Caspase-dependent and -independent pathways in neuronal apoptosis after spinal cord injury Kay L.H. Wu, Chin Hsu and Julie Y.H. Chan

Graduate Institute of Medicine, Kaohsiung Medical university, and Department o Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Introduction

1. Spinal cord injury (SCI) results in irreversible neuronal damage leading to apoptotic cell death.2. Accumulated evidence indicates that mitochondria initiate apoptosis via cellular events that

include caspases-dependent and caspases-independent pathways.3. We study the hypotheses that mitochondrial respiratory impairment promotes neuronal apoptosis

and the impairment is induced via early stage of free radical production after SCI.

Aims of Study

1. To identify the temporal profiles of apoptosis in the spinal cord after injury.2. To delineate the relationship between mitochondrial respiratory dysfunction

and neuronal apoptosis after spinal cord injury.3. To investigate the involvement of free radical in mitochondrial dysfunction in

the spinal cord after injury.

Materials and Methods

1 52 3 4 7 10

Tissue fixation

Electromicroscopy

Genomic DNAProteins

Mitochondria

DNA LadderingWestern Blot

T8

14

Summary and ConclusionFree radical

NeuronNucleus

AIF

DNAfragmentation

Caspase-3activation

Apoptosis

PARPactivation

I

IIIII

IV

Caspase-9activation cyt. c

Figure 1. DNA fragmentation detected in the spinal cord after T8 transection (SCT) alone or with addition of coenzyme Q10 (CoQ 10) treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. baseline control (C) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test.

M C 1 2 3 4 5 7 10 14

SCT only

SCT with CoQ10

Post operation time(day)

0 1 2 3 4 5 7 10 14

Rat

io

0

20

40

60

80

100

120

SCT onlySCT with CoQ10

**

#

*

#

#

#

*

**

*

#

##

*

Post operation time(day)

0 1 2 3 4 5 7 10 14

Rat

io

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

SCT onlySCT with CoQ10

*

*

* **

**

*

#

#

#

#

#

Figure 3. Time-course of changes in the mitochondrial respiratory complex I activity, ATP production in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative electron microscopic photomicrographs illustrating swelling of the mitochondrial cristae after SCT.

Normal control (50000 X) Post SCT 1 day (50000 X)Mitochondria complex I activityin the spinal cord after injury

ATP concentrationin the spinal cord after injury

Figure 5. Time-course changes of iNOS expression or NOx levels in the spinal cord after SCI alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating the distribution of dihydroethidium or expression of NADPH oxidase subunits in the injured spinal cord.

Post operation time(day)

0 1 2 3 4 5 7 10 14

Opt

ical

den

sity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

SCT onlySCT with CoQ10

** *

*

*

# ##

# #

Post operation time(day)

0 1 2 3 4 5 7 10 14

Rat

io

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

SCT onlySCT with CoQ10

*

#

* * *

##

Cytosolic iNOS expressionin the spinal cord after injury

NOx concentrationin the spinal cord after injury

gp91phox

p47phox

p67phox

C 1 2 3 4 5 7 10 14Dihydroethidium stain

in 3rd day after SCI

Post operation time(day)

0 1 2 3 4 5 7 10 14

Opt

ical

den

sity

0

200

400

600

800

1000

1200

1400

1600

1800

2000SCT onlySCT with CoQ10

#

***

***

*

#

## #

#

#

#

*

Post operation time(day)

0 1 2 3 4 5 7 10 14

Ra

tio o

f o

ptic

al d

en

sity

0.0

0.2

0.4

0.6

0.8

1.0

SCT onlySCT with CoQ10

*

**

*

*

*

#

##

# #

Nucleus AIF expression

(NeuN, AIF and TUNEL triple-labeled cells, 1000 X)

3rd day after SCI

NeuN AIF

TUNEL Merge

Figure 4. Time-course of changes in the expression of apoptosis inducing factor (AIF) in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating co-localization of AIF-immunoreactivity with NeuN-immunoreactivity in the TUNEL-positive cells.

0 1 2 3 4 5 7 10 14

Figure 2. Time-course of changes in the expression of caspase-dependent cytosolic proteins in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or #P<0.05 vs. SCT groups in the Scheffé multiple-range test.

Cytosolic cleaved caspase-9 expression

Cytosolic cleaved caspase-3 expression

Cytosolic cytochrome c expression Cleaved PARP expression

Post operation time(day)

0 1 2 3 4 5 7 10 14

Ratio

of optic

al d

ensi

ty

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

SCT only

SCT with CoQ10

*

*

#

##

*

Post operation time(day)

0 1 2 3 4 5 7 10 14

Ratio

of optic

al d

ensi

ty

0.0

0.2

0.4

0.6

0.8

1.0

1.2

SCT onlySCT with CoQ10

* *

**

##

#

#

*

Post operation time(day)

0 1 2 3 4 5 7 10 14

Ratio

of o

ptic

al d

en

sity

0

1

2

3

4

5

6

7

SCT onlySCT with CoQ10

*

*

*

*

#

* *

*# #

# #

#

#

Post operation time(day)

0 1 2 3 4 5 7 10 14

Ratio

of optic

al d

ensi

ty

0.0

0.5

1.0

1.5

2.0

2.5

SCT onlySCT with CoQ10

**

*

* *

#

* **

#

##

#

C 1 2 3 4 5 7 10 14 C 1 2 3 4 5 7 10 14C 1 2 3 4 5 7 10 14 C 1 2 3 4 5 7 10 14