Abstract
BackgroundCalreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This ‘ecto-CRT’ may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Demyelination was induced in the spinal cord by intradermal injection of recombinant mouse MOG mixed with incomplete Freund’s adjuvant (IFA) at the base of the tail. Tissue samples were analysed by semi-quantitative scoring of immunohistochemically stained frozen tissue sections. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. CRT present in rat serum and in a cohort of human serum derived from 14 multiple sclerosis patients and 11 healthy controls was measured by ELISA.ResultsStained tissue scores revealed significantly (p<0.05) increased amounts of CRT, CHOP and p-eIF2 alpha in the lesion, lesion edge and normal-appearing white matter when compared to controls. CHOP and p-eIF2 alpha were also significantly raised in regions of grey matter and the central canal (p<0.05). Immunofluorescent dual-label staining confirmed expression of these markers in astrocytes, microglia or neurons. Dual staining of rat and human spinal cord lesions with Oil Red O and CRT antibody showed co-localisation of CRT with the rim of myelin fragments. ELISA testing of sera from control and EAE rats demonstrated significant down-regulation (p<0.05) of CRT in the serum of EAE animals, compared to saline and IFA controls. This contrasted with significantly increased amounts of CRT detected in the sera of MS patients (p<0.05), compared to controls.ConclusionThis data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.
Highlights
Calreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER)
CRT achieved a semi-quantitative score of 4.3 ± 0.4 arbitrary units (AU), in lesion centres, against scores of 0.7 AU (± 0.1-0.2, p
The chief outcomes from this study have been the demonstration for the first time that: (i) following MOG-induced spinal cord demyelination in the Dark Agouti (DA) rat, CRT, C/EBP homologous binding protein (CHOP) and p-EIF2α were present at significantly increased levels within spinal cord lesions; (ii) significantly increased amounts of CRT, CHOP, X-box binding protein 1 (XBP1) and Phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) were detectable in the region of the central canal of diseased animals; (iii) levels of circulating CRT were significantly lower in EAE rat sera, when compared to control samples; (iv) CRT in human blood sera was present at significantly higher levels in individuals with multiple sclerosis (MS), than in healthy controls
Summary
Calreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This ‘ecto-CRT’ may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. On the other hand, increased expression of transcription factor CHOP is widely considered to indicate that cells may have activated a pro-apoptotic response following the failure to restore normal ER function [6]
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