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Cell Stress and Chaperones

, Volume 19, Issue 3, pp 311–320 | Cite as

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

  • Intan N. F. Shafie
  • Mark McLaughlin
  • Richard Burchmore
  • Mary Ann A. Lim
  • Paul Montague
  • Pamela E. J. Johnston
  • Jacques Penderis
  • Thomas J. Anderson
Original Paper

Abstract

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD.

Keywords

Dog Spinal cord disease Clusterin Biomarkers Sod

Notes

Acknowledgments

This study was funded by the Ministry of Higher Education of Malaysia (MOHE), University Putra Malaysia and the British Small Animal Veterinary Association PetSavers. The authors are grateful to Professor David Eckersall for supplying haptoglobin antibody and also clinicians and staff of the neurology service in SAHGUVS for their help and cooperation with this study.

Declaration for conflict of interest

The authors of this manuscript do not have any financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the manuscript.

Supplementary material

12192_2013_457_Fig8_ESM.jpg (7 kb)
Supplementary 1

We have demonstrated that the RLFP technique could be used to differentiate three Sod1 genotypes; wild type (WT), heterozygous (het) and homozygous (homo). Mixtures of wild type and homozygous PCR products were generated to represent each of the genotypes: wild type (100% WT: 0% homozygous), heterozygous (50% WT: 50% homozygous), and homozygous (0% WT: 100% homozygous). Intermediate ratios were also included; 75% WT: 25% homozygous and 25% WT: 75% homozygous. The heterozygous genotype displayed two bands at equal intensities (236 bp and 204 bp) in a mixture containing 50% wild type and 50% homozygous. In 100% wild type mixture, a prominent band was observed at 204bp, suggesting DNA fragments are completely digested whereas in 100% homozygous sample, a single band was observed at 236bp, which is comparable with undigested (UD) PCR product. (JPEG 6 kb)

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High resolution image (TIFF 354 kb)
12192_2013_457_Fig9_ESM.jpg (9 kb)
Supplementary 2

Sod1 genotyping of canine spleen and blood-derived DNA. HpyAV digestion for 30 minutes in spleen and blood-derived DNA are depicted below. Partial digestion observed in control sample from spleen (C1 and C2), a single band (204 bp) consistent with wild type profile was detected in B1 blood sample, indicating a complete digestion had occurred with 100 ng of PCR product. The heterozygous profile was detected in B3 blood sample, demonstrating two bands at almost equal intensities (236bp and 204bp) and therefore showing a clear-cut differentiation between partially digested DNA products as observed in spleen-derived DNA. Two blood samples (B2 and B4) and a spleen sample demonstrated a homozygous profile, which represented a single band with size corresponding to undigested (UD) sample (236bp). (JPEG 8 kb)

12192_2013_457_MOESM2_ESM.tif (556 kb)
High resolution image (TIFF 555 kb)
12192_2013_457_MOESM3_ESM.docx (15 kb)
ESM 1 (DOCX 14 kb)

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Copyright information

© Cell Stress Society International 2013

Authors and Affiliations

  • Intan N. F. Shafie
    • 1
    • 4
  • Mark McLaughlin
    • 1
  • Richard Burchmore
    • 2
    • 3
  • Mary Ann A. Lim
    • 5
  • Paul Montague
    • 2
  • Pamela E. J. Johnston
    • 1
  • Jacques Penderis
    • 1
  • Thomas J. Anderson
    • 1
  1. 1.School of Veterinary Medicine, College of Medical, Veterinary and Life SciencesUniversity of GlasgowGlasgowUK
  2. 2.Institute of Infection, Immunity and InflammationUniversity of GlasgowGlasgowUK
  3. 3.Glasgow Polyomics, Wolfson Wohl Cancer Research CentreUniversity of GlasgowGlasgowUK
  4. 4.Faculty of Veterinary MedicineUniversity Putra MalaysiaSerdangMalaysia
  5. 5.School of Life Sciences and Chemical Technology, Ngee Ann PolytechnicSingaporeSingapore

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