CMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: a cross-sectional analysis.

BACKGROUND: The international Inherited Neuropathy Consortium (INC) was created with the goal of obtaining much needed natural history data for patients with Charcot-Marie-Tooth (CMT) disease. We analysed clinical and genetic data from patients in the INC to determine the distribution of CMT subtypes and the clinical impairment associated with them. METHODS: We analysed data from 1652 patients evaluated at 13 INC centres. The distribution of CMT subtypes and pathogenic genetic mutations were determined. The disease burden of all the mutations was assessed by the CMT Neuropathy Score (CMTNS) and CMT Examination Score (CMTES). RESULTS: 997 of the 1652 patients (60.4%) received a genetic diagnosis. The most common CMT subtypes were CMT1A/PMP22 duplication, CMT1X/GJB1 mutation, CMT2A/MFN2 mutation, CMT1B/MPZ mutation, and hereditary neuropathy with liability to pressure palsy/PMP22 deletion. These five subtypes of CMT accounted for 89.2% of all genetically confirmed mutations. Mean CMTNS for some but not all subtypes were similar to those previously reported. CONCLUSIONS: Our findings confirm that large numbers of patients with a representative variety of CMT subtypes have been enrolled and that the frequency of achieving a molecular diagnosis and distribution of the CMT subtypes reflects those previously reported. Measures of severity are similar, though not identical, to results from smaller series. This study confirms that it is possible to assess patients in a uniform way between international centres, which is critical for the planned natural history study and future clinical trials. These data will provide a representative baseline for longitudinal studies of CMT. CLINICAL TRIAL REGISTRATION: ID number NCT01193075.

CMT1X phenotypes represent loss of GJB1 gene function.

OBJECTIVE: To investigate possible genotype-phenotype correlations and to evaluate the natural history of patients with Charcot-Marie-Tooth disease type 1X (CMT1X). BACKGROUND: CMT1X is caused by over 260 distinct mutations in the gap junction beta 1 (GJB1) gene, located on the X chromosome, which encodes the gap junction protein connexin 32 (Cx32). The natural history of CMT1X is poorly understood, and it remains unknown whether particular mutations cause more severe neuropathies through abnormal gain-of-function mechanisms. METHODS: We evaluated 73 male patients with CMT1X, who each have 1 of 28 different GJB1 mutations predicted to affect nearly all domains of Cx32. Disability was evaluated quantitatively by the CMT Neuropathy Score (CMTNS) as well as by the CMT Symptom Score (CMTSS) and the CMT Examination Score (CMTES), which are both based on the CMTNS. Patients were also evaluated by neurophysiology. RESULTS: In all patients, disability increased with age, and the degree of disability was comparable with that observed in patients with a documented GJB1 deletion. Disability correlated with a loss of motor units as assessed by motor unit number estimates. CONCLUSIONS: Taken together, these data suggest that most GJB1 mutations cause neuropathy by a loss of normal connexin 32 function. Therefore, treatment of male patients with Charcot-Marie-Tooth disease type 1X may prove amenable to gene replacement strategies.

Mutation of a putative protein degradation gene LITAF/SIMPLE in Charcot-Marie-Tooth disease 1C.

BACKGROUND: Charcot-Marie-Tooth (CMT) neuropathy is a heterogeneous group of inherited disorders of the peripheral nervous system. The authors recently mapped an autosomal dominant demyelinating form of CMT type 1 (CMT1C) to chromosome 16p13.1-p12.3. OBJECTIVE: To find the gene mutations underlying CMT1C. METHODS: The authors used a combination of standard positional cloning and candidate gene approaches to identify the causal gene for CMT1C. Western blot analysis was used to determine relative protein levels in patient and control lymphocyte extracts. Northern blotting was used to characterize gene expression in 1) multiple tissues; 2) developing sciatic nerve; and 3) nerve-crush and nerve-transection experiments. RESULTS: The authors identified missense mutations (G112S, T115N, W116G) in the LITAFgene (lipopolysaccharide-induced tumor necrosis factor-alpha factor) in three CMT1C pedigrees. LITAF, which is also referred to as SIMPLE, is a widely expressed gene encoding a 161-amino acid protein that may play a role in protein degradation pathways. The mutations associated with CMT1C were found to cluster, defining a domain of the LITAF protein having a critical role in peripheral nerve function. Western blot analysis suggested that the T115N and W116G mutations do not alter the level of LITAF protein in peripheral blood lymphocytes. The LITAF transcript is expressed in sciatic nerve, but its level of expression is not altered during development or in response to nerve injury. This finding is in stark contrast to that seen for other known genes that cause CMT1. CONCLUSIONS: Mutations in LITAF may account for a significant proportion of CMT1 patients with previously unknown molecular diagnosis and may define a new mechanism of peripheral nerve perturbation leading to demyelinating neuropathy.

Differential cyclin D1 requirements of proliferating Schwann cells during development and after injury.

