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1.  Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy 
Brain  2009;132(7):1741-1752.
Dominant intermediate Charcot-Marie-Tooth neuropathy type B is caused by mutations in dynamin 2. We studied the clinical, haematological, electrophysiological and sural nerve biopsy findings in 34 patients belonging to six unrelated dominant intermediate Charcot-Marie-Tooth neuropathy type B families in whom a dynamin 2 mutation had been identified: Gly358Arg (Spain); Asp551_Glu553del; Lys550fs (North America); Lys558del (Belgium); Lys558Glu (Australia, the Netherlands) and Thr855_Ile856del (Belgium). The Gly358Arg and Thr855_Ile856del mutations were novel, and in contrast to the other Charcot-Marie-Tooth-related mutations in dynamin 2, which are all located in the pleckstrin homology domain, they were situated in the middle domain and proline-rich domain of dynamin 2, respectively. We report the first disease-causing mutation in the proline-rich domain of dynamin 2. Patients with a dynamin 2 mutation presented with a classical Charcot-Marie-Tooth phenotype, which was mild to moderately severe since only 3% of the patients were wheelchair-bound. The mean age at onset was 16 years with a large variability ranging from 2 to 50 years. Interestingly, in the Australian and Belgian families, which carry two different mutations affecting the same amino acid (Lys558), Charcot-Marie-Tooth cosegregated with neutropaenia. In addition, early onset cataracts were observed in one of the Charcot-Marie-Tooth families. Our electrophysiological data indicate intermediate or axonal motor median nerve conduction velocities (NCV) ranging from 26 m/s to normal values in four families, and less pronounced reduction of motor median NCV (41–46 m/s) with normal amplitudes in two families. Sural nerve biopsy in a Dutch patient with Lys558Glu mutation showed diffuse loss of large myelinated fibres, presence of many clusters of regenerating myelinated axons and fibres with focal myelin thickenings—findings very similar to those previously reported in the Australian family. We conclude that dynamin 2 mutations should be screened in the autosomal dominant Charcot-Marie-Tooth neuropathy families with intermediate or axonal NCV, and in patients with a classical mild to moderately severe Charcot-Marie-Tooth phenotype, especially when Charcot-Marie-Tooth is associated with neutropaenia or cataracts.
doi:10.1093/brain/awp115
PMCID: PMC2724916  PMID: 19502294
intermediate CMT; dynamin 2; neutropaenia; hereditary neuropathy; cataracts
2.  Clinicopathologic analysis of 124 biopsy-proven peripheral nerve diseases. 
Journal of Korean Medical Science  2000;15(2):211-216.
We reviewed dinical, histological and ultrastructural findings of 124 cases of sural nerve biopsy specimens to delineate the trends of peripheral nerve diseases in our institute. Eighty-one were men and 43 were women. We categorized them into five groups: specific diagnosis (66 cases, 53.2%), axonal degeneration type (47 cases, 37.9%), demyelinating type (4 cases, 3.2%), mixed axonal degeneration-demyelinating type (6 cases, 4.8%) and normal (1 case, 0.9%). Cases with specific diagnosis included 21 inflammatory demyelinating polyneuropathy (15 chronic inflammatory demyelinating polyradiculoneuropathy, 6 Guillain-Barre disease), 13 hereditary motor and sensory neuropathy (7 Charcot-Marie-Tooth type I, 6 Charcot-Marie-Tooth type II), 10 vasculitis, 6 toxic neuropathy, 4 leprosy, 3 diabetic neuropathy, 2 alcoholic neuropathy, 1 Fabry's disease and other specific diseases (5 cases). In our cases, the proportion of specific diagnoses was higher, while the proportion of demyelinating peripheral neuropathies and normal were lower than those of Western series. The results of this study indicate that 1) a dose clinicopathologic correlation is important to make a precise diagnosis of peripheral nerve biopsy, 2) Biopsy under strict indication may reduce unnecessary histologic examination, 3) There is no difference in disease pattern of peripheral neuropathy between Western people and Koreans.
PMCID: PMC3054620  PMID: 10803700
3.  Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot–Marie–Tooth disease type 1X 
Brain  2011;135(1):88-104.
Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot–Marie–Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot–Marie–Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot–Marie–Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of β-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell–cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot–Marie–Tooth type 1, we also found frequent cell–cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot–Marie–Tooth type 1 disease in humans. Thus, colony-stimulating factor-1 or its cognate receptor are promising target molecules for treating the detrimental, low-grade inflammation of several inherited neuropathies in humans.
doi:10.1093/brain/awr283
PMCID: PMC3267979  PMID: 22094537
inflammation; endoneurial fibroblasts; myelin, axonopathy; neuromuscular junction
4.  Genetic Interaction between MTMR2 and FIG4 Phospholipid Phosphatases Involved in Charcot-Marie-Tooth Neuropathies 
PLoS Genetics  2011;7(10):e1002319.
