The role of mutations in Connexin47 (Cx47) in myelinatingcells of the central nervous system (CNS).

Abstract

Cx47 forms homotypic gap junction communication channelsbetween oligodendrocytes (OLs), the myelinating cells of the central nervoussystem (CNS), as well as heterotypic channels with Cx43 expressed in astrocytes(As). Pelizaeus-Merzbacher-like disease type 1 (PMLD1) arises in patients withmutations in GJC2, encoding Cx47; PMLD1 causes nystagmus, cerebral ataxia,spasticity and changes in CNS white matter. One mutation (p.Ile33Met) has beenassociated with a much milder phenotype, spastic paraplegia type 44 (SPG44).Prior studies in cell lines have shown that PMLD1 mutants show defective proteintrafficking, intracellular retention in the ER and loss-of-function, without evidenceof activation of the unfolded protein response. We hypothesize that in primary mouseOLs, PMLD1 mutations cause activation of the cellular stress response compared tothe milder SPG44 associated mutation. To study this, pure OL cultures were lentivirallytransduced to express Cx47WT and mutants. Immunofluorescence (IF) studiesshowed that PMLD1 associated mutants exhibit a diffuse cytoplasmic staining thatcolocalize with the staining of the ER resident chaperone Grp94; on the otherhand of Cx47WT and Cx47I33M both show a punctate staining. In addition, cellsexpressing PMLD1 associated mutations showed increased staining for C/EBPhomologous protein (CHOP) -a component of the UPR mediated apoptosis pathway-compared to Cx47WT and Cx47I33M, as well as an increased activation ofCaspase-3, a protein that is activated during apoptosis and changes in cellularmorphology suggestive of apoptosis. These results indicate that in primary OLsexpression of PMLD1 mutants, but not SPG44, lead to ER protein retention,activation of the UPR pathway and apoptosis, providing insight into themechanism of PMLD1.