Schwann cells ER-associated degradation contributes to myelin maintenance in adult nerves and limits demyelination in CMT1B mice

In the peripheral nervous system (PNS) myelinating Schwann cells synthesize large amounts of myelin protein zero (P0) glycoprotein, an abundant component of peripheral nerve myelin. In humans, mutations in P0 cause the demyelinating Charcot-Marie-Tooth 1B (CMT1B) neuropathy, one of the most diffused genetic disorders of the PNS. We previously showed that several mutations, such as the deletion of serine 63 (P0-S63del), result in misfolding and accumulation of P0 in the endoplasmic reticulum (ER), with activation of the unfolded protein response (UPR). In addition, we observed that S63del mouse nerves display the upregulation of many ER-associated degradation (ERAD) genes, suggesting a possible involvement of this pathway in the clearance of the mutant P0. In ERAD in fact, misfolded proteins are dislocated from the ER and targeted for proteasomal degradation. Taking advantage of inducible cells that express the ER retained P0, here we show that the P0-S63del glycoprotein is degraded via ERAD. Moreover, we provide strong evidence that the Schwann cell-specific ablation of the ERAD factor Derlin-2 in S63del nerves exacerbates both the myelin defects and the UPR in vivo, unveiling a protective role for ERAD in CMT1B neuropathy. We also found that lack of Derlin-2 affects adult myelin maintenance in normal nerves, without compromising their development, pinpointing ERAD as a previously unrecognized player in preserving Schwann cells homeostasis in adulthood. Finally, we provide evidence that treatment of S63del peripheral nerve cultures with N-Acetyl-D-Glucosamine (GlcNAc), known to enhance protein quality control pathways in C.elegans, ameliorates S63del nerve myelination ex vivo. Overall, our study suggests that potentiating adaptive ER quality control pathways might represent an appealing strategy to treat both conformational and age-related PNS disorders. Author summary Charcot-Marie-Tooth neuropathies are a large family of peripheral nerve disorders, showing extensive clinical and genetic heterogeneity. Although strong advances have been made in the identification of genes and mutations involved, effective therapies are still lacking. Intracellular retention of abnormal proteins has been recently suggested as one of the pathogenetic events that might underlie several conformational neuropathies. To limit the toxic effects of accumulated mutant proteins, cells have developed efficient protein quality control systems aimed at optimizing both protein folding and degradation. Here we show that ER-associated degradation limits Schwann cells stress and myelin defects caused by the accumulation of a mutant myelin protein into the ER. In addition, we also describe for the first time the importance of Schwann cells ERAD in preserving myelin integrity in adult nerves, showing that genetic ERAD impairment leads to a late onset, motor-predominant, peripheral neuropathy in vivo. Effort in the design of strategies that potentiate ERAD and ER quality controls is therefore highly desirable.

