National Centre for Biomedical Engineering Science, Galway Neuroscience Centre, National University of Ireland, Galway, Ireland.
Acta Neuropathol Commun. 2013 Jul 15;1:37. doi: 10.1186/2051-5960-1-37.
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. 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.
Stained 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.
This data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.
钙网织蛋白（CRT）是一种伴侣蛋白，有助于内质网（ER）中糖基化蛋白的正确折叠。在 ER 应激条件下，CRT 上调，并可能在细胞表面表达或分泌。这种“外显 CRT”可能激活先天免疫反应，也可能有助于清除凋亡细胞。我们和其他研究已经证明，在活检和死后的人类多发性硬化症（MS）样本中，内质网应激标志物 CHOP、BiP、ATF4、XBP1 和磷酸化 eIF2α（p-eIF2α）的上调。我们通过分析炎症性脱髓鞘大鼠模型中 CRT、BiP、CHOP、XBP1 和 p-eIF2α 的表达变化来扩展这项工作。通过尾根部皮内注射重组小鼠 MOG 与不完全弗氏佐剂（IFA）混合，在脊髓中诱导脱髓鞘。通过对冷冻组织切片进行免疫组织化学染色的半定量评分分析组织样本。从脊髓病变动物取样后生成的数据与使用来自 IFA 或盐水注射对照的组织获得的数据进行了比较。通过 ELISA 测量来自 14 名多发性硬化症患者和 11 名健康对照者的血清中的 CRT 含量。
染色组织评分显示，与对照组相比，在病变、病变边缘和正常外观的白质中 CRT、CHOP 和 p-eIF2α 的含量显著增加（p<0.05）。在灰质和中央管区域，CHOP 和 p-eIF2α 也显著升高（p<0.05）。免疫荧光双重标记染色证实这些标志物在星形胶质细胞、小胶质细胞或神经元中表达。用油红 O 和 CRT 抗体对大鼠和人脊髓病变进行双重染色显示 CRT 与髓磷脂碎片的边缘共定位。ELISA 检测 EAE 大鼠和对照大鼠的血清表明，与盐水和 IFA 对照相比，EAE 动物血清中的 CRT 显著下调（p<0.05）。相比之下，与对照组相比，MS 患者血清中检测到的 CRT 量显著增加（p<0.05）。
该数据强调了 CRT 和其他内质网应激蛋白在炎症性脱髓鞘中的潜在重要性。
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