Direct relationship between increased expression and mistrafficking of the Charcot–Marie–Tooth–associated protein PMP22

Justin T Marinko,Bruce D Carter,Charles R Sanders

doi:10.1074/jbc.ac120.014940

Abstract

Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ∼1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third WT PMP22 allele. In rodent models overexpressing the PMP22 (peripheral myelin protein 22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum quality control system, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of WT or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells and PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mistrafficking of PMP22, which is likely a contributor to disease pathology and progression.

Highlights

Charcot–Marie–Tooth disease (CMT) is an eponym for a large range of related neuropathies that occur with a prevalence of ;1:2500 in the human population [1, 2]
In three biological replicates, interrogating 2500 individual HEK293 cells per experiment, we found that WT peripheral myelin protein 22 (PMP22) traffics to the plasma membrane (PM) with a mean trafficking efficiency of 0.27 6 0.01
Previous studies have shown that PMP22 trafficking efficiency is related to the stability of its folded structure

Summary

Introduction

Charcot–Marie–Tooth disease (CMT) is an eponym for a large range of related neuropathies that occur with a prevalence of ;1:2500 in the human population [1, 2]. Patients afflicted with CMT1A present slowed nerve conduction velocity and axonal loss, accompanied by a shortening of internodal length [1] Beyond these morphological changes in compact myelin, overexpression of PMP22 has been shown to cause Schwann cell apoptosis [21, 22]. One hypothesis for CMT1A pathology is that under normal conditions, expression of PMP22 occurs at levels that approach saturation of the ER protein folding quality control system, such that introduction of a third copy of PMP22 overwhelms the system, leading to ER stress and accumulation of cytotoxic aggregates This is supported by the fact that stimulation of autophagy leads to increased degradation of such. We quantitatively examine the question of whether increased expression of PMP22 in model cell lines results in increased formation of intracellularly trapped protein and a decrease in PM trafficking efficiency

Methods

Results

Discussion

Conclusion