Insights into wild-type dynamin 2 and the consequences of DNM2 mutations from transgenic zebrafish.

Dynamin 2 (DNM2) encodes a ubiquitously expressed large GTPase with membrane fission capabilities that participates in the endocytosis of clathrin-coated vesicles. Heterozygous mutations in DNM2 are associated with two distinct neuromuscular disorders, Charcot-Marie-Tooth disease (CMT) and autosomal dominant centronuclear myopathy (CNM). Despite extensive investigations in cell culture, the role of dynamin 2 in normal muscle development is poorly understood and the consequences of DNM2 mutations at the molecular level in vivo are not known. To address these gaps in knowledge, we developed transgenic zebrafish expressing either wild-type dynamin 2 or dynamin 2 with either a CNM or CMT mutation. Taking advantage of the live imaging capabilities of the zebrafish embryo, we establish the localization of wild-type and mutant dynamin 2 in vivo, showing for the first time distinctive dynamin 2 subcellular compartments. Additionally, we demonstrate that CNM-related DNM2 mutations are associated with protein mislocalization and aggregation. Lastly, we define core phenotypes associated with our transgenic mutant fish, including impaired motor function and altered muscle ultrastructure, making them the ideal platform for drug screening. Overall, using the power of the zebrafish, we establish novel insights into dynamin 2 localization and dynamics and provide the necessary groundwork for future studies examining dynamin 2 pathomechanisms and therapy development.

S1P/S1P Receptor Signaling in Neuromuscolar Disorders.

The bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), and the signaling pathways triggered by its binding to specific G protein-coupled receptors play a critical regulatory role in many pathophysiological processes, including skeletal muscle and nervous system degeneration. The signaling transduced by S1P binding appears to be much more complex than previously thought, with important implications for clinical applications and for personalized medicine. In particular, the understanding of S1P/S1P receptor signaling functions in specific compartmentalized locations of the cell is worthy of being better investigated, because in various circumstances it might be crucial for the development or/and the progression of neuromuscular diseases, such as Charcot-Marie-Tooth disease, myasthenia gravis, and Duchenne muscular dystrophy.

Defective membrane remodeling in neuromuscular diseases: insights from animal models.

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.

Community exercise is feasible for neuromuscular diseases and can improve aerobic capacity.

OBJECTIVE: The aim of this phase 2 trial was to ascertain the feasibility and effect of community-based aerobic exercise training for people with 2 of the more common neuromuscular diseases: Charcot-Marie-Tooth disease type 1A (CMT) and inclusion body myositis (IBM). METHODS: A randomized single-blinded crossover trial design was used to compare a 12-week aerobic training program using recombinant exercise bicycles compared to a control period. The training occurred 3 times per week in community gyms local to the participants. Support was available from trained gym staff and a research physiotherapist. The 2 disease groups were analyzed separately. The primary outcome measure was peak oxygen uptake (VO2 peak) during a maximal exercise test, with secondary measures of muscle strength, function, and patient-reported measures. RESULTS: Data from 23 people with CMT and 17 people with IBM were included in the analysis. Both disease groups had high levels of participation and demonstrated improvements in VO2 peak, with a moderate effect size in the CMT participants (Cohen d = 0.53) and a strong effect size in the IBM group (Cohen d = 1.72). No major changes were observed in the secondary outcome measures. Qualitative interviews revealed that participants valued the support of gym instructors and the research physiotherapists in overcoming challenges to participation. CONCLUSION: Twelve weeks of aerobic training in community gyms was feasible, safe, and improved aerobic capacity in people with CMT and IBM. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with CMT type 1A and IBM, an aerobic training program increases aerobic capacity.

Neuromuscular implications in left ventricular hypertrabeculation/noncompaction.

