Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot-Marie-Tooth disease type 1B.

Charcot-Marie-Tooth disease type 1B (CMT1B) is genetically linked to chromosome 1q21-23. The major peripheral myelin protein gene, P0, has been cloned and localized to the same chromosomal region. P0 is a 28 kDa glycoprotein involved in the compaction of the multilamellar myelin sheet and accounts for more than half of the peripheral myelin protein content. We checked whether P0 is altered in CMT1B, and show here that a 3 basepair deletion in exon 2 of the P0 gene is present in all affected individuals of a CMT1B family. The mutation results in the deletion of serine 34 in the extracellular domain of P0, suggesting that alterations of P0 cause CMT1B.

Oxidative phosphorylation in human muscle in patients with ocular myopathy and after general anaesthesia.

The fuel preference of human muscle mitochondria has been given. Substrates which are oxidized with low velocity cannot be used to detect defects in oxidative phosphorylation. After general anaesthesia, the oxygen uptake with the different substrates is much lower than after local analgesia. The latter was therefore used in the subsequent study. In 15 out of 18 patients with ocular myopathy, defects in oxidative phosphorylation could be detected in isolated muscle mitochondria prepared from freshly biopsied tissue. Measurement of the activity of segments of the respiratory chain in homogenate from frozen muscle showed no, or minor defects. In two of these patients showing exercise intolerance, decreased oxidation of NAD(+)-linked substrates and apparently normal mitochondrial DNA, further study revealed deficiency of pyruvate dehydrogenase in a girl with ptosis and a high Km of complex I for NADH in a man. Both patients responded to vitamin therapy.

[The practice guideline 'Dermatomyositis, polymyositis and sporadic inclusion body myositis']. / Richtlijn 'dermatomyositis, polymyositis en sporadische "inclusion body"-myositis'.

This guideline presents recommendations for the diagnosis and treatment of dermatomyositis, polymyositis and sporadic inclusion body myositis (sIBM) according to the best available evidence. Characteristic skin abnormalities can be sufficient for the diagnosis of dermatomyositis. In case of doubt, a skin biopsy is advisable. A muscle biopsy is indicated when other examinations are inconclusive and the musculature is involved. The working group considers screening for cancer to be required in adults with dermatomyositis and presents recommendations for the way that this should be done. At least one-third of all patients with polymyositis has, or will develop, an associated inflammatory connective tissue disease. If a patient with a connective tissue disease develops symmetrical, proximal muscle weakness in the course of weeks or months, this may be assumed to be due to polymyositis. In the absence ofpre-existing connective tissue disease, demonstration of a mononuclear cell infiltrate in muscle tissue is a prerequisite for the diagnosis ofpolymyositis. The histopathology of muscle tissue is used as the gold standard for the diagnosis of sIBM. The practice guideline presents criteria for the concept 'activity' of myositis. Disease activity serves as a guideline for the treatment of polymyositis and dermatomyositis. The treatment of choice for dermatomyositis and polymyositis is high-dose prednisone. Physical activity does not have a negative effect on the course of these diseases. The long-term prognosis ofdermatomyositis and polymyositis is not well known. The clinical course of sIBM is slowly progressive.

Polyneuropathies and CNS protein metabolism. III. Changes in protein synthesis rate induced by acrylamide intoxication.

A defect in neuronal protein metabolism has been proposed as one of the primary, molecular events underlying the development of polyneuropathies of the dying back type. Using acrylamide-intoxication as an experimental model to study these polyneuropathies, changes in leucine-incorporation into proteins disability. Proteinsynthesis rates were determined in vivo using flooding concentrations of [1-14C]valine as the precusor. Under conditions of acute and of chronic intoxication, a decrease in synthesis rate was measured preceding the loss of functional ability. Similar changes in protein synthesis rate were observed in peripheral tissues such as heart muscle and liver showing the general toxicity of acrylamide. Methylene bisacrylamide, that was used to discriminate between the neurotoxic action of acrylamide and its systemic effects, interfered with protein synthesis rates in a comparable way. No change in protein synthesis rate was observed under in vitro conditions suggesting that the interference of acrylamide with the synthetic machinery for protein synthesis in vivo is mediated by one or more as yet unknown indirect factors.

Autosomal recessive form of hereditary motor and sensory neuropathy type I.