Neurons regulate Schwann cell proliferation, but little is known about the molecular basis of this interaction. We have examined the possibility that cyclin D1 is a key regulator of the cell cycle in Schwann cells. Myelinating Schwann cells express cyclin D1 in the perinuclear region, but after axons are severed, cyclin D1 is strongly upregulated in parallel with Schwann cell proliferation and translocates into Schwann cell nuclei. During development, cyclin D1 expression is confined to the perinuclear region of proliferating Schwann cells and the analysis of cyclin D1-null mice indicates that cyclin D1 is not required for this type of Schwann cell proliferation. As in the adult, injury to immature peripheral nerves leads to translocation of cyclin D1 to Schwann cell nuclei and injury-induced proliferation is impaired in both immature and mature cyclin D1-deficient Schwann cells. Thus, our data indicate that the molecular mechanisms regulating proliferation of Schwann cells during development or activated by axonal damage are fundamentally different.

Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.

Axon-Schwann cell interactions regulate the expression of fibroblast growth factor-5 (FGF-5).

We screened for genes whose expression is significantly up- or downregulated during Wallerian degeneration in adult rat sciatic nerve with cDNA arrays. Fibroblast growth factor-5 (FGF-5) mRNA seemed to be induced. This was confirmed by northern blotting and in situ hybridization, as well as Western blotting for FGF-5 in axotomized nerve. Axon-Schwann cell interactions decreased the steady-state level of FGF-5 mRNA in regenerating sciatic nerves, and forskolin diminished its expression in cultured Schwann cells. We conclude that denervated Schwann cells synthesize FGF-5, which is a secreted, neuronotrophic member of the FGF family.

Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling.

In peripheral nerves, Schwann cells (SCs) form contacts with axons, other SCs, and extracellular matrix components that are critical for their migration, differentiation, and response to injury. Here, we report that lysophosphatidic acid (LPA), an extracellular signaling phospholipid, regulates the morphology and adhesion of cultured SCs. Treatment with LPA induces f-actin rearrangements resulting in a "wreath"-like structure, with actin loops bundled peripherally by short orthogonal filaments. The latter appear to anchor the SC to a laminin substrate, because they colocalize with the focal adhesion proteins, paxillin and vinculin. SCs also respond to LPA treatment by forming extensive cell-cell junctions containing N-cadherin, resulting in cell clustering. Pharmacological blocking experiments indicate that LPA-induced actin rearrangements and focal adhesion assembly involve Rho pathway activation via a pertussis toxin-insensitive G-protein. The transcript encoding LP(A1), the canonical G-protein-coupled receptor for LPA, is upregulated after sciatic nerve transection, and SCs cultured from lp(A1)-null mice exhibit greatly diminished morphological responses to LPA. Cultured SCs can release an LPA-like factor implicating SCs as a potential source of endogenous, signaling LPA. These data, together with the previous demonstration of LPA-mediated SC survival, implicate endogenous receptor-mediated LPA signaling in the control of SC development and function.

Internodal specializations of myelinated axons in the central nervous system.

We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated beta subunit, Kvbeta2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvbeta2 and Caspr2 to the juxtaparanodal membrane. In addition to the paranodal staining, an internodal strand of Caspr staining apposes the inner mesaxon of the myelin sheath. Unlike myelinated axons in the peripheral nervous system, there was no internodal strand of Kv1.1, Kv1.2, Kvbeta2, or Caspr2. Thus, the organization of the nodal, paranodal, and juxtaparanodal axonal membrane is similar in the central and peripheral nervous systems, but the lack of Kv1.1/Kv1.2/Kvbeta2/Caspr2 internodal strands indicates that the oligodendrocyte myelin sheaths lack a trans molecular interaction with axons, an interaction that is present in Schwann cell myelin sheaths.

Alpha4 integrin is expressed during peripheral nerve regeneration and enhances neurite outgrowth.

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.

Molecular organization of the nodal region is not altered in spontaneously diabetic BB-Wistar rats.

We examined the organization of the molecular components of the nodal region in spontaneously diabetic BB-Wistar rats. Frozen sections and teased fibers from the sciatic nerves were immunostained for nodal (voltage-gated Na(+) channels, ankyrin(G), and ezrin), paranodal (contactin, Caspr, and neurofascin 155 kDa), and juxtaparanodal (Caspr2, the Shaker-type K(+) channels Kv1.1 and Kv1.2, and their associated subunit Kvbeta2) proteins. All of these proteins were properly localized in myelinated fibers from rats that had been diabetic for 15-44 days, compared to age-matched, nondiabetic animals. These results demonstrate that the axonal membrane is not reorganized, so nodal reorganization is not likely to be the cause of nerve conduction slowing in this animal model of acute diabetes.

Ezrin, radixin, and moesin are components of Schwann cell microvilli.