We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P2, thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P2 and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P2 homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P2 is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.
Author Summary
Charcot-Marie-Tooth type 4B1 (CMT4B1) and Charcot-Marie-Tooth type 4J (CMT4J) are severe autosomal recessive demyelinating neuropathies with childhood onset. We previously demonstrated that loss of the phospholipid phosphatase MTMR2 causes CMT4B1 with myelin outfoldings in human and mouse and that loss of the phospholipid phosphatase FIG4 causes CMT4J and neurodegeneration in the mouse. MTMR2 has a predicted role in membrane trafficking, which is crucial for myelin membrane biogenesis and homeostasis. However, the biochemical activity of MTMR2 in vivo and the role of MTMR2 in myelination still remain to be assessed. MTMR2 and FIG4 act on the same phospholipid substrate PtdIns(3,5)P2, but with predicted opposite effects. We generated a double Mtmr2/Fig4-null mouse which showed that Mtmr2 and Fig4 interact in neurons and Schwann cells to control phospholipid metabolism. Moreover, Mtmr2-null myelin outfoldings are rescued by Fig4 heterozygosity, suggesting that imbalance of PtdIns(3,5)P2 is at the basis of the excessive myelin growth and hypermyelination.
doi:10.1371/journal.pgen.1002319
PMCID: PMC3197679  PMID: 22028665
5.  Rapid genetic screening of Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies patients★ 
Neural Regeneration Research  2012;7(32):2522-2527.
We used the allele-specific PCR-double digestion method on peripheral myelin protein 22 (PMP22) to determine duplication and deletion mutations in the proband and family members of one family with Charcot-Marie-Tooth disease type 1 and one family with hereditary neuropathy with liability to pressure palsies. The proband and one subclinical family member from the Charcot-Marie-Tooth disease type 1 family had a PMP22 gene duplication; one patient from the hereditary neuropathy with liability to pressure palsies family had a PMP22 gene deletion. Electron microscopic analysis of ultrathin sections of the superficial peroneal nerve from the two probands demonstrated demyelination and myelin sheath hyperplasia, as well as an ‘onion-like’ structure in the Charcot-Marie-Tooth disease type 1A patient. We observed an irregular thickened myelin sheath and ‘mouse-nibbled’-like changes in the patient with hereditary neuropathy with liability to pressure palsies. In the Charcot-Marie-Tooth disease type 1A patient, nerve electrophysiological examination revealed moderate-to-severe reductions in the motor and sensory conduction velocities of the bilateral median nerve, ulnar nerve, tibial nerve, and sural nerve. Moreover, the compound muscle action potential amplitude was decreased. In the patient with hereditary neuropathy with liability to pressure palsies, the nerve conduction velocity of the bilateral tibial nerve and sural nerve was moderately reduced, and the nerve conduction velocity of the median nerve and ulnar nerve of both upper extremities was slightly reduced.
doi:10.3969/j.issn.1673-5374.2012.32.006
PMCID: PMC4200708  PMID: 25337104
Charcot-Marie-Tooth disease; hereditary neuropathy with liability to pressure palsies; peripheral myelin protein 22; gene mutation; PCR-double digestion method; myelin sheath; action potential; neuropathology; neural regeneration
6.  Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice 
Molecular Medicine  2013;19(1):43-53.
We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.
doi:10.2119/molmed.2012.00248
PMCID: PMC3646097  PMID: 23508572
7.  Axonal Pathology Precedes Demyelination in a Mouse Model of X-Linked Demyelinating/ Type I Charcot-Marie Tooth (CMT1X) Neuropathy 
X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the gene that encodes the gap junction protein connexin32 (Cx32). Cx32 is expressed by myelinating Schwann cells and forms gap junctions in non-compact myelin areas but axonal involvement is more prominent in X-linked compared to other forms of demyelinating Charcot-Marie-Tooth disease. To clarify the cellular and molecular mechanisms of axonal pathology in CMT1X, we studied Gjb1-null mice at early stages (i.e. 2- to 4-month-old) of the neuropathy, when there is minimal or no demyelination. The diameters of large myelinated axons were progressively reduced in Gjb1-null mice compared to those in wild type littermates. Furthermore, neurofilaments were relatively more dephosphorylated and more densely packed starting at 2 months of age. Increased expression of β-amyloid precursor protein, a marker of axonal damage, was also detected in Gjb1-null nerves. Finally, fast axonal transport, assayed by sciatic nerve ligation experiments, was slower in distal axons of Gjb1-null vs. wild type animals with reduced accumulation of synaptic vesicle-associated proteins. These findings demonstrate that axonal abnormalities including impaired cytoskeletal organization and defects in axonal transport precede demyelination in this mouse model of CMT1-X.