We studied pathologic changes in sural nerve biopsies from four patients with probable autosomal recessive (AR) hereditary motor and sensory neuropathy (HMSN) type I with a median motor nerve conduction velocity greater than 10 m/sec, comparing them with the pathologic features in autosomal dominant (AD) HMSN type I. The four recessive and two sporadic cases showed segmental demyelination. However, the classic onion bulbs of concentric Schwann cell processes, which occur in AD type I, were rare; many axons, also of a smaller size, were surrounded by onion bulbs of basal laminae. Schwann cells of the myelinated and unmyelinated types were involved in these onion bulb formations. Patients with HMSN type I who have many basal lamina onion bulbs should be considered as having AR inheritance.

Congenital paucity of secondary synaptic clefts (CPSC) syndrome in 2 adult sibs.

We studied 2 elderly sibs with a congenital form of myasthenia who had ptosis since early childhood. The extraocular muscles were weak and the proximal limb muscles became slowly weaker throughout life. Laboratory investigations of biopsies of intercostal muscle from these patients showed the following abnormalities: the amplitude of miniature end-plate potentials was small and the binding of 125I-alpha-bungarotoxin at the end-plate area was reduced, suggesting a considerable reduction of acetylcholine receptors (AChRs). Secondary synaptic clefts were scarce, whereas the number of end-plates per muscle fiber was increased. There was no indication of impaired transmitter release as the quantal content was within the range of controls. We conclude that these patients suffered from the congenital paucity of secondary synaptic clefts (CPSC) syndrome, described recently in 2 cases of myasthenic children, and suggest that the CPSC syndrome is a developmental disorder in which a deficiency of AChRs may be caused by a decreased clustering or insertion of AChRs. The increased number of end-plates per muscle fiber in both patients could serve as a compensatory mechanism.

Intermittent cyclophosphamide and prednisone treatment of polyneuropathy associated with monoclonal gammopathy of undetermined significance.

In an open prospective study, we analyzed the effect of cyclophosphamide (300 mg/m2 body surface daily for 4 days) combined with prednisone (40 mg/m2 body surface daily for 5 days) at 4-week intervals during 6 months in 16 patients with polyneuropathy associated with monoclonal gammopathy of undetermined significance (MGUS). Eleven patients had an IgM-MGUS and five an IgG-MGUS. During a follow-up period of 3 years, eight patients had improvement and six patients stabilized, based on quantitative neurologic examination, the Rankin disability scale, and electrophysiologic studies. These 14 patients had neuropathy with demyelinating and axonal features. One patient with a purely axonal neuropathy had deterioration despite therapy. One other patients developed severe leukopenia as side effect of cyclophosphamide, necessitating withdrawal of treatment. A difference in response was not present in patient with IgM- or IgG-MGUS, nor in patients with or without autoantibodies against myelin-associated glycoprotein. Nine patients had a bone marrow biopsy before and 1 year after treatment. In eight patients, the monoclonal lymphoid IgM or plasma cell IgG infiltration decreased, while in four the monoclonality disappeared after treatment. In the patient who had neurologic deterioration, repeated bone marrow biopsy showed deposits of amyloid. In conclusion, short-term treatment with intermittent cyclophosphamide and prednisone may have a long-term favorable effect in patients with demyelinating polyneuropathy associated with MGUS.

Two divergent types of nerve pathology in patients with different P0 mutations in Charcot-Marie-Tooth disease.

In seven unrelated patients with a demyelinating motor and sensory neuropathy, we found mutations in exons 2 and 3 of the P0 gene. Morphologic examination of sural nerve biopsy specimens showed a demyelinating process with onion bulb formation in all cases. In four patients, ultrastructural examination demonstrated uncompacted myelin in 23 to 68% of the myelinated fibers, which is in agreement with the widely accepted function of P0 as a homophilic adhesion molecule. Three patients showed normal compact myelin, but morphology was dominated by the abundant occurrence of focally folded myelin. The two divergent pathologic phenotypes exemplify that some mutations act differently on P0 protein formation or function than others, which is probably determined by site and nature of the mutation in the P0 gene.

Calcitonin gene-related peptide-like immunoreactivity, in botulinum toxin-paralysed rat muscles.

