A novel mouse model of CMT1B identifies hyperglycosylation as a new pathogenetic mechanism

Abstract

Mutations in the Myelin Protein Zero gene (MPZ), encoding P0, the major structural glycoprotein of peripheral nerve myelin, are the cause of Charcot–Marie-Tooth (CMT) type 1B neuropathy, and most P0 mutations appear to act through gain-of-function mechanisms. Here we investigated how misglycosylation, a pathomechanism encompassing several genetic disorders, may affect P0 function. Using in vitro assays, we showed that gain of glycosylation is more damaging for P0 trafficking and functionality as compared to loss of glycosylation. Hence, we generated, via CRISPR/Cas9, a mouse model carrying the MPZD61N mutation, predicted to generate a new N-glycosylation site in P0. In humans, MPZD61N causes a severe early onset form of CMT1B, suggesting that hyperglycosylation may interfere with myelin formation, leading to pathology. We show here that MPZD61N/+ mice develop a tremor as early as P15 which worsens with age and correlates with a significant motor impairment, reduced muscular strength and with substantial alterations in neurophysiology. Pathological analysis confirmed a dysmyelinating phenotype characterized by diffuse hypomyelination and focal hypermyelination. We find that the mutant P0D61N does not cause significant endoplasmic reticulum stress, a common pathomechanism in CMT1B, but is properly trafficked to myelin where it causes myelin uncompaction. Finally, we show that myelinating DRG cultures from MPZD61N mice replicate some of the abnormalities seen in vivo, suggesting that they may represent a valuable tool to investigate therapeutic approaches. Collectively our data indicate that the MPZD61N/+ mouse represents an authentic model of severe CMT1B affirming gain-of-glycosylation in P0 as a novel pathomechanism of disease.

Case report: A novel variant (H49N) in Myelin Protein Zero gene is responsible for a patient with Charcot–Marie–Tooth disease

“…Therefore, there may be a relationship between CMTDID disease and residue contact of these two positions. In addition, Veneri et al ( 21 ) found that the increase of glycosylation sites in MPZ can impair its function and lead to loosen myelin. Mutations in H49N produce an NCS sequence that belongs to the glycosylation motif (N-X-S/T), resulting in excessively glycosylation of MPZ.…”

A new mouse model of Charcot-Marie-Tooth 2J neuropathy replicates human axonopathy and suggest alteration in axo-glia communication

“…Moreover, in vitro experiments have shown that the T124M mutation does not interfere with P0 adhesiveness [ 67 ]. Interestingly, the P0T124A and P0N122S mutations, which similarly abolish native N -glycosylation, are responsible for late-onset CMT1B with only moderate reduction of NCV [ 7 , 68 , 69 ], and like P0T124M, these mutated P0 proteins retain their adhesive propriety in vitro [ 70 ]. This suggests that the P0 protein backbone is generally sufficient for P0 homophilic adhesion and for myelin compaction.…”

Charcot–Marie–Toothneuropathies: Current gene therapy advances and the route toward translation

“…Most Mpz protein mutants fail to reach the cell membrane or the myelin sheath and are instead accumulated in the ER, causing ER stress, stimulation of the unfolded protein response (UPR), hyperglycosylation, and apoptosis. [193][194][195][196][197][198][199][200] There are a great number of different Mpz mutations that cause a spectrum of phenotypes associated with CMT1B or CMT2 types. This variability raises the possibility that some Mpz mutations result in a gain of function while others result in a loss of function cellular effects; however, dominant-negative mechanisms are likely responsible in most cases.…”

“…Mpz is a major compact myelin transmembrane protein responsible for the proper compaction of myelin layers. Most Mpz protein mutants fail to reach the cell membrane or the myelin sheath and are instead accumulated in the ER, causing ER stress, stimulation of the unfolded protein response (UPR), hyperglycosylation, and apoptosis 193–200 . There are a great number of different Mpz mutations that cause a spectrum of phenotypes associated with CMT1B or CMT2 types.…”