Characterising PMP22 proximal partners in a Schwann cell model of Charcot-Marie-Tooth disease type1A

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary condition caused by duplication of the PMP22 gene. Overexpression of peripheral myelin protein 22 in Schwann cells leads to myelin sheath defects and axonal loss. We have produced a cell model to facilitate studies of the molecular mechanisms involved in PMP22 accumulation and clearance. Our model is a stably transfected, clonal, immortalized human Schwann cell line with over-expressed levels of PMP22 fusion protein. A control-transfected cell line (vector lacking PMP22) was also produced. PMP22-transfected cells had reduced levels of mitosis, with the PMP22 fusion protein concentrated in punctate aggregates in the cytoplasm and expressed at the plasma membranes, which were often irregular and spindly. In contrast, control cells (control-transfected and parent cell lines) generally had smooth and regular plasma membrane morphology. Culturing in the presence of NRG1 and forskolin lead to upregulation of markers of myelination potential in the control cells. These markers were more variable in the cells stably transfected with PMP22, including decreased levels of transcripts of SOX10, S100B and NGFR, but increased levels of MPZ andEGR2 compared to controls. Using proximity-dependent biotin identification (BioID2), several hundred proteins were identified in the proximity of the overexpressed PMP22, of which, 291 significant proteins were only detected in the PMP22 and not in control pull-downs. Among the most significantly enriched PMP22-interacting proteins were integrins alpha-2 (ITGA2) and alpha-7 (ITGA7), which play a role in myelination via their interactions with the extracellular matrix. The presence of ITGA2 in just the PMP22-transfected fraction was confirmed by western blot. Some of the proteins were associated with several enriched molecular pathways including molecular transport and protein trafficking and may represent potential therapeutic targets for CMT1A by promoting degradation and enhanced trafficking of PMP22.