Ezrin, radixin, and moesin (ERM proteins), as well as the neurofibromatosis 2 (NF2) tumor suppressor merlin/schwannomin, all belong to the protein 4.1 family, yet only merlin is a tumor suppressor in Schwann cells. To gain insight into the possible functions of ERM proteins in Schwann cells, we examined their localization in peripheral nerve, because we have previously shown that merlin is found in paranodes and in Schmidt-Lanterman incisures. All three ERM proteins were highly expressed in the microvilli of myelinating Schwann cells that surround the nodal axolemma as well as in incisures and cytoplasmic puncta in the vicinity of the node. In all of these locations, ERM proteins were colocalized with actin filaments. In contrast, ERM proteins did not surround nodes in the CNS. The colocalization of ERM proteins with actin indicates that they have functions different from those of merlin in myelinating Schwann cells.

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin-treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine-1-phosphate receptor edg3/lp(B3), may play important roles in mediating Schwann cell differentiation.

A unique role for Fyn in CNS myelination.

We analyzed the role of Fyn tyrosine kinase in CNS myelination by using fyn(-/-) null mutant mice, which express no Fyn protein. We found a severe myelin deficit in forebrain at all ages from 14 d to 1 year. The deficit was maximal at 1 month of age and was similar regardless of mouse strain background or whether it was determined by bulk isolation of myelin or by quantitation of myelin basic protein. To determine the cellular basis of the myelin deficit, we counted oligodendrocytes in tissue sections of mice expressing oligodendrocyte-targeted beta-galactosidase, and we used light and electron microscopy to examine the number and morphology of myelinated fibers and size of myelinated CNS structures. All of these parameters were reduced in fyn(-/-) mice. Unexpectedly, there were regional differences in the myelin deficit; in contrast to forebrain, fyn(-/-) cervical spinal cord exhibited no reduction in myelin content, number of oligodendrocytes, or number of myelinated fibers, nor was myelination delayed developmentally. We found that oligodendrocytes express Src, but there was no significant reduction of myelin content in null mutants lacking the Fyn-related kinases Src, Yes, or Lyn. Finally, we investigated the molecular features of Fyn that are required for myelination and found that a single amino acid substitution, which abolishes the tyrosine kinase activity of Fyn, resulted in a myelin deficit as great as that observed in the complete absence of Fyn protein. These results demonstrate that Fyn plays a unique role in myelination, one that requires its kinase activity.

Chronic multiple paraneoplastic syndromes.

A patient presented with symptoms of limbic and brainstem encephalitis, motor and sensory neuronopathy, cerebellar dysfunction, and highly positive anti-Hu antibodies. He also harbored P/Q-type calcium channel antibodies and manifested the Lambert-Eaton myasthenic syndrome (LEMS). Small-cell lung cancer was found, and he received both antineoplastic therapy and intravenous immunoglobulin (IVIg). Remission of the malignancy was achieved. Although the anti-Hu-related manifestations improved after therapy, LEMS has persisted, leading to IVIg dependency.

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

On the molecular architecture of myelinated fibers.

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a "radial component" composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of "reflexive" adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin(G). The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated beta2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions.

Charcot-Marie-Tooth disease type 1: molecular pathogenesis to gene therapy.

Charcot-Marie-Tooth disease type 1 (CMT1) is caused by mutations in the peripheral myelin protein, 22 kDa (PMP22) gene, protein zero (P0) gene, early growth response gene 2 (EGR-2) and connexin-32 gene, which are expressed in Schwann cells, the myelinating cells of the peripheral nervous system. Although the clinical and pathological phenotypes of the various forms of CMT1 are similar, including distal muscle weakness and sensory loss, their molecular pathogenesis is likely to be quite distinct. In addition, while demyelination is the hallmark of CMT1, the clinical signs and symptoms of the disease are probably produced by axonal degeneration, not demyelination itself. In this review we discuss the molecular pathogenesis of CMT1, as well as approaches to an effective gene therapy for this disease.

X-linked Charcot-Marie-Tooth disease and connexin32.

X-linked Charcot-Marie-Tooth disease is caused by mutations in the gene for the gap junction protein connexin32. This protein is expressed in peripheral nerve and present in noncompacted myelin, where it likely forms channels around and across the myelin sheath. Studies in cell culture and in transgenic mice show that connexin32 mutations can cause a loss of channel function or a gain of toxic effects on myelinating Schwann cells or both, with resulting peripheral nerve degeneration.

Nodes, paranodes, and incisures: from form to function.

The exquisite molecular architecture of myelinated fibers is the basis for saltatory conduction. The nodal axolemma contains high concentrations of voltage-dependent sodium channels as well as the cell adhesion molecules neurofascin and Nr-CAM, all of which are probably linked to the axonal cytoskeleton by ankyrin. At paranodes, the axonal membrane contains paranodin/Caspr, which may be a Ca(2+)-dependent cell adhesion molecule with a heterophilic partner on the apposed glial cell membrane. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, as well as the associated beta 2 subunit, which together may function to dampen re-entrant excitation. The paranodes and incisures of the Schwann cell myelin sheath contain "reflexive" adherens junctions and gap junctions. The adherens junctions are composed of E-cadherin as well as alpha- and beta-catenin, which together probably join the adjacent layers of noncompact myelin together. Reflexive gap junctions, comprising connexin32 and at least one other connexin protein, form a radial pathway for the diffusion of ions and small molecules directly across the myelin sheath.