Peripheral neuropathies caused by mutations in the myelin protein zero

Abstract

Charcot-Marie-Tooth disease type 1B (CMT1B) is caused by mutations in the major PNS myelin protein myelin protein zero (MPZ). MPZ is a member of the immunoglobulin supergene family and functions as an adhesion molecule helping to mediate compaction of PNS myelin. Mutations in MPZ appear to either disrupt myelination during development, leading to severe early onset neuropathies, or to disrupt axo–glial interactions leading to late onset neuropathies in adulthood. Identifying molecular pathways involved in early and late onset CMT1B will be crucial to understand how MPZ mutations cause CMT1B so that rational therapies for both early and late onset neuropathies can be developed.

Introduction

In 1962, Bird et al. evaluated a 2.5-year-old girl and her two brothers, who presented with foot drop, distal leg weakness and a loss of deep tendon reflexes. Motor nerve conduction velocities (NCV) were 5 to 15 m/s. The original handwritten pedigree led to one of the first linkage studies in neurological disease. By 1982, these studies had revealed linkage to the Duffy (Fy) blood group gene localized on chromosome 1 [1]. In 1993, Hayasaka and colleagues localized the gene encoding the major peripheral nervous system (PNS) myelin protein P0 (MPZ) to a region on chromosome 1 near the Duffy locus, making MPZ a candidate gene for hereditary motor sensory neuropathy (HMSN) IB. Subsequent research identified point mutations in MPZ in families with linkage to Duffy, and MPZ mutations were confirmed to be the cause of the inherited demyelinating neuropathies collectively known as Charcot-Marie-Tooth disease type 1B (CMT1B) [2].

Many of the early reported cases with MPZ mutations had NCVs between 10 and 15 m/s, similar to those reported in the initial CMT1B family. Thus, it was hypothesized that CMT1B patients could be distinguished from patients with CMT1A by nerve conduction testing, because the more common CMT1A patients typically have faster NCV in the range of 20–25 m/s [3], [4]. However, this hypothesis has not proven to be correct. Presently, more than 95 distinct MPZ mutations have been shown to cause neuropathy (see http://www.molgen.ua.ac.be/CMTMutations) and many of the affected patients have widely disparate nerve conductions, as well as widely different clinical phenotypes. Some patients have even had normal or near normal NCV with reduced motor and sensory evoked amplitudes, leading to a clinical diagnosis of CMT2 despite the fact that their neuropathy is caused by a mutation in a myelin-specific gene [5]. At present, a fundamental question is why some mutations cause severe neuropathies and other mutations cause milder or later onset symptoms.

MPZ is the major myelin protein expressed by Schwann cells, comprising approximately 50% of all PNS myelin proteins [6], [7] and is necessary for both normal myelin function and structure [8]. The gene encoding MPZ in rats, mice [9] and humans [10] is divided into six exons, distributed over 7 kb of DNA [9], is located on 1q22–q23 of chromosome 1 in humans [10], and is expressed exclusively by myelinating Schwann cells. Thus, MPZ is not found in other tissues, including CNS myelin [11]. The six exons encode a protein of 248 amino acids; the 29 amino acid signal peptide encoded by exon 1 is cleaved prior to the insertion of the final 219 amino acid protein into the myelin sheath [9]. Because the cleaved amino acids are not present in the myelin sheath, they are not included in the subsequent numbering of amino acids in this review. The mature protein has an extracellular domain (amino acids 1 through 124), a single transmembrane domain (amino acids 125 through 150) and a cytoplasmic domain (amino acids 151 through 219) [9], [12], [13]. The amino acid sequence of MPZ is more than 95% identical in human, rat and cow [10].