doi:10.1097/NEN.0b013e3181efa658
PMCID: PMC3034224  PMID: 20720503
Axonal degeneration; Axonal transport; Charcot-Marie Tooth; Connexin32; Cx32; Gap junctions; Neurofilaments
8.  Clinical syndromes associated with tomacula or myelin swellings in sural nerve biopsies 
OBJECTIVES—To describe the neuropathological features of clinical syndromes associated with tomacula or focal myelin swellings in sural nerve biospies and to discuss possible common aetiopathological pathways leading to their formation in this group of neuropathies.
METHODS—Fifty two patients with sural nerve biopsies reported to show tomacula or focal myelin swellings were reviewed, light and electron microscopy were performed, and tomacula were analysed on teased fibre studies. Molecular genetic studies were performed on those patients who were available for genetic testing.
RESULTS—Thirty seven patients were diagnosed with hereditary neuropathy with liability to pressure palsies (HNPP), four with hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1), four with HMSN with myelin outfolding (CMT4B), three with IgM paraproteinemic neuropathy, three with chronic inflammatory demyelinating polyneuropathy (CIDP), and one with HMSN III (CMT3).
CONCLUSIONS—Most of these syndromes were shown to be related to genetic or immunological defects of myelin components such as peripheral myelin protein 22 (PMP22), myelin protein zero (P0), or myelin associated glycoprotein (MAG). These proteins share the HNK-1 epitope which has been implicated in cell adhesion processes. Impaired myelin maintenance may therefore contribute to the formation of tomacula and subsequent demyelination.


doi:10.1136/jnnp.68.4.483
PMCID: PMC1736856  PMID: 10727485
9.  How do Mutations in GJB1 Cause X-linked Charcot-Marie-Tooth Disease? 
Brain research  2012;1487:198-205.
The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive weakness, atrophy, and sensory abnormalities that are most pronounced in the distal extremities. Some patients have CNS manifestations. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and length-dependent axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. Mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32) cause CMT1X; more than 400 different mutations have been described. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. Effective therapies remain to be developed.
doi:10.1016/j.brainres.2012.03.068
PMCID: PMC3488165  PMID: 22771394
CMT; neuropathy; connexin32; gap junctions; Schwann cells; oligodendrocytes; myelin
10.  CMT2C with vocal cord paresis associated with short stature and mutations in the TRPV4 gene 
Neurology  2010;75(22):1968-1975.
Background:
Recently, mutations in the transient receptor potential cation channel, subfamily V, member 4 gene (TRPV4) have been reported in Charcot-Marie-Tooth Type 2C (CMT2C) with vocal cord paresis. Other mutations in this same gene have been described in separate families with various skeletal dysplasias. Further clarification is needed of the different phenotypes associated with this gene.
Methods:
We performed clinical evaluation, electrophysiology, and genetic analysis of the TRPV4 gene in 2 families with CMT2C.
Results:
Two multigenerational families had a motor greater than sensory axonal neuropathy associated with variable vocal cord paresis. The vocal cord paresis varied from absent to severe, requiring permanent tracheotomy in 2 subjects. One family with mild neuropathy also manifested pronounced short stature, more than 2 SD below the average height for white Americans. There was one instance of dolichocephaly. A novel S542Y mutation in the TRPV4 gene was identified in this family. The other family had a more severe, progressive, motor neuropathy with sensory loss, but less remarkable short stature and an R315W mutation in TRPV4. Third cranial nerve involvement and sleep apnea occurred in one subject in each family.
Conclusion:
CMT2C with axonal neuropathy, vocal cord paresis, and short stature is a unique syndrome associated with mutations in the TRPV4 gene. Mutations in TRPV4 can cause abnormalities in bone, peripheral nerve, or both and may result in highly variable orthopedic and neurologic phenotypes.