Changes in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) at the motor endplates of botulinum toxin-paralysed rat muscles were investigated using immunohistochemistry. One day following toxin injection, a dramatic increase in CGRP-LI was detected at the motor endplates and within preterminal axons of the soleus and gastrocnemius muscles. The upregulation of CGRP-LI persisted throughout the period during which muscle fibres were paralysed and new neuromuscular junctions were being formed by the growing sprouts. Decline of CGRP-LI at the motor endplates coincided with clinical recovery. Both up- and down-regulation of CGRP-LI took place earlier in the soleus than in the gastrocnemius muscle. Up-regulation of CGRP-LI was also detected in a subpopulation of motor axons in the sciatic nerves and in the spinal motor neurons innervating the paralysed muscles. These results indicate that levels of CGRP are regulated, at least partly, by changes in the target innervation. They also suggest an important role for CGRP in the regenerative processes following muscle paralysis.

The status of HMSN type III.

The indistinctness of the HMSN type III concept of Dyck (1975) prompted us to evaluate the diagnostic criteria. Based on a literature review and the observations in five of our own cases, restricted criteria are formulated. We conclude that at present the diagnosis of AR HMSN type III can be applied reasonably to the condition of "congenital hypomyelination", which shows a congenital or early childhood onset, extremely slow motor nerve conduction velocities of less than 6-7 m s-1 in upper limbs, and in nerve biopsy only fibres with no or hardly any myelin and "onion bulbs" of basal lamina. Amyelination might be the most severe or earliest expression of congenital hypomyelination. The existence of an inherited type III with mainly classical onion bulbs is uncertain, as only sporadic cases have been described.

Evaluation of electrophysiological and clinical tests in an exploratory trial of Org 2766 in motor neuron disease.

Twenty four patients with motor neuron disease (MND) participated in a double-blind, placebo-controlled trial with the ACTH 4-9 analog, Org 2766. Patients were examined three times during an 8 week treatment period, using a summated score for several manually and functionally tested muscles (sum score), myometry, jitter, fibre density (FD), macro motor unit potential (MUP), and supramaximal evoked muscle action potentials. No differences were found between Org 2766 and placebo treated patients. In an open 1 yr follow-up study, 5 out of 13 patients treated with Org 2766 died; the others showed continued progression of weakness. The methods used for assessment of muscle function were compared. The highest interest reliability was obtained in the sum score and myometry. Mean differences that might be detectable were relatively small for the sum score and myometry, and large for FD and MUP. We concluded that clinical function testing and myometry are superior to electromyographic measurements for assessment of changes in MND patients.

A novel gamma-sarcoglycan mutation causing childhood onset, slowly progressive limb girdle muscular dystrophy.

Limb girdle muscular dystrophy is a heterogeneous group of disorders. One autosomal recessive subtype, LGMD2C, has been linked to chromosome 13, and is caused by gamma-sarcoglycan deficiency in muscle. This report describes a novel missense mutation identified in a large consanguineous Dutch family with LGMD. This mutation leads to reduction of gamma-sarcoglycan, and gives rise to a childhood-onset, slowly-progressive dystrophy.

Immunohistochemical localization of acetylcholine receptors at human endplates using a monoclonal antibody.

We describe a simple indirect immunohistochemical method for localization of acetylcholine receptors (AChR) in motor endplates at the light and electron microscopic level. This method involves the use of a monoclonal antibody directed against the main immunogenic region (MIR) of AChRs and is applicable to periodate-lysine-paraformaldehyde (PLP)-fixed tissue. We discuss the advantages of this method, as compared with the alpha-bungarotoxin-immunoperoxidase technique, and stress its value for diagnostic investigations of motor point biopsies from patients with neuromuscular transmission disorders.

ACTH/MSH like peptides in the treatment of cisplatin neuropathy.

