute SCI in micepain after acute SCI in mice

0

2

4

6

8

10

12

14

On

set

of

Gro

om

ing

(D

ays)

CAPN1 KO

WT

**

0

.5

1

1.5

2

2.5

3

3.5

4

Severi

ty o

f G

roo

min

g

CAPN1 KO

WT

CAPN1 deletion showed CAPN1 deletion showed CAPN1 deletion showed CAPN1 deletion showed earlier onset of grooming earlier onset of grooming earlier onset of grooming earlier onset of grooming

CAPN1 deletion worsenedCAPN1 deletion worsenedCAPN1 deletion worsenedCAPN1 deletion worsenedSCISCISCISCI----pain after SCI in mice pain after SCI in mice pain after SCI in mice pain after SCI in mice

10

0.

0.

——————————————————————————

Group n Grooming Behavior Incidence Onset

____________________________________________________

CAPN-KO 5 4 80% 6

WT 4 3 75% 12.5

____________________________________________________

CAPN1 deletion exacerbated CAPN1 deletion exacerbated CAPN1 deletion exacerbated CAPN1 deletion exacerbated SCISCISCISCI----pain after SCI in micepain after SCI in micepain after SCI in micepain after SCI in mice

CAPN1reduction or deletion

ERK2 Activation

ERK1 Activation

Summary for Part 1: Targeting calpain 1 and ERK2 Cross-talk may provide synergistic effects after SCI

ERK2 Inhibition

Spared ERK1

11

Worsens SCI-Pain

Improves Locomotion

Improves locomotionReduces pain

ERK1ERK1ERK1ERK1----////---- mice exhibit mice exhibit mice exhibit mice exhibit exacerbated paralysis in EAE. exacerbated paralysis in EAE. exacerbated paralysis in EAE. exacerbated paralysis in EAE. ((((Agrawal A, 2006)Agrawal A, 2006)Agrawal A, 2006)Agrawal A, 2006)

ERK1 plays a critical protective role against ERK1 plays a critical protective role against ERK1 plays a critical protective role against ERK1 plays a critical protective role against NMDA injury.NMDA injury.NMDA injury.NMDA injury. (Nakazawa T, 2008)

NeuroprotectiveNeuroprotective

Fenbendazole (FBZ)

• A benzimidazole anthelminth for animal use only

• FBZ inhibits microtubule formations, and thereby blocking mitosis in

nematodes

Part 2: FBZ/FluBZ Therapy Targeting Multiple Signal Pathways for SCI

nematodes

• FBZ has greater sensitivity for nematodes as compared to mammalian

tubulin

• Its influence on the outcomes of ongoing experiments are a concern

12

Villar et al., 2007; Friedman et al., 1978

FBZ improves locomotor function and tissue sparing after moderate SCI in mice

LocomotorFunction

T950 kdynIH SCI DeviceC57BL/6 mice

13Yu CG et al., 2014 FBZ-medicated feed (8 mg/kg/day) for 4 wks prior to SCI, n=7/group

Tissue Sparing

Eriochrome cyanine (EC) staining for myelin

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 kdynIH SCI DeviceSD rats

SCI+Vehicle SCIFluBZ+

Total Tissue Sparing

FluBZ IP treatment, 3 hrs post-injury for 2 wks (10 mg/kg/day, n=10 per group)

0

5

10

15

20

25

30

35

40

45

50

To

tal

Tis

su

e S

pari

ng

(m

m

)

.

SCI-Vehicle

SCI-FluBZ

***3

0

5

10

15

20

25

30

35

To

tal

Wh

ite M

att

er

Sp

ari

ng

(m

m

)

.

SCI-Vehicle

SCI-FluBZ

***

3 3

0

2

4

6

8

10

12

14

To

tal

Gra

y M

att

er

Sp

ari

ng

(m

m

)

.

SCI-Vehicle

SCI-FluBZ

*

Total Tissue Sparing Gray Matter SparingWhite Matter Sparing

SCI+Vehicle SCIFluBZ+

J Clin Invest. 2009 Oct;119(10):2990-9.

B cells produce pathogenic antibodies and impair recovery after B cells produce pathogenic antibodies and impair recovery after B cells produce pathogenic antibodies and impair recovery after B cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice.spinal cord injury in mice.spinal cord injury in mice.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 Effects of Effects of Effects of fenbendazolefenbendazolefenbendazolefenbendazole on the on the on the on the murinemurinemurinemurine humoralhumoralhumoralhumoral immune system.immune system.immune system.immune system.Landin AM, Frasca D, Zaias J, Van der Put E, Riley RL, Altman NH,

15

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)

FBZ reduced IgG levels and CD45R-positive B cells

at lesion site six weeks after SCI in mice

Sham SCI-Control SCI+FBZ

CD45R DAPI

Immunohistochemistry for IgG Immunofluorescence staining for CD45R-B cells

16Yu CG et al., 2014 16

SCI-Control SCI-FBZ Sham

Yu CG et al., 2014

R3: 30.27% R3: 35.39%

c

ba

FLuBZ improves recovery of B cell population 4 weeks after SCI

Sham

SCI+FluBZ

SCI+Vehicle

17

0

5

10

15

20

25

30

35

40

B-C

ell

Po

pu

lati

on

(%

)

.

