Endoplasmic Reticulum Stress as a Component of Demyelination in Animal Models of Multiple Sclerosis

Abstract

Endoplasmic reticulum (ER) stress is a homeostatic signalling pathway, linked to many neurological diseases including multiple sclerosis (MS), which can lead to cell death when prolonged. Increasing evidence suggests that Calreticulin (CRT), a multifunctional ER resident chaperone protein, plays a role in apoptotic cell clearance and it is implicated in autoimmunity. Studies in human tissue, in our lab, have shown ER stress-associated molecules at increased levels, in white and grey matter MS lesions. To complement this work we have set up EAE animal models of inflammatory white and grey matter lesions in female Dark Agouti (DA) rats and report here the profile of expression of ER stress signalling molecules in lesioned tissue. White matter demyelination was induced in rats by immunisation with 25-50 µg of recombinant myelin oligodendrocyte glycoprotein (rmMOG) emulsified in incomplete Freund's adjuvant (IFA) and control rats were injected with IFA or saline alone. EAE spinal cord white matter lesions were present in the dorsal, ventral and lateral funiculi. Cortical demyelinated lesions were produced by immunising DA rats with a sub-clinical dose of rmMOG (5 µg) to prime animals and then 21 days later proinflammatory TNFalpha and IFN gamma were stereotactically injected into the subarachnoid space. Semiquantitative analysis of immunostained tissue revealed CHOP (p<0.001), XBP1 (p<0.05), p-EIF2 alpha (p<0.001) and CRT (p<0.01) expression were significantly increased in EAE spinal cord lesions when compared to healthy control tissue. Interestingly, there was also a significant up-regulation of CHOP (p<0.05), XBP1 (p<0.01) and p-eIF2 alpha (p<0.05) in the epithelial cell lining of the central canal of diseased spinal cord tissue. These same stress markers (CHOP (p<0.05), XBP1 (p<0.05) and p-eIF2 alpha (p<0.01)) were also significantly up-regulated in the inner grey matter of the spinal cord. Analysis of spinal cord tissue was carried out by real time PCR and histological staining to determine expression profiles of ER-stress associated molecules BiP/Grp78, CHOP, CRT, XBP-1 and p-eIF2 alpha. Grp78 and CHOP transcripts showed a trend towards up-regulation whereas spliced XBP1 mRNA displayed a downward trend in EAE, compared to controls, but this was not statistically-significant. Preliminary results have shown the expression of CRT and CHOP in cortical grey matter lesions induced in the second EAE model. In spinal cord and cortical lesions, ER stress proteins were detected in a variety of cell types including oligodendrocytes, astrocytes, microglia and neurons. To address if an acute inflammatory response in the cortex had an impact on cognitive behaviour we used the novel object recognition test. However, there were no significant alterations on cognition. As part of a new biomarker project, ELISA was used to determine if secreted markers of ER stress (BiP, CRT or hepcidin) were detectable in spinal cord EAE and MS patient samples were analysed by ELISA. Overall, CRT was significantly decreased in the serum of EAE diseased animals compared to controls. This molecule showed a trend towards upregulation in serum samples from MS patients. There was no significant difference found for hepcidin in EAE samples. This data highlights the potential importance of ER stress in inflammatory demyelination.