GLOSSARY
= compound muscle action potential;
= Charcot-Marie-Tooth;
= Charcot-Marie-Tooth Type 2C;
= hereditary motor and sensory neuropathy;
= nerve conduction velocity;
= restriction fragment length polymorphism;
= spinal muscular atrophy;
= sensory nerve action potential;
= scapuloperoneal spinal muscular atrophy.
doi:10.1212/WNL.0b013e3181ffe4bb
PMCID: PMC3014233  PMID: 21115951
11.  A Laminin-2, Dystroglycan, Utrophin Axis is Required for Compartmentalization and Elongation of Myelin Segments 
Animal and plant cells compartmentalize to perform morphogenetic functions. Compartmentalization of myelin-forming Schwann cells may favor elongation of myelin segments to the size required for efficient conduction of nerve impulses. Compartments in myelinated fibers were described by Ramon-y-Cajal and depend on periaxin, mutated in the hereditary neuropathy Charcot-Marie-Tooth 4F. Lack of periaxin in mice causes loss of compartments, formation of short myelin segments (internodes) and reduced nerve conduction velocity. How compartments are formed and maintained, and their relevance to human neuropathies is largely unknown. Here we show that formation of compartments around myelin is driven by the actin cytoskeleton, and maintained by actin and tubulin fences through linkage to the dystroglycan complex. Compartmentalization and establishment of correct internodal length requires the presence of glycosylated dystroglycan, utrophin and extracellular laminin-2/211. A neuropathic patient with reduced internodal length and nerve conduction velocity due to absence of laminin-2/211 (congenital muscular dystrophy 1A) also shows abnormal compartmentalization. These data link formation of compartments through a laminin2-dystroglycan-utrophin-actin axis to internodal length, and provide a common pathogenetic mechanism for two inherited human neuropathies. Other cell types may exploit dystroglycan complexes in similar fashions to create barriers and compartments.
doi:10.1523/JNEUROSCI.5672-08.2009
PMCID: PMC2940832  PMID: 19321787
Cajal-bands; Schwann cell compartments; Dystroglycan complex; internodal length; myelin; inherited peripheral neuropathies
12.  Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A 
ASN NEURO  2013;5(5):e00128.
Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin–proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.
In peripheral nerves of neuropathic C22 mice the frequency of cytosolic PMP22 aggregates increases with age, which elicits a response from protein quality control mechanisms. The combined effects of aging and neuropathic genotype exacerbate disease progression leading to nerve defects.
doi:10.1042/AN20130024
PMCID: PMC3848555  PMID: 24175617
autophagy; chaperone; ubiquitin; myelin; protein aggregation; Schwann cell.; AMC, amino-methyl coumarin; CathD, Cathepsin D; CMT1A, Charcot–Marie–Tooth disease type 1A; di-8-ANEPPS, 4-[2-(6-dibutylamino)-2-naphthalenyl)ethenyl]-1-(3-sulfopropyl) hydroxide; Egr2, early growth response 2; endoH, endoglycosidase H; ER, endoplasmic reticulum; HRP, horseradish peroxidase; HSF1, heat-shock factor 1; Hsp, heat-shock protein; IgG, immunoglobulin; LAMP1, lysosomal membrane-associated protein 1; LC3, light chain 3; MCP-1, monocyte chemoattractant protein 1; MS, multiple sclerosis; Oct6, octamer-binding transcription factor 6; PMP22, peripheral myelin protein 22; PNGaseF, N-glycosidase F; pUb, polyubiquitinated; TFEB, transcription factor EB; UPS, ubiquitin–proteasome system; Wt, wild-type
13.  Remodelling of motor nerve terminals in demyelinating axons of periaxin null mutant mice 
Glia  2008;56(4):471-479.
Myelin formation around axons increases nerve conduction velocity and regulates phenotypic characteristics of the myelinated axon. In the peripheral nervous system, demyelinating forms of hereditary Charcot-Marie-Tooth (CMT) diseases, due to Schwann-cell intrinsic molecular defects, leads to reduced nerve conduction velocity and changes in the axonal phenotype. Several mouse models of CMT diseases have been generated, allowing the study of consequences of demyelination in peripheral nerve fibres. Nevertheless, the effect of demyelination at the level of the neuromuscular synapse has been largely overlooked. Here we show that in the periaxin knock-out mice, a model of CMT condition, neuromuscular junctions develop profound morphological changes in pre-terminal region of motoraxons. These changes include extensive preterminal branches which originate in demyelinated regions of the nerve fibre and axonal swellings associated with residually-myelinated regions of the fibre. Using intracellular recording from muscle fibres we detected asynchronous failure of action potential transmission at high but not low stimulation frequencies, a phenomenon consistent with branch point failure. Taken together, our morphological and electrophysiological findings suggest that preterminal branching due to segmental demyelination near the neuromuscular synapse in periaxin KO mice may underlie phenotypic disabilities present in this mouse model of CMT disease. These results opens a new avenue of research in order to understand the cellular changes responsible for clinical disabilities in demyelinating conditions.
doi:10.1002/glia.20620
PMCID: PMC4335188  PMID: 18205176
14.  Large Kindred Evaluation of Mitofusin 2 Novel Mutation, Extremes of Neurologic Presentations, and Preserved Nerve Mitochondria 
Archives of neurology  2011;68(10):1295-1302.
