Differential activation of ER stress pathways in myelinating cerebellar tracts

Abstract

Myelin production during brain development requires an increase in membrane protein and lipid production in oligodendrocytes and this primarily occurs in the endoplasmic reticulum (ER), an organelle which initiates the Unfolded Protein Response (UPR) when under stress. We hypothesise that the UPR is activated in white matter tracts during myelination in order to expand the ER capacity of oligodendrocytes. Using early and late stage markers, critical myelination time points were identified by immunohistochemistry in developing rat cerebellum. These were correlated to peaks in ER stress signalling by staining for activated UPR transducers (pIRE1, ATF6 and pPERK) and associated downstream molecules (peIF2α, PDI, GRP78, GRP94, CHOP and calreticulin) in cerebellar tracts III and IV. Gene expression in developing cerebellum was assessed by qPCR. Actively myelinating tracts were shown to have differential expression of pIRE1, PERK and ATF6 as well as UPR targets GRP94, GRP78 and PDI. Activated pIRE1-positive cells were widespread at P14 and P17 and at significantly higher numbers during myelination than at other stages. Nuclear-localised ATF6 (indicative of the active transcription factor) peaked at P10, concurrent with the initial phase of myelination. The percentage of cells positive for pPERK was less than 1% at postnatal ages but increased significantly in adult tissue. The downstream targets GRP78, GRP94 and PDI were significantly up-regulated at P17 compared to P7 and remained significantly elevated in adults. The majority of cells positive for these markers and ATF6 were oligodendrocytes as confirmed by dual-labelling. Although gene expression in the cerebellum for GRP78, GRP94 and PDI did not change significantly over time, ATF6 and XBP1s both showed significant fold changes between early and late timepoints. This data helps promote understanding of events occurring during developmental myelination and may have implications for the development of reparative treatments in diseases such as multiple sclerosis.

Keywords: ATF6; Endoplasmic reticulum stress; IRE1; Myelination; UPR.