SCI+Vehicle

Sham

SCI+FluBZ 10mg/kg

* #

d

R3: 26.56%

c SCI+FluBZ

Flow cytometry analysis for CD45RA-positive B cell population

FluBZFluBZFluBZFluBZ inhibited ERK1/2, inhibited ERK1/2, inhibited ERK1/2, inhibited ERK1/2, cyclincyclincyclincyclin B1, and B1, and B1, and B1, and astroglialastroglialastroglialastroglial activation 4 activation 4 activation 4 activation 4 wkswkswkswks after SCI after SCI after SCI after SCI

Sham SCI+Vehicle SCI+FluBZ

pERK1 44 KDapERK2 42 KDa

eIF4E (25 KDa)(loading control)

.6

.7

.8

.9

Cycli

n B

1 U

pre

gu

lati

on

(Cycli

n B

1/G

AP

DH

)

SCI+FluBZ

SCI+Vehicle

Sham

***

##Cyclin B1(58 kDa)

ERK1/2 Activation

Cyclin B1 upregulation

18

0

.1

.2

.3

.4

.5

Cycli

n B

1 U

pre

gu

lati

on

(Cycli

n B

1/G

AP

DH

)

.

(58 kDa)

GAPDH(36 kDa)

Sham SCI+Vehicle SCI+FluBZ

0

10

20

30

40

50

60

GF

AP

Exp

ressio

n (

GF

AP

/GA

PD

H)

.

SCI+FluBZ

SCI+Vehicle

Sham

***

##

GFAP

(43-45 kDa)

GAPDH

(36 kDa)

Sham SCI+Vehicle SCI+FluBZ

A B

upregulation

Astroglial Activation

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.MAP kinase and pain.MAP kinase and pain.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 MAPK activation in nociceptive neurons and pain MAPK activation in nociceptive neurons and pain MAPK activation in nociceptive neurons and pain hypersensitivity.hypersensitivity.hypersensitivity.hypersensitivity.Obata K1, Noguchi K.

19

Rev Med Suisse. 2013 Jun 26;9(392):1342-5.[Glial cells and chronic pain: from the laboratory to clinical hope].[Glial cells and chronic pain: from the laboratory to clinical hope].[Glial cells and chronic pain: from the laboratory to clinical hope].[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.AstrocytesAstrocytesAstrocytesAstrocytes--------multitaskersmultitaskersmultitaskersmultitaskers in chronic pain.in chronic pain.in chronic pain.in chronic pain.Hansen RR1, Malcangio M.

FluBZ attenuates pain behaviorsafter excitotoxic SCI in rats

0

2.5

5

7.5

10

12.5

15

17.5

20

22.5

Gro

om

ing

On

set

(Days)

.

QUIS+Vehicle

QUIS+FluBZ

0

.1

.2

.3

.4

.5

.6

.7

.8

.9

Gro

om

ing

Are

a (

cm

)

.

QUIS+Vehicle

QUIS+FluBZ

0

.5

1

1.5

2

2.5

3

3.5

4

4.5

Gro

om

ing

Severi

ty

QUIS+Vehicle

QUIS+FluBZ

Grooming Onset Grooming Area Grooming Severity

20

. 0.

Vehicle-treated FluBZ-treated

Grooming incidence 75% 40%

Onset grooming (days) 11 19

Summary for Part 2 (FBZ/FluBZ therapy)

• Fenbendazole and flubendazole Improve experimental outcomesfollowing SCI

• Flubendazole inhibitspERK1/2pERK1/2cyclin B1B-cell responseastroglial activation

• Flubendazole is clinically approved and could easily be translated tohuman clinical trials.

21

Future Direction

SCI primary injury

pERK1/2

Cyclin B1-CDK1

cell cycle progression at G2/M

FluBZ

inhibit

microtubule formation

G1G1

M

G2

S

Molecularmechanisms

M

G2

S

G1

22

cell cycle progression at G2/M

B cells Astrocytes MicrogliaCell nucleus

Inflammation, Nociceptionastroglial scarAutoimmune

Neuronal/axonal

damage

Failed axonal

regeneration

Locomotor

deficits

Chronic

Pain

Cellular

mechanisms

Pathologicalmechanisms

Dysfunction

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Funding Sources• University of Florida Seed Grant:

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.

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

Dr. James W. Geddes, Ph.D.University of Kentucky

Ms. Zel Frye,

Ms. Liz Jones,

Ms. Julie Combs

Dr. Jeanie F. Kincer

• 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

Charles Mashburn, Ph.D.

Lauren Thompson

Allie Zeller

Lauren Power

Mackenzie Jones

Jessica Jones

Dr. Robert P. Yezierski, Ph.D.University of Florida