This review focuses on recent advances in the association between left ventricular hypertrabeculation/noncompaction (LVHT), a form of unclassified cardiomyopathy, and neuromuscular disorders (NMD). So far, LVHT has been found in single patients with dystrophinopathy, dystrobrevinopathy, laminopathy, zaspopathy, myotonic dystrophy, infantile glycogenosis type II (Pompe's disease), myoadenylate-deaminase deficiency, mitochondriopathy, Barth syndrome, Friedreich ataxia, and Charcot-Marie-Tooth disease. Most frequently LVHT is found in patients with Barth syndrome and mitochondrial disorders. The prevalence of LVHT in NMD patients is not known. On the contrary, NMD can be detected in up to four fifths of the patients with LVHT. Because LVHT is associated with an increased risk of rhythm abnormalities and heart failure, it is essential to detect LVHT as soon as possible. Because of adequate therapeutic options, all patients with NMD should undergo a comprehensive cardiological examination as soon as their neurological diagnosis is established. In reverse, all patients with LVHT should undergo a comprehensive neurological investigation following the detection of LVHT.

Pyruvate oxidation in Charcot-Marie-Tooth disease.

Significant differences were found in pyruvate oxidation of disrupted skin fibroblasts from patients with Charcot-Marie-Tooth disease (CMT) or neuromuscular disease controls and normal controls, but there was a similar oxidative defect in both disease groups. "Heritable heterogeneity in situ" and additional enzyme faults may explain the normal activity of pyruvate dehydrogenase in some explants, and the lack of correlation between enzyme activity and CMT symptoms. Since experimental, histochemical, and microscopic studies support the concept of localized hypoxia in perineural or neural cells in CMT, the peripheral location of the defect suggests an inherited susceptibility to environmental influences in these cells.

Immunochemical study of connectin (titin) in neuromuscular diseases using a monoclonal antibody: connectin is degraded extensively in Duchenne muscular dystrophy.

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elastic property of myofibrils. In the present study, the degradation state of connectin in biopsied skeletal muscles from various neuromuscular diseases was investigated by Western blot analysis using a monoclonal antibody which reacts extensively with the degradation products of connectin. In Duchenne muscular dystrophy (DMD), connectin was degraded progressively and relentlessly after 5 years of age. In Becker muscular dystrophy, degradation of connectin was much less than in DMD. Connectin was well preserved in normal controls, and was only minimally degraded in Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, limb girdle muscular dystrophy and myotonic dystrophy, even when the biopsied muscles showed a similar degree of weakness as those of DMD. The degradation of connectin, even though secondary, is presumed to play an important role in the pathogenesis of myofibrillar degeneration in DMD.

The uptake of 3H(G)L leucine into single muscle fibers in Charcot-Marine-Tooth disease.

In previous studies, the incorporation of 3H(G)L-leucine into muscles of patients with Charcot-Marie-Tooth (CMT) disease was shown to be increased in comparison with that observed in motor neuron disease (MND). To determine the cause of the increased uptake in CMT, studies of single fiber leucine incorporations have been undertaken. The results of this study indicate that the increased incorporation is into those muscle fibers which are undergoing regeneration following reinnervation. These results do not support the thesis that there is an associated myopathic process in CMT.

An autoradiographic study of muscular dystrophy, motor neuron disease and Charcot-Marie-Tooth disease.

The autoradiographic findings using tritiated leucine are described in muscle biopsy material from five patients with progressive muscular dystrophy (P.M.D.), three with motor neuron disease (M.N.D.) and four with Charcot-Marie-Tooth disease (C.M.T.). In progressive muscular dystrophy there is a marked increase in uptake of leucine into cytoplasmic proteins and precursors, and reduced incorporation into structural protein. In Charcot-Marie Tooth disease muscle there is a significantly increased uptake into cytoplasmic elements and a normal uptake into structural protein. In motor neuron disease the uptake into cytoplasmic elements appears normal but is reduced into structural proteins. The abnormal uptake in C.M.T. could be explained as a product of regenerative efforts associated with reinnervation. However, the abnormal uptake may represent the primary effects of gene action in the muscle, as seems probable in progressive muscular dystrophy.

The effects of partial chronic denervation on forearm metabolism.

