To express wild-type MPZ and mutant MPZ genes, the RT4 cells (2105) seeded on 6-well culture plates were transfected with MPZ-containing vectors [pCMV-Myc-MPZ WT/V169fs/R98C and pEGFP(C1)-MPZ WT/V169fs], as well as their control vectors [pCMV-Myc and pEGFP(C1)] using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions

To express wild-type MPZ and mutant MPZ genes, the RT4 cells (2105) seeded on 6-well culture plates were transfected with MPZ-containing vectors [pCMV-Myc-MPZ WT/V169fs/R98C and pEGFP(C1)-MPZ WT/V169fs], as well as their control vectors [pCMV-Myc and pEGFP(C1)] using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. efficacy of 3 different aminosalicylic acids (ASAs; 4-ASA, sodium 4-ASA and 5-ASA) in alleviating pathological phenotypes. FACS analysis indicated that the number of apoptotic rat SCs, RT4 cells, induced by mutant MPZ overexpression was significantly reduced following treatment with each ASA. In particular, treatment with 4-ASA reduced the levels of ER stress markers in RT4 cells induced by V169fs MPZ mutant overexpression and relieved the retention of V169fs mutant proteins in the ER. Additionally, the level of an apoptotic signal mediator (p-JNK) was only decreased in the RT4 cells expressing R98C MPZ mutant protein following treatment with 4-ASA. Although 4-ASA is known as a free radical scavenger, treatment with 4-ASA in the model did not moderate the level of reactive oxygen species, which was elevated by the expression of mutant MPZ proteins. On the whole, the findings of this study indicate that treatment with 4-ASA reduced the ER stress and SC death caused by 2 different MPZ mutants and suggest that ASA may be a potential therapeutic agent for CMT. models expressing mutant MPZ protein that caused ER stress and Schwann cell death and investigated whether the 3 ASAs can alleviate these pathological effects. Materials and methods Cell culture and transfection Rat Schwann cells, RT4 cells (RT4-D6P2T, CRL-2768, ATCC), were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Biowest) containing 10% fetal bovine serum and 1% penicillin-streptomycin (Biowest) at 37C in a 5% CO2 atmosphere. The MPZ gene was amplified from the pCMV6-entry-MPZ vector (Origene). The amplified PCR product was TRAILR-1 cloned into the pCMV-Myc or p-EGFP(C1) vector (Clontech). Mutant genes (V169fs, L184fs, R185fs, S226fs and R98C) were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). To express wild-type MPZ and mutant MPZ genes, the RT4 cells (2105) seeded on 6-well culture plates were transfected with MPZ-containing vectors [pCMV-Myc-MPZ WT/V169fs/R98C and pEGFP(C1)-MPZ WT/V169fs], as well as their control vectors [pCMV-Myc and pEGFP(C1)] using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Based on western blot analysis and immunocytochemistry, the transcription efficiency was >90%. The Mcl1-IN-12 transfected cells were incubated at 37C for 48 h. The RT4 cells (2105) were transfected with the MPZ expression vectors and treated with the drugs (1-100 and (10,21). In particular, the R98C mutant has been reported to activate the IRE1 pathway, leading to apoptosis (21). The V169fs mutant has also been shown to induce ER stress and cell death by being retained in the ER compartments of non-Schwann cells (HeLa or 293 cell lines) (10). To validate the induction of Schwann cell death or ER stress by MPZ mutant proteins, we generated wild-type (WT) and 5 mutant MPZ (V169fs, L184fs, R185fs, S226fs or R98C) expression vectors by site-directed mutagenesis. From western blot analysis and immunocytochemistry, we confirmed the effective expression of MPZ proteins by the transient transfection of WT and mutant MPZ vectors into the RT4 cells with a >90% transfection efficiency (Figs. 1, Mcl1-IN-12 ?,22 and Mcl1-IN-12 S1). In addition, we observed that the levels of ER stress markers, such as BiP and CHOP were altered by either MPZ-V169fs or MPZ-R98C Mcl1-IN-12 overexpression. The CHOP expression levels were elevated by the overexpression of MPZ-V169fs and MPZ-R98C mutants, while the BiP level was elevated only by the overexpression of MPZ-V169fs mutant (Fig. S1). Thus we proceeded with further experiments using only the MPZ-V169fs and MPZ-R98C mutant. Open in a separate window Figure 1 Schwann cell death is induced by MPZ mutant overexpression. (A) Following the overexpression of mutant MPZ proteins (V169fs and R98C) in RT4 cells for 48 h, the number of live rat Schwann cells was reduced (n=3; *P<0.05; ***P<0.001). (B) FACS analysis showing the quantities of live cells, apoptotic cells (Annexin V+ cells), and late-stage apoptotic/necrotic cells (Annexin V+ and PI+ cells) by Annexin V and PI staining (n=3; ***P<0.001). (C) Western blot analysis indicating the levels of cleaved and total caspase-3 following the overexpression of MPZ mutant. Quantification of the western blot analysis data indicating an increase in the level of cleaved caspase-3 following the overexpression of MPZ mutant (n=3; *P<0.05; ***P<0.001). MPZ, myelin protein zero. Open in a separate window Figure 2 ER stress in Schwann cells is induced by MPZ mutant overexpression. (A) Western blot analysis demonstrating changes in the levels of ER stress markers (BiP and CHOP) following transfection with wild-type, V169fs and R98C mutants of MPZ in RT4 cells. Quantitative analyses of the western blot analysis data were performed for BiP and CHOP normalized to ?-actin (n=3; *P<0.05). (B) Immunocytochemistry Mcl1-IN-12 for overexpressed wild-type MPZ and the mutants (Myc), and an ER marker (PDI) indicating the localization of MPZ proteins in relation to the ER in RT4 cells (n=3; scale bar, 10 or (7,10,21-23). In this study, to determine whether Schwann.