Background
Mitofusin 2 (MFN2) is a mitochondrial membrane protein mediating mitochondrial fusion and function. Mutated MFN2 is responsible for Charcot-Marie-Tooth type 2A2. In small kindreds, specific MFN2 mutations have been reported to associate with severity of axonal neuropathy, optic atrophy, and involvement of the central nervous system. The results of the nerve biopsy specimens suggested that the mitochondria are structurally abnormal in patients with MFN2 mutations.
Objective
To study a newly identified MFN2 mutation, Leu146Phe, and the associated phenotypes in a large kindred.
Patients
An American kindred of Northern European and Cherokee American Indian descent.
Results
Genetic analysis revealed a novel GTPase domain MFN2 mutation Leu146Phe that associated with clinical status of 15 studied persons (10 affected and 5 unaffected) and not found in 800 control persons. Clinical manifestations were markedly different. In 1 affected person, optic atrophy and brain magnetic resonance imaging abnormalities led to multiple sclerosis diagnosis and interferon β-1a treatment when neuropathy was initially unrecognized. Age of onset ranged from 1 to 45 years. In some affected family members, severe and rapid-onset motor sensory neuropathy led to early loss of ambulation, whereas other family members experienced minimal neuropathic sensory symptoms. Despite histologically significant loss of nerve fibers, the mitochondria were not distinguishable from diseased sural nerve biopsy specimens and healthy controls.
Conclusions
Novel MFN2 mutation Leu146Phe causes Charcot-Marie-Tooth type 2A2. Intrafamilial clinical phenotype variability is emphasized and has important implications in genetic counseling. The clinical phenotype may mimic multiple sclerosis when optic atrophy and the characteristic brain lesions of MFN2 on magnetic resonance imaging are present and neuropathy is mild or unrecognized. The predicted molecular pathogenesis may occur without evident histological abnormalities of mitochondria in nerve.
doi:10.1001/archneurol.2011.225
PMCID: PMC3543870  PMID: 21987543
15.  An essential role of MAG in mediating axon-myelin attachment in Charcot-Marie-Tooth 1A disease 
Neurobiology of disease  2012;0:221-231.
Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy caused by the duplication of the PMP22 gene. Demyelination precedes the occurrence of clinical symptoms that correlate with axonal degeneration. It was postulated that a disturbed axon-glia interface contribute to altered myelination consequently leading to axonal degeneration. In this study, we examined the expression of MAG and Necl4, two critical adhesion molecules that are present at the axon-glia interface, in sural nerve biopsies of CMT1A patients and in peripheral nerves of mice overexpressing human PMP22, an animal model for CMT1A. We show an increase in the expression of MAG and a strong decrease of Necl4 in biopsies of CMT1A patients as well as in CMT1A mice. Expression analysis revealed that MAG is strongly upregulated during peripheral nerve maturation, whereas Necl4 expression remains very low. Ablating MAG in CMT1A mice results in separation of axons from their myelin sheath. Our data show that MAG is important for axon-glia contact in a model for CMT1A, and suggest that its increased expression in CMT1A disease has a compensatory role in the pathology of the disease. Thus, we demonstrate that MAG together with other adhesion molecules such as Necl4 is important in sustaining axonal integrity.
doi:10.1016/j.nbd.2012.08.009
PMCID: PMC3612363  PMID: 22940629
Myelin associated protein (MAG); Nectin-like protein (Necl4); peripheral neuropathy; CMT1A; axonal pathology
16.  X-linked Charcot-Marie-Tooth disease 
The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive muscle atrophy and weakness, areflexia, and variable sensory abnormalities; central nervous system manifestations occur, too. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and distal axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. More than 400 different mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32), cause CMT1X. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. An effective therapy remains to be developed.