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

Hyaline Fibromatosis Syndrome (HFS) is a rare inherited disease that is characterized by an accumulation of an unidentified hyaline material, largely affecting connective tissues. Patients afflicted with HFS present a wide range of clinical symptoms such as nodules, papules, hyperplasia of the gingiva, joint contractions, thickness and hyperpigmentation of the skin, and osteopenia. Depending of the severity and the delay of appearance of these symptoms, the life expectancy varies between 2 years and > 30 years. Death earlier in life is due to recurrent diarrhea and chest infections of unknown origin. In 2003, the gene responsible for HFS was identified to be Capillary Morphoqenesis Gene 2 (CMG2), a gene upregulated during capillary morphogenesis in three-dimensional collagen matrices. This gene encodes a type I transmembrane glycoprotein composed of a von Willebrand (vWA) domain followed by an immunoglobulin (Ig)-like domain in its extracellular part, a single α-helix spanning transmembrane domain, and a 148 amino acids cytoplasmic tail predicted as unstructured. Even though the physiological function of CMG2 remains to be elucidated, it has been shown that this protein induces endothelial proliferation, and dictates cell polarization during embryogenesis in a zebrafish model. Besides its physiological function, CMG2 was found to be an anthrax toxin receptor, thus its alternative name ANTXR2, and has been extensively studied in this context. Genetic analysis has led to the identification of 34 CMG2 mutations in HFS patients. The molecular mechanisms underlying the disease have however not yet been identified. During the five years of my thesis, we were interested in understanding the cellular consequences of these mutations and in getting a better understanding of the physiological function of CMG2. I first showed that most of the HFS mutations in the vWA domain lead to alterations in folding of the domain, either in terms of kinetics or of structure, leading to recognition of the protein by the ER quality control (ERQC) and subsequent degradation by the ERAD pathway. A direct consequence of this retention is the loss of function at the cell surface or other cellular sites. In a second study, I studied the effect of novel HFS mutations that our collaborators and we identified in the CMG2 Ig-like domain. Since the structure of this domain was uncharacterized, we first generated a model of the domain and found that it has an Ig-like fold, despite the very low sequence homology, and harbors two disulfide bonds essential for folding and stabilization of the domain. HFS mutations localized to this domain led to aberrant intermolecular disulfide binding, ER retention and enhanced ERAD when compared to the vWA domain HFS mutations as shown in patients fibroblasts. The consequential loss of CMG2 could be partly alleviated by treating cells with proteasome inhibitor, such as Bortezomib, illustrating that ERAD components are potential therapeutic targets for HFS. As most of the HFS mutations in the ectodomain of CMG2 trigger this ER retention during the CMG2 biosynthesis, we then addressed this process by studying one of the most described post-translational modifications occurring in the ER for protein folding, namely the N-glycosylation. We found that the two potential CMG2 N-glycosylation sites, N250 and N260, are both occupied. Neither of the two sites is essential for ER exit. Glycosylation of CMG2 on N260 is however important since the expression levels of the N260A mutant were significant lower that for the WT protein, despite equivalent or higher synthesis, suggesting that glycosylation at this site is important for efficient folding. Finally, in order to better understand the CMG2 function, we investigated what the physiological ligands could be, assuming it is a receptor as suggested by its role in anthrax infection. We identified type VI collagen as a potential ligand in-vitro that activates CMG2 in-vivo, by phosphorylation of CMG2 cytoplasmic tail. This CMG2 activation upon type VI collagen binding could be a signal for ligand-triggered endocytosis, as is has been described with the protective antigen (PA) of anthrax toxin. The results of this work allow us to gain insights in the molecular mechanisms underlying HFS. Our studies indicate that the identification of the genotype-phenotype correlation of this disease is important for proper diagnosis and clinical approaches, and also that a therapeutic strategy based on proteasome inhibitors, such as Bortezomib, could be envisioned to treat some HFS symptoms in order to improve the life conditions of the patients.

EPFL2011

Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

Julie Deuquet Ariosa, Laurence Gouzi Abrami, Nicolas Jean Philippe Guex, Suzanne Salvi, Françoise Gisou van der Goot Grunberg

Hyaline Fibromatosis Syndrome (HIS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized lg-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the lg-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Wiley Open Access2011

Systemic hyalinosis mutations in the CMG2 ectodomain leading to loss of function through retention in the endoplasmic reticulum

Julie Deuquet Ariosa, Laurence Gouzi Abrami, Françoise Gisou van der Goot Grunberg

Systemic hyalinosis is an autosomal recessive disease that encompasses two allelic syndromes, infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF), which are caused by mutations in the CMG2 gene. Here we have analyzed the cellular consequences of five patient-derived point mutations in the extracellular von Willebrand domain or the transmembrane domain of the CMG2 protein. We found that four of the mutations led to retention of the protein in the endoplasmic reticulum (ER), albeit through different mechanisms. Analysis of recombinant CMG2 von Willebrand factor A (vWA) domains, to which three of the mutations map, indicated that the mutations did not prevent proper folding and ligand binding, suggesting that, in vivo, slow folding, rather than misfolding, is responsible for ER retention. Our work shows that systemic hyalinosis can be qualified as a conformational disease, at least for the mutations that have been mapped to the extracellular and transmembrane domains. The long ER half-life and the ligand binding ability of the mutated von Willebrand domains suggest that treatments based on chemical chaperones could be beneficial.

Wiley-Blackwell2009

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

EPFL2011