PHENOTYPE OF PMP22 TRANSGENIC SCHWANN CELLS IN CULTURE

Abstract

Charcot‐Marie‐Tooth type 1A (CMT1A) neuropathy is due to a duplication of the peripheral myelin protein 22 (PMP22) gene. In the sciatic nerves of PMP22 transgenic (PMP22tg) animals, Schwann cells (SC) switch on the myelinating program by producing detectable levels of myelin related proteins (PMP22, P0, MBP), but express also early maturational markers and antigens typical of non‐myelinating SC (p75NGFR).To further investigate PMP22tg SC behaviour, we established short‐term (five days) SC cultures from P30 normal and PMP22tg rats. Semiquantitative RT‐PCR was used to study PMP22, P0, MBP and p75NGFR mRNA expression, both in basal conditions and after forskolin (FSK) addition to mimic the axonal contact. SC proliferation rate was studied using a colorimetric assay (MTT based). Moreover, a gold particle motility assay was used to evaluate cell motility and shaping. Finally, preliminary experiments on SC migration in presence of IGFI were performed using a Boyden chamber.Homozygous and heterozygous PMP22tg SC, compared to normal ones, showed higher basal levels of PMP22 (0.82 vs 0.6 vs 0.09), P0 (2.24 vs 1.92 vs 0.53) and p75NGFR (1.33 vs 1.38 vs 0.34) mRNAs. FSK treatment (24 hrs) further increased PMP22 levels in homozygous (1.57 vs 0.82), heterozygous (0.93 vs 0.6) and normal (0.194 vs 0.09) cultures. PMP22tg SC showed a lower proliferation rate than normal ones. In basal conditions no differences were found between PMP22tg and normal SC in terms of cell area and motility, whereas FSK treatment determined, in PMP22tg SC, a significant decrease in cell area and motility. Finally, cell migration in the presence of a chemoattractant molecule did not differ between PMP22tg and normal SC.Cultured PMP22tg SC, besides maintaining the ability to overexpress PMP22, further increase its expression after FSK treatment. As PMP22tg SC in the presence of FSK also show a decreased motility and area, but migration seems to be identical between normal and transgenic cells, we conclude that axonal contact may increase PMP22 expression at a level that affects SC ability of forming myelin.