MPZ is expressed in myelinating Schwann cells as part of the coordinated program of myelin gene expression. As with other myelin-specific genes, MPZ expression is induced by as yet unidentified axonal signals [14]. When axons have established a one-to-one relationship with developing Schwann cells, the Schwann cells increase messenger RNA (mRNA) expression of a series of genes including MPZ, PMP22, myelin basic protein (MBP) and myelin associated glycoprotein (MAG), as well as the mRNAs encoding the cholesterol biosynthetic enzymes, hydroxymethylglutaryl (HMG) Co-A reductase and oleoyl-coenzyme A synthase so that sufficient amounts of both myelin structural proteins and membrane constituents are available during myelin synthesis [15], [16]. Once myelination has been completed, its maintenance also depends on continued Schwann cell–axonal interactions. If a peripheral nerve is cut, severing the axon and its Schwann cells below the cut from the neuronal cell body, axons degenerate and demyelination occurs, initiating the process of Wallerian degeneration. During Wallerian degeneration, myelinating Schwann cells change their pattern of gene expression, turning off expression of MPZ and the other myelin-specific genes, and turning on a series of genes, such as the low affinity nerve growth factor receptor (NGFR), previously expressed prior to myelination in immature Schwann cells. If the nerve is crushed, however, allowing regeneration to occur after Wallerian degeneration, Schwann cell differentiation and myelination are restored as the axons regenerate through the crushed segment, recontacting denervated Schwann cells (reviewed by Kamholz et al. [17]).

The coordinate program of myelination also involves proper localization of molecules in the developing myelin sheath. As shown in the cartoon of a myelinated axon and its node of Ranvier in Fig. 1, the mature myelin sheath has two regions, compact and noncompact, each of which contains a unique non-overlapping set of protein constituents. MPZ is normally targeted to the compact region along with PMP22 and MBP, which participate in forming the highly organized myelin sheath and in electrically insulating axons. The noncompact region, which does not contain MPZ, is composed of two subdomains, the paranode and the juxtaparanode.

MPZ is a member of the immunoglobulin supergene family with a region of the extracellular domain similar in structure to a single immunoglobulin variable region domain [15]. In vitro studies have shown that MPZ can act as a homophilic adhesion molecule [18], as is the case with other members of the immunoglobulin supergene family. Supporting this hypothesis, crystallographic analysis of the MPZ extracellular domain demonstrates that it interacts in cis to form homotetramers, which then interact in trans with tetramers of MPZ extracellular domains from the opposing myelin wrap to form the intraperiod line of compact myelin [19]. Cis interactions occur by the formation of a doughnut structure by four MPZ extracellular domains around a large central hole. The “four-fold interface” (FFI) where these domains meet requires direct interaction between nine “critical” amino acids. Trans interactions, between opposing MPZ molecules occur at two additional interfaces of the extracellular domain, termed putative “adhesive interfaces” (AIs) and “head to head interfaces” (HHIs). AIs and HHIs require specific interactions between 10 and 7 critical amino acids, respectively. Taken together, these data thus suggest that MPZ plays an essential adhesive role in myelination by holding together adjacent wraps of myelin membrane through homotypic interactions of the extracellular domain.

The highly basic intracellular domain of P0 is also necessary for mediating adhesion by the extracellular domain of the molecule. Mutations in this region have been shown to interfere with homotypic adhesion in vitro [20] and can cause particularly severe forms of demyelinating peripheral neuropathy in patients (see below) [21], [22]. Moreover, co-expression of both wild-type and cytoplasmically truncated MPZ causes a loss of in vitro adhesion, presumably as a result of a dominant-negative effect of the truncated protein [23]. Deletion of a 14 amino acid sequence in the cytoplasmic domain of MPZ also abolished adhesion in in vitro assays [24]. Point mutations in this sequence that alters a PKCα substrate motif (RSTK, between amino acids 198 and 201) and mutation of an adjacent serine residue (204) also abolish adhesion in this system. Furthermore, PKCα and the activated C kinase receptor (RACK1) are coimmunoprecipitated from cells expressing wild type MPZ but not MPZ bearing a deletion of the 14 amino acids between residues 192 and 206. Taken together, these results indicate that PKC-mediated phosphorylation is an important component of the regulation of MPZ-mediated adhesion. An affected patient with a mutation of the initial arginine of the PKC substrate domain (R198S) has also been identified suggesting that disrupting adhesion is associated with demyelination [24].

The cytoplasmic domain of MPZ also plays an important role in mediating compaction of the Schwann cell cytoplasm and the formation of the major dense line in PNS myelin. This role appears to require an interaction between MPZ and MBP, because mice doubly deficient in the genes for MPZ and MBP lack major dense lines [25], while some Schwann cells form major dense lines in MPZ knockout mice [8]. Moreover, the PNS is normal in shiverer mutant mice that cannot express MBP [11]. How MPZ affects the compaction of the cytoplasmic domain is not known. Early speculation hypothesized that basic residues in the cytoplasmic domain of MPZ might interact with phospholipids of adjacent cytoplasmic aspects of Schwann cell membranes [11], [26].