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

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.

Fig. 1

SDS-PAGE analysis of IE and DM CSF. SDS-PAGE analysis of IE (n = 4) and DM CSF (n = 4) followed by Coomassie Blue staining revealed an additional protein band at approximately 38 kDa, which was consistently visible in DM CSF (as shown by black arrow), but present at a lower intensity in the IE cases. The comparatively low densities of staining in lane 5 may have been due to a loading error. Mkr pre-stained molecular weight marker, IE idiopathic epilepsy, DM degenerative myelopathy

Fig. 2

Haptoglobin levels in IE and DM CSF. a Western blot analysis of CSF haptoglobin levels in IE (n = 8) and DM (n = 5). Considerable signal intensity variations were detected between samples, and samples marked X were considered to be unquantifiable. Samples marked C and H were also excluded due to acute disease and heterozygosity for the Sod1 (118G > A) mutation. b Vertical scattered graph of data distribution. There was no statistically significant difference between groups. Data presented as median and interquartile range. std reference standard, IE idiopathic epilepsy, DM degenerative myelopathy. Filled upright triangle represents individuals with heterozygosity for Sod1 mutation in IE group

Fig. 3

Clusterin levels in IE and DM CSF. a Western blot analysis of CSF clusterin levels in IE (n = 9) and DM (n = 7). b Vertical scattered graph of data distribution. Statistical analysis revealed a significant elevation in clusterin between the IE and DM groups (p < 0.001). Samples marked C and H were excluded due to acute disease and heterozygosity for the Sod1 (118G > A) mutation. Sample marked L was collected from lumbar CSF and the protein value from this sample is represented as open square in the vertical scatter graph. Data presented as median and interquartile range. ***p < 0.001; std reference standard, IE idiopathic epilepsy, DM degenerative myelopathy. Filled upright triangle represents individuals with heterozygosity for Sod1 mutation in IE group

Fig. 4

Plasma clusterin levels in control (non-neurological disorders) and DM samples. a Western blot analysis of plasma clusterin levels in control (n = 8) and DM (n = 8) cases b Plasma clusterin signals were plotted in vertical scatter plot. Statistical analysis revealed no significant difference. Data presented as median and interquartile range. std reference standard, Ctrl control, DM degenerative myelopathy

Fig. 5

Analysis of clusterin mRNA levels and cellular distribution in control and DM spinal cords. a The relative signal of clusterin and cyclophilin RT-PCR amplicons observed on ethidium bromide stained agarose gels are shown in the top panels. The signals for clusterin mRNA were normalised relative to cyclophilin (cyclophilin:clusterin) and shown graphically. The statistical analysis revealed no significant difference between two groups (exact p value = 0.05); however, the mean of clusterin mRNA in the DM group (n = 4) was found to be elevated by 42 % compared to the control group (n = 4). Data presented as median and interquartile range. b Clusterin immunostaining in T12 spinal cord sections in a representative control and DM case demonstrated a dark, punctate staining pattern localised in the neuronal cytoplasm (as marked by arrow) but not in the nucleus as seen at ×60 magnification (see top right insert). The staining intensity of clusterin in neuronal cell bodies was assessed by a subjective scoring system, but no significant difference was evident between control (n = 4) and DM (n = 5) groups. Mkr molecular weight marker, Ctrl control, DM degenerative myelopathy

Fig. 6

The comparative analysis of clusterin CSF in various neurological disorders. a Clusterin signals obtained from Western blot analyses. b Signals were quantified and shown graphically. Statistical analysis found that clusterin was significantly elevated in DM (n = 4) and cIVDD (n = 4) compared to IE (n = 7) (DM vs. IE, p < 0.001; cIVDD vs. IE, p < 0.01) and meningitis (n = 8) (DM vs. meningitis, p < 0.05; cIVDD vs. meningitis, p > 0.05). There was no significant difference in CSF clusterin between DM and cIVDD. Samples marked X were excluded from the statistical analysis. The sample marked L was collected from the lumbar cistern and the protein value from this sample is represented as open square in the vertical scatter graph. Data presented as median and interquartile range. *p < 0.05, **p < 0.01, ***p < 0.001; std reference standard, IE idiopathic epilepsy, DM, degenerative myelopathy, MEN meningoencephalitis, cIVDD chronic intervertebral disc disease. Filled upright triangle represents individuals with heterozygosity for Sod1 mutation in control groups

Fig. 7

The potential underlying mechanisms leading to CSF clusterin elevation in DM. a A cartoon illustrating the blood, CSF and brain interfaces. CSF clusterin elevation may reflect changes in the blood clusterin levels. The protein may leave the blood vessels and enter the CSF pathways through the tight junctions between the ependymal cells of the choroid plexus. b Compartment model of CSF and spinal cord parenchyma interfaces. Increased clusterin mRNA expression with a concomitant increase of clusterin (CLU) distribution in DM motor neurons may lead to an elevation in CSF clusterin. The potential mechanism involves the movement of clusterin from motor neurons or potentially astrocytes into the subarachnoid space via the Virchow–Robin spaces. Clusterin is subsequently disseminated throughout the CSF pathway