Effects of chronic denervation upon in vivo forearm metabolism were studied in six patients and six controls. The diagnosis was amyotrophic lateral sclerosis in four patients, the neuronal form of Charcot-Marie Tooth disease in one patient, and an unclassified chronic disease of the lower motor neurons in one patient. In all cases the forearm muscles showed clinical weakness and electrical evidence of denervation, while muscle biopsy from a proximal muscle of the upper limb showed typical denervation atrophy. At rest there was increased oxygen utilization and lactate output as well as a tendency for increased uptake of glucose and long chain fatty acids from arterial blood per 100 ml of forearm tissue. During exercise the abnormally high lactate output increased further. An increased arterial lactate concentration was present during rest and exercise. Oxidation of fatty acids was not impaired. It is suggested that these abnormalities are consistent with an augmented utilization of blood borne fuels at rest by denervated muscles. A concurrent regional ischemia of muscles during rest and exercise, possibly due to defective autoregulation of skeletal muscle blood flow, may explain the abnormally high lactate generation.

[Cardiac involvement in neuromuscular diseases]. / Repercusión cardíaca de las enfermedades neuromusculares.

Many neuromuscular disorders involve the heart, occasionally with overt clinical disease. Muscular dystrophies (dystrophinopathies, limb girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, Steinert's myotonic dystrophy), congenital myopathies, inflammatory myopathies and metabolic diseases (glycogenosis, periodic paralysis, mitochondrial diseases) may produce dilated or hypertrophic cardiomyopathy and heart rhythm or conduction disturbances. Furthermore the heart is commonly involved in some hereditary and degenerative diseases (Friedreich's ataxia and Kugelberg-Welander syndrome) and acquired (Guillain-Barré syndrome) or inherited (Refsum's disease and Charcot-Marie-Tooth syndrome) polyneuropathies. A cardiologist's high clinical suspicion and a simple but systematic skeletal muscle and peripheral nerve investigation, including muscle enzymes quantification, neurophysiological study and muscle biopsy, are necessary for an accurate diagnosis. In selected patients, more sophisticated biochemical and genetic analysis will be necessary. In most cases, endomyocardial biopsy is not essential for the diagnosis.

Mitochondrial dysfunction in neuromuscular disorders.

This review deciphers aspects of mitochondrial (mt) dysfunction among nosologically, pathologically, and genetically diverse diseases of the skeletal muscle, lower motor neuron, and peripheral nerve, which fall outside the traditional realm of mt cytopathies. Special emphasis is given to well-characterized mt abnormalities in collagen VI myopathies (Ullrich congenital muscular dystrophy and Bethlem myopathy), megaconial congenital muscular dystrophy, limb-girdle muscular dystrophy type 2 (calpainopathy), centronuclear myopathies, core myopathies, inflammatory myopathies, spinal muscular atrophy, Charcot-Marie-Tooth neuropathy type 2, and drug-induced peripheral neuropathies. Among inflammatory myopathies, mt abnormalities are more prominent in inclusion body myositis and a subset of polymyositis with mt pathology, both of which are refractory to corticosteroid treatment. Awareness is raised about instances of phenotypic mimicry between cases harboring primary mtDNA depletion, in the context of mtDNA depletion syndrome, and established neuromuscular disorders such as spinal muscular atrophy. A substantial body of experimental work, derived from animal models, attests to a major role of mitochondria (mt) in the early process of muscle degeneration. Common mechanisms of mt-related cell injury include dysregulation of the mt permeability transition pore opening and defective autophagy. The therapeutic use of mt permeability transition pore modifiers holds promise in various neuromuscular disorders, including muscular dystrophies.

Myelin and axon pathology in a long-term study of PMP22-overexpressing mice.