doi:10.1111/j.1529-8027.2012.00424.x
PMCID: PMC3779456  PMID: 23279425
CMT; connexin32; gap junctions; myelin; neuropathy; oligodendrocytes; Schwann cells
17.  A Case of Cauda Equina Syndrome in Early-Onset Chronic Inflammatory Demyelinating Polyneuropathy Clinically Similar to Charcot-Marie-Tooth Disease Type 1 
To present a case of cauda equina syndrome (CES) caused by chronic inflammatory demyelinating polyneuropathy (CIDP) which seemed clinically similar to Charcot-Marie-Tooth disease type1 (CMT1). CIDP is an immune-mediated polyneuropathy, either progressive or relapsing-remitting. It is a non-hereditary disorder characterized by symmetrical motor and sensory deficits. Rarely, spinal nerve roots can be involved, leading to CES by hypertrophic cauda equina. A 34-year-old man presented with low back pain, radicular pain, bilateral lower-extremity weakness, urinary incontinence, and constipation. He had had musculoskeletal deformities, such as hammertoes and pes cavus, since age 10. Lumbar spine magnetic resonance imaging showed diffuse thickening of the cauda equina. Electrophysiological testing showed increased distal latency, conduction blocks, temporal dispersion, and severe nerve conduction velocity slowing (3 m/s). We were not able to find genetic mutations at the PMP 22, MPZ, PRX, and EGR2 genes. The pathologic findings of the sural nerve biopsy revealed thinly myelinated nerve fibers with Schwann cells proliferation. We performed a decompressive laminectomy, intravenous IgG (IV-IgG) and oral steroid. At 1 week after surgery, most of his symptoms showed marked improvements except foot deformities. There was no relapse or aggravation of disease for 3 years. We diagnosed the case as an early-onset CIDP with cauda equine syndrome, whose initial clinical findings were similar to those of CMT1, and successfully managed with decompressive laminectomy, IV-IgG and oral steroid.
doi:10.3340/jkns.2014.55.6.370
PMCID: PMC4166336  PMID: 25237436
Cauda equina syndrome; Chronic inflammatory demyelinating polyneuropathy; Charcot-Marie-Tooth disease; Laminectomy
18.  Motor and sensory neuropathy due to myelin infolding and paranodal damage in a transgenic mouse model of Charcot–Marie–Tooth disease type 1C 
Human Molecular Genetics  2013;22(9):1755-1770.
Charcot–Marie–Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt–Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.
doi:10.1093/hmg/ddt022
PMCID: PMC3613163  PMID: 23359569
19.  Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function 
eLife  2013;2:e01064.
The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function.
DOI: http://dx.doi.org/10.7554/eLife.01064.001
eLife digest
Charcot-Marie-Tooth disease is an inherited disorder of the nervous system with symptoms that typically begin in adolescence or early adulthood. The sensory and motor nerves gradually degenerate, causing muscles to waste away and leading to the loss of touch sensation across the body. One subtype of the disease—Charcot-Marie-Tooth 2B—is caused by mutations in a gene called rab7, which codes for a protein that helps to regulate the breakdown of waste proteins inside cells.
Charcot-Marie-Tooth 2B is described as a genetically dominant disorder because all patients have one wild type copy and one mutant copy of the rab7 gene. Overexpression of the mutant gene in cells grown in culture alters many of the signaling pathways inside the cells, but it is unclear whether these alterations cause the pathology seen in the disease.
Now, Cherry et al. have obtained new insights into the genetics of Charcot-Marie-Tooth 2B by creating the first animal model of the disorder. Fruit flies that did not have the rab7 gene in the light-sensitive sensory neurons in their eyes were used to compare normal and mutant cells. While the two cell types were initially similar, the mutant cells gradually degenerated in the adult animal. By contrast, cells that overexpressed a mutant form of the rab7 gene continued to function normally throughout adulthood. Moreover, when mutant Rab7 proteins were introduced into the cells that lacked the rab7 gene, the proteins restored the cells’ sensitivity to light. These results suggest that mutant Rab7 proteins do not cause degeneration; instead, it is the loss of normal Rab7 function that causes problems.
At present, most research into treatment is aimed at finding ways to reduce the activity of mutant Rab7 proteins. However, the work of Cherry et al. suggests that increasing the activity of normal Rab7 proteins—or increasing the activity of alternative pathways that degrade waste proteins—may help to restore nerve function in this, and possibly other, neurodegenerative diseases.
DOI: http://dx.doi.org/10.7554/eLife.01064.002
doi:10.7554/eLife.01064
PMCID: PMC3857549  PMID: 24327558
endosome; neuropathy; genetics; synapse; D. melanogaster
20.  Polytherapy with a combination of three repurposed drugs (PXT3003) down-regulates Pmp22 over-expression and improves myelination, axonal and functional parameters in models of CMT1A neuropathy 
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model. Based on these observations in preclinical models, a clinical trial of PTX3003 in CMT1A, a neglected orphan disease, is warranted. If the efficacy of PTX3003 is confirmed, rational polytherapy based on novel combinations of existing non-toxic drugs with pleiotropic effects may represent a promising approach for rapid drug development.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-014-0201-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13023-014-0201-x
PMCID: PMC4279797  PMID: 25491744
Systems Biology; Repurposing; Combination therapy; Baclofen; Naltrexone; Sorbitol; Synergy; CMT1A; Low dose
21.  A novel transgenic mouse model of Chinese Charcot-Marie-Tooth disease type 2L 
Neural Regeneration Research  2014;9(4):413-419.
