Elevated Peripheral Myelin Protein 22, Reduced Mitotic Potential, and Proteasome Impairment in Dermal Fibroblasts from Charcot-Marie-Tooth Disease Type 1A Patients

Abstract

A common form of hereditary autosomal dominant demyelinating neuropathy known as Charcot-Marie-Tooth disease type 1A (CMT1A) is linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Although studies from animal models have led to better understanding of the pathobiology of these neuropathies, there continues to be a gap in the translation of findings from rodents to humans. Because PMP22 was originally identified in fibroblasts as growth arrest specific gene 3 (gas3) and is expressed broadly in the body, it was tested whether skin cells from neuropathic patients would display the cellular pathology observed in Schwann cells from rodent models. Dermal fibroblasts from two CMT1A pedigrees with confirmed PMP22 gene duplication were studied. Samples from age-matched non-neuropathic individuals were used as controls. CMT1A patient-derived cultures contain approximately 1.5-fold elevated levels of PMP22 mRNA, exhibit reduced mitotic potential, and display intracellular protein aggregates as compared to cells from unaffected individuals. The presence of cytosolic PMP22 coincides with a decrease in proteasome activity and an increase in autophagy-lysosomal proteins, including LC3-II and LAMP1. These results indicate that the abnormalities in the subcellular processing of excess PMP22 elicit a detectable response in human CMT1A fibroblasts, a phenotype that resembles Schwann cells from neuropathic mice. These findings support the use of human CMT1A fibroblasts as a platform for therapy testing.

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

“…WT PMP22 is overexpressed in the most common form of CMT disease, CMT1A. This has led to interest in the possibility that misfolded, nondegraded, PMP22 is a source of cytotoxicity contributing to disease etiology (13,23,24,30,31). Here, we examined expression of PMP22 in both transiently transfected model cell lines and in stable cells.…”

“…Indeed, when PMP22 is reconstituted into lipid bilayers, it is sufficient to induce wrapping of the membranes to produce myelin-like assemblies (12). However, PMP22 has also been implicated in a number of other processes within myelin-producing Schwann cells (1,2,(13)(14)(15)(16). In vitro studies revealed that PMP22 is only modestly stable in detergent micelles, with the native conformation favored over the denatured ensemble by only 1.5 6 0.1 kcal mol 21 (17,18).…”

“…Beyond these morphological changes in compact myelin, overexpression of PMP22 has also been shown to cause Schwann cell apoptosis (21,22). Mouse models with multiple copies of WT PMP22 or expressing disease mutant forms of PMP22, as well as CMT1A patientderived dermal fibroblasts, display the presence of cytosolic PMP22 aggregates coinciding with a decrease in proteasomal activity (1,8,13,23,24). Under normal conditions, PMP22 that is not able to mature beyond the endoplasmic reticulum (ER) is removed from the membrane by ER quality control and degraded in the cytosol via proteosomal and/or lysosomal pathways (23).…”

Subcellular diversion of cholesterol by gain‐ and loss‐of‐function mutations in PMP22

“…While mostly known for its linkage to neuropathies, PMP22 is expressed in a variety of cell types, including fibroblasts (Amici et al, 2006; Grossi et al, 1998; Lee et al, 2018; Notterpek et al, 2001). In dermal fibroblasts from CMT1A patients with PMP22 gene duplication, the overproduced PMP22 forms aggregates and activates the autophagy–lysosomal pathway, similarly to Schwann cells from neuropathic mice (Fortun et al, 2006; Lee et al, 2018). Here, we also discovered altered lipid metabolism in CMT1A fibroblasts, as indicated by downregulation of genes linked to sterol synthesis (Figure 2e).…”

“…Human skin fibroblasts from four CMT1A patients (GM05148 [P1], GM05167 [P2], GM05146 [P3], and GM05165 [P4]) were purchased from the Coriell Institute (Camden, New Jersey). Skin fibroblasts from nonneuropathic individuals were obtained under an IRB‐approved protocol by Dr Guang‐Bin Xia (Department of Neurology, University of Florida) and were provided to us for the described studies (Lee et al, 2018). All procedures involving the human cells were carried out in compliance with IRB approval.…”