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p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

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Abstract

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser392. Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

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Abbreviations

ALS:

Amyotrophic lateral sclerosis

ANOVA:

Analysis of variance

APAF1:

Apoptotic protease activating factor 1

ATM:

Ataxia telangiectasia mutated

ATR:

Ataxia telangiectasia and Rad3 related

BRCA2:

Breast cancer 2, early onset

BSA:

Bovine serum albumin

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid

CHK2:

Checkpoint homolog (S. pombe)

CNS:

Central nervous system

DAB:

Diaminobenzidine

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

DNA:

Deoxyribonucleic acid

DSB:

Double-strand breaks

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

GFAP:

Glial fibrillary acidic protein

H2AX:

Histone H2A.X

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HRP:

Horseradish peroxidase

hUCB:

Human umbilical cord blood derived stem cells

Olig2:

Oligodendrocyte transcription factor 2

PAGE:

Poly acrylamide gel electrophoresis

PBS:

Phosphate buffered saline

RT-PCR:

Reverse Transcription based Polymerase chain reaction

PMSF:

Phenyl methane sulfonyl fluoride

SCI:

Spinal cord injury

SDS:

Sodium dodecyl sulfate

SSB:

Single-strand breaks

STP:

Staurosporine

TAE:

Tris-acetate-EDTA

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick end labeling

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Acknowledgments

We are thankful to Noorjehan Ali for technical assistance. We thank Shellee Abraham for manuscript preparation, and Sushma Jasti and Diana Meister for review of the manuscript. This study was funded by a grant from Caterpillar Inc., Peoria, IL and OSF Saint Francis Inc., Peoria, IL (J.S.R.).

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Authors and Affiliations

  1. Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL, 61656, USA

    Ramaprasada Rao Kotipatruni, Venkata Ramesh Dasari, Krishna Kumar Veeravalli & Jasti S. Rao

  2. Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA

    Dzung H. Dinh, Daniel Fassett & Jasti S. Rao

Authors
  1. Ramaprasada Rao Kotipatruni
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  2. Venkata Ramesh Dasari
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  3. Krishna Kumar Veeravalli
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  4. Dzung H. Dinh
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  5. Daniel Fassett
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  6. Jasti S. Rao
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Corresponding author

Correspondence to Jasti S. Rao.

Additional information

Ramaprasada Rao Kotipatruni and Venkata Ramesh Dasari contributed equally.

Electronic supplementary material

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11064_2011_530_MOESM1_ESM.tif

Supplementary Figure 1. Activation of p53 in spinal neurons in vitro . (A) Immunoblot analysis of p53 and phospho-p53 in STP (2µM)-treated spinal neurons. GAPDH was used as a control. (B) Quantitation of (A). Error bars indicate mean ± SE. Significant at P < 0.05. (C) Confocal scanning microscopic images demonstrate p53 expression in STP-treated spinal neurons. (D) Immunocytochemistry of phospho-p53 in STP-treated spinal neurons. DAPI was used for nuclear staining. Bar = 200µm. (TIFF 7831 kb)

11064_2011_530_MOESM2_ESM.tif

Supplementary Figure 2. Mitochondrial Bax-mediated apoptosis in injured spinal neurons. (A) Fluorescent microscopic images demonstrate p53 expression with Bax co-localization in STP-treated spinal neurons. Bax is conjugated with Alexa Fluor 488 secondary antibody and p53 is conjugated with Alexa Fluor 594 secondary antibodies. (B) Bax translocation into the mitochondria. Bax expression showed by green fluorescence and HSP60 used to recognize the mitochondrial location. Bax is conjugated with Alexa Fluor 488 secondary antibody and HSP60 is conjugated with Alexa Fluor 594 secondary antibodies. Bar = 200µm. n=3. (TIFF 6926 kb)

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Kotipatruni, R.R., Dasari, V.R., Veeravalli, K.K. et al. p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord. Neurochem Res 36, 2063–2074 (2011). https://doi.org/10.1007/s11064-011-0530-2

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