We previously found that the K141N mutation in heat shock protein B8 (HSPB8) was responsible for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the present study was to generate a transgenic mouse model bearing the K141N mutation in the human HSPB8 gene, and to determine whether this K141NHSPB8 transgenic mouse model would manifest the clinical phenotype of Charcot-Marie-Tooth disease type 2L, and consequently be suitable for use in studies of disease pathogenesis. Transgenic mice overexpressing K141NHSPB8 were generated using K141N mutant HSPB8 cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. PCR and western blot analysis confirmed integration of the K141NHSPB8 gene and widespread expression in tissues of the transgenic mice. The K141NHSPB8 transgenic mice exhibited decreased muscle strength in the hind limbs and impaired motor coordination, but no obvious sensory disturbance at 6 months of age by behavioral assessment. Electrophysiological analysis showed that the compound motor action potential amplitude in the sciatic nerve was significantly decreased, but motor nerve conduction velocity remained normal at 6 months of age. Pathological analysis of the sciatic nerve showed reduced myelinated fiber density, notable axonal edema and vacuolar degeneration in K141NHSPB8 transgenic mice, suggesting axonal involvement in the peripheral nerve damage in these animals. These findings indicate that the K141NHSPB8 transgenic mouse successfully models Charcot-Marie-Tooth disease type 2L and can be used to study the pathogenesis of the disease.
doi:10.4103/1673-5374.128248
PMCID: PMC4146190  PMID: 25206829
nerve regeneration; peripheral nerve injury; axonal injury; animal models; Charcot-Marie-Tooth disease type 2L; gene mutation; pronuclear injection; transgenic model; small heat shock protein B8; NSFC grant; neural regeneration
22.  Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice 
Brain  2012;135(12):3551-3566.
Charcot–Marie–Tooth disease type 1B is caused by mutations in myelin protein zero. R98C mice, an authentic model of early onset Charcot–Marie–Tooth disease type 1B, develop neuropathy in part because the misfolded mutant myelin protein zero is retained in the endoplasmic reticulum where it activates the unfolded protein response. Because oral curcumin, a component of the spice turmeric, has been shown to relieve endoplasmic reticulum stress and decrease the activation of the unfolded protein response, we treated R98C mutant mice with daily gastric lavage of curcumin or curcumin derivatives starting at 4 days of age and analysed them for clinical disability, electrophysiological parameters and peripheral nerve morphology. Heterozygous R98C mice treated with curcumin dissolved in sesame oil or phosphatidylcholine curcumin performed as well as wild-type littermates on a rotarod test and had increased numbers of large-diameter axons in their sciatic nerves. Treatment with the latter two compounds also increased compound muscle action potential amplitudes and the innervation of neuromuscular junctions in both heterozygous and homozygous R98C animals, but it did not improve nerve conduction velocity, myelin thickness, G-ratios or myelin period. The expression of c-Jun and suppressed cAMP-inducible POU (SCIP)—transcription factors that inhibit myelination when overexpressed—was also decreased by treatment. Consistent with its role in reducing endoplasmic reticulum stress, treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin was associated with decreased X-box binding protein (XBP1) splicing. Taken together, these data demonstrate that treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin improves the peripheral neuropathy of R98C mice by alleviating endoplasmic reticulum stress, by reducing the activation of unfolded protein response and by promoting Schwann cell differentiation.
doi:10.1093/brain/aws299
PMCID: PMC3577101  PMID: 23250879
Charcot-Marie-Tooth disease 1B; curcumin; myelin protein zero; peripheral neuropathy; unfolded protein response
23.  Electrophysiological Evaluation of Chronic Inflammatory Demyelinating Polyneuropathy and Charcot-Marie-Tooth Type 1: Dispersion and Correlation Analysis 
Journal of Physical Therapy Science  2013;25(10):1265-1268.