We analyzed clinical and pathological disease in 2 peripheral myelin protein-22 (PMP22) overexpressing mouse models for 1.5 years. C22 mice have 7 and C3-PMP mice have 3 to 4 copies of the human PMP22 gene. C3-PMP mice showed no overt clinical signs at 3 weeks and developed mild neuromuscular impairment; C22 mice showed signs at 3 weeks that progressed to severe impairment. Adult C3-PMP mice had very similar, stable, low nerve conduction velocities similar to adults with human Charcot-Marie-Tooth disease type 1A (CMT1A); velocities were much lower in C22 mice. Myelination was delayed, and normal myelination was not reached in either model but the degree of dysmyelination in C3-PMP mice was considerably less than that in C22 mice; myelination was stable in the adult mice. Numbers of myelinated, fibers were reduced at 3 weeks in both models, suggesting that normal numbers of myelinated fibers are not reached during development in the models. In adult C3-PMP and wild-type mice, there was no detectable loss of myelinated fibers,whereas there was clear loss of myelinated fibers in C22 mice.In C3-PMP mice, there is a balance between myelination status and axonal function early in life, whereas in C22 mice, early reduction of axons is more severe and there is major loss of axons in adulthood. We conclude that C3-PMP mice may be an appropriate model for most CMT1A patients, whereas C22 mice may be more relevant to severely affected patients in the CMT1 spectrum.

Quantitative and qualitative fat analysis in human leg muscle of neuromuscular diseases by 1H MR spectroscopy in vivo.

1H MR spectra were recorded from human gastrocnemius muscle at 63.86 MHz using the body coil of the Signa scanner as transmitter and a 3-in. surface coil as receiver. The fat content of the muscle was quantified relative to that of water in a selected volume or slice. The fat/water ratio was 0.05-0.07 for normal muscle but increased to 0.5-6.0 in primary and secondary muscular disorders such as Duchenne and myotonic dystrophy, Charcot-Marie-Tooth and polio muscular atrophy, cerebral palsy, and spina bifida. In Werdnig-Hoffmann spinal atrophy the ratio was above 10. Water-suppressed and slice-selective 1H spectroscopy was used for qualitative analysis of fat. The 1H profile of gastrocnemius muscles between healthy individuals and patients with neuromuscular diseases showed two major differences. In the normal muscle spectra, the resonance from the -(CH2)n- protons at 1.6 ppm was the most pronounced, whereas in the diseased muscle spectra resonances also appeared between 1.1 and 1.4 ppm. Some diseased muscle spectra showed multiple resonances from -CH = CH- in polyunsaturated fatty acids between 5.5 and 7.0 ppm. The corresponding resonances from = CH-CH2-, 1.9-2.0 ppm, and = CH-CH2-CH =, 2.7-2.9 ppm, were also seen. These peaks are usually not detected in normal muscle.

Cyanide metabolism in motor neuron disease.

Cyanide concentrations in whole blood, saliva and urine were measured in 83 patients with motor neuron disease (MND) and age-, sex-matched control subjects consisting of 62 patients with and 49 without neurological disorders. Cyanide levels in whole blood and urine of MND patients were significantly higher than the non-neurological control groups in smokers and non-smokers. Cyanide levels in whole blood of MND patients were also higher than the neurologic control group in smokers, but not in non-smokers. There was no significant difference between the cyanide level and either the clinical types or degree of disability of MND. The results suggest that MND patients possess a disorder in cyanide metabolism.

Nuclear envelope proteins and neuromuscular diseases.

Several neuromuscular diseases are caused by mutations in emerin and A-type lamins, proteins of the nuclear envelope. Emery-Dreifuss muscular dystrophy is caused by mutations in emerin (X-linked) or A-type lamins (autosomal dominant). Mutations in A-type lamins also cause limb-girdle muscular dystrophy type 1B, dilated cardiomyopathy with conduction defect, and Charcot-Marie-Tooth disorder type 2B1. They also cause partial lipodystrophy syndromes. The functions of emerin and A-type lamins and the mechanisms of how mutations in these proteins cause tissue-specific diseases are not well understood. The mutated proteins may cause structural damage to cells but may also affect processes such as gene regulation. This review gives an overview of this topic and describes recent advances in identification of disease-causing mutations, studies of cells and tissues from subjects with these diseases, and animal and cell culture models.