# Characterising PMP22-Proximal Partners in a Schwann Cell Model of Charcot–Marie–Tooth Disease Type1A

## TL;DR

This study creates a Schwann cell model to investigate proteins interacting with PMP22, a key factor in Charcot–Marie–Tooth disease type 1A, potentially aiding in understanding and treating the condition.

## Contribution

A novel Schwann cell model overexpressing PMP22 was developed to identify proximal protein partners and study disease mechanisms in CMT1A.

## Key findings

- A stable Schwann cell line overexpressing PMP22 was developed, showing irregular plasma membranes and reduced mitosis.
- Proximity-dependent biotin identification revealed 291 proteins uniquely associated with PMP22, including integrins ITGA2 and ITGA7.
- Altered gene expression and protein trafficking pathways suggest potential therapeutic targets for CMT1A.

## Abstract

Charcot–Marie–Tooth disease (CMT) encompasses a group of progressive and variable genetic diseases in which the peripheral nerves controlling movement and sensation in the legs and arms become damaged. Symptoms include muscle weakness and sensation loss in the feet and legs and sometimes in the hands, which can be detrimental to patient mobility. Schwann cells form insulating sheaths which protect nerve fibres that carry electrical impulses. The most common form of CMT is type 1A (CMT1A), caused by the overproduction of the PMP22 protein by Schwann cells, leading to nerve insulation loss and damage to peripheral nerves. Current models of CMT1A have limitations including high cost, variability and being time-consuming to perform. To address this, we developed a stable Schwann cell line which overexpresses the PMP22 protein. Using this cell model, we identified proteins which interact with, or are in very close proximity to, the overexpressed PMP22 protein. Some of these proteins are found on the surface of Schwann cells, which anchor the cells to the surrounding supportive material and are important for the maintenance of the insulating sheath. This cell model may complement existing models and be used to study the mechanisms of action of the disease and potentially to identify therapies for CMT1A.

Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary condition caused by the 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, immortalised human Schwann cell line with overexpressed 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, JUN, S100B and NGFR, but increased levels of MPZ and EGR2 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 proximity of PMP22 and not in that of 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 the degradation and enhanced trafficking of PMP22.