[Purpose] The purpose of this study was to analyze and compare electrophysiological characteristics observed in nerve conduction studies (NCS) of chronic inflammatory demyelinating polyneuropathy (CIDP) and Charcot-Marie-Tooth disease type 1 (CMT 1). [Subjects] A differential diagnosis of acquired and congenital demyelinating neuropathies was based on a study of 35 patients with NCS-confirmed CIDP and 30 patients with CMT 1 genetically proven by peripheral myelin protein-22 (PMP-22) gene analysis, pulsed-field gel electrophoresis (PFGE), and Southern blot analysis. [Methods] We analyzed values collected in motor nerve conduction studies. We conducted dispersion analysis of the amplitudes of the compound muscle action potential (CMAP) of various nerve types and correlation coefficient analysis of the motor nerve conduction velocity (MNCV). [Results] We found that CIDP and CMT 1 were clearly attributable to severe polyneuropathy. In dispersion analysis, CIDP showed greater differences in proximal-to-distal amplitude ratios. Moreover, CMT 1 showed relatively high correlations compared to CIDP based on correlation coefficient analysis of MNCV. [Conclusion] The results of this study suggest that CIDP showed greater asymmetry than CMT 1 in MNCV and CMAP amplitudes.
doi:10.1589/jpts.25.1265
PMCID: PMC3820196  PMID: 24259772
Chronic inflammatory demyelinating polyneuropathy; Charcot-Marie-Tooth disease type 1; Dispersion and correlation analysis
24.  Oral high dose ascorbic acid treatment for one year in young CMT1A patients: a randomised, double-blind, placebo-controlled phase II trial 
BMC Medicine  2009;7:70.
Background
High dose oral ascorbic acid substantially improved myelination and locomotor function in a Charcot-Marie-Tooth type 1A mouse model. A phase II study was warranted to investigate whether high dose ascorbic acid also has such a substantial effect on myelination in Charcot-Marie-Tooth type 1A patients and whether this treatment is safe.
Methods
Patients below age 25 years were randomly assigned to receive placebo or ascorbic acid (one gram twice daily) in a double-blind fashion during one year. The primary outcome measure was the change over time in motor nerve conduction velocity of the median nerve. Secondary outcome measures included changes in minimal F response latencies, compound muscle action potential amplitude, muscle strength, sensory function, Charcot-Marie-Tooth neuropathy score, and disability.
Results
There were no significant differences between the six placebo-treated (median age 16 years, range 13 to 24) and the five ascorbic acid-treated (19, 14 to 24) patients in change in motor nerve conduction velocity of the median nerve (mean difference ascorbic acid as opposed to placebo treatment of 1.3 m/s, confidence interval -0.3 to 3.0 m/s, P = 0.11) or in change of any of the secondary outcome measures over time. One patient in the ascorbic acid group developed a skin rash, which led to discontinuation of the study medication.
Conclusion
Oral high dose ascorbic acid for one year did not improve myelination of the median nerve in young Charcot-Marie-Tooth type 1A patients. Treatment was relatively safe.
Trial registration
Current Controlled Trials ISRCTN56968278, ClinicalTrials.gov NCT00271635.
doi:10.1186/1741-7015-7-70
PMCID: PMC2784478  PMID: 19909499
25.  MpzR98C arrests Schwann cell development in a mouse model of early-onset Charcot–Marie–Tooth disease type 1B 
Brain  2012;135(7):2032-2047.
Mutations in myelin protein zero (MPZ) cause Charcot–Marie–Tooth disease type 1B. Many dominant MPZ mutations, including R98C, present as infantile onset dysmyelinating neuropathies. We have generated an R98C ‘knock-in’ mouse model of Charcot–Marie–Tooth type 1B, where a mutation encoding R98C was targeted to the mouse Mpz gene. Both heterozygous (R98C/+) and homozygous (R98C/R98C) mice develop weakness, abnormal nerve conduction velocities and morphologically abnormal myelin; R98C/R98C mice are more severely affected. MpzR98C is retained in the endoplasmic reticulum of Schwann cells and provokes a transitory, canonical unfolded protein response. Ablation of Chop, a mediator of the protein kinase RNA-like endoplasmic reticulum kinase unfolded protein response pathway restores compound muscle action potential amplitudes of R98C/+ mice but does not alter the reduced conduction velocities, reduced axonal diameters or clinical behaviour of these animals. R98C/R98C Schwann cells are developmentally arrested in the promyelinating stage, whereas development is delayed in R98C/+ mice. The proportion of cells expressing c-Jun, an inhibitor of myelination, is elevated in mutant nerves, whereas the proportion of cells expressing the promyelinating transcription factor Krox-20 is decreased, particularly in R98C/R98C mice. Our results provide a potential link between the accumulation of MpzR98C in the endoplasmic reticulum and a developmental delay in myelination. These mice provide a model by which we can begin to understand the early onset dysmyelination seen in patients with R98C and similar mutations.
doi:10.1093/brain/aws140
PMCID: PMC3381724  PMID: 22689911
Charcot–Marie–Tooth type 1B; demyelination; neuromuscular disorders; glial cells; neuropathy

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