The neurological toxicity seen in patients treated with cisplatin in most cases concerns ototoxicity and peripheral neuropathy. Thus far, the pathogenesis of cisplatin neuropathy remains obscure. Yet the fact that cisplatin affects mainly the sensory peripheral nerve fibers points towards an involvement of the dorsal root ganglia. In a rat model of cisplatin neuropathy, following a cumulative dose of approx. 12 mg/kg cisplatin the sensory nerve conduction velocity began to slow as compared to age-matched controls. Peptides derived from ACTH and MSH are known to exert neurotrophic effects. In vivo they facilitate postlesion repair mechanisms in the peripheral nervous system by enhancing the early sprouting response of the damaged nerve. Surprisingly, chronic treatment with a synthetic ACTH4-9 analog not only prevented cisplatin neurotoxicity following a low or high dose regimen, but also counteracted already existing cisplatin-induced neurotoxicity. Stimulated by these findings a randomized, double blind, placebo-controlled study was performed to assess the efficacy of the peptide in the prevention of cisplatin neuropathy in women suffering from ovarian cancer. The threshold of vibration perception (VPT) was used as the principal measure of neurotoxicity. Following 6 cycles of chemotherapy the VPT had increased more than 8-fold in women receiving placebo as co-medication. Whereas the VPT in women receiving 1 mg/m2 body surface ACTH4-9 analog before and after each cisplatin cycle only increased less than 2-fold. No side effects of the peptide treatment were observed and the clinical response to the chemotherapy was similar in all treatment groups. Collectively these preclinical and clinical data suggest that treatment based on non-endocrine fragments of ACTH/MSH may be a therapeutic option in the treatment of cisplatin neuropathy.

No evidence for enhancement of recovery from a crush lesion of a motor nerve in rats by Org2766.

The present study was undertaken to investigate the effect of Org2766 on the recovery of lesioned motor nerve fibres. The facial nerve of Wistar rats was crush lesioned and the process of recovery was assessed by functional and histological methods. Functional recovery was examined in two experiments and was assessed by quantifying blind the return of eyelid and whisker movements. No difference could be demonstrated between Org2766-treated and saline-treated groups of animals. The histological investigations involved quantifying the number of reinnervated motor endplates in the whisker muscle. On days 10 to 12 in male rats and on days 12 and 13 in female rats, there was no difference in reinnervation between the two treatment groups. It was concluded that with the methods used in this study, no significant effect of Org2766 on the axonal regeneration of motor nerve fibres could be established.

Upregulation of B-50/GAP-43 in Schwann cells at denervated motor endplates and in motoneurons after rat facial nerve crush.

Crush or transection of peripheral nerves of the adult rat is accompanied by changes in protein expression, including the growth associated protein (GAP-43) B-50. Following peripheral nerve crush in rat enhanced B-50 immunoreactivity was observed in regenerating nerve fibres and in newly formed axon terminals. However, before reinnervation was apparent, an unexpected transient increase in B-50 immunoreactivity was observed at denervated motor endplates [J. Neurosci. 8 (1988) 1759]. This study was performed to clarify this observation. Four days following facial nerve crush B-50 immunoreactivity was detected by double immunofluorescence microscopy in Sl00-positive Schwann cells covering the denervated endplates. Using diluted polyclonal and monoclonal B-50 antibodies we found that B-50 immunoreactivity at the denervated motor endplates was strongly increased in comparison to innervated motor endplates in which B-50 immunoreactivity was hardly detectable. However, when a high concentration of B-50 antibodies was applied the normal innervated motor endplates were also B-50 immunoreactive. Muscle fibres did not display B-50 immunoreactivity. Northern blot analysis revealed elevated B-50 mRNA in denervated muscle and in degenerating nerve with respect to the controls. The B-50 mRNA levels in these non-neuronal tissues were very low compared to the intact and injured facial nucleus containing the neuronal cell bodies. Electron microscopy demonstrated that the B-50 protein was localized in the processes of Schwann cells covering axon terminals of intact and vacant motor endplates and in axon varicosities of sympathetic nerves. This study has confirmed that prior to reinnervation B-50 immunoreactivity is increased at denervated motor endplates and shows that B-50 is co-localized with S100 in Schwann cells. Therefore, upregulation of B-50 expression in Schwann cells may explain the early occurrence of B-50 immunoreactivity at the motor endplate.

B-50/GAP-43 in neuronal development and repair.

The protein kinase C substrate B-50 is identical to the growth-associated protein GAP-43. Although as yet no causal relationship has been established between B-50/GAP-43 and neurite outgrowth, evidence accumulates that the function of the protein relates to neuronal plasticity. Stimulation of PC12 cells by NGF results in translocation of the protein from cytosolic stores to the membrane of newly formed neurite-like extensions. The protein is associated with the inner leaflet of the growth cone membrane isolated from neonatal rat brain and was used as a marker to study the development of the rat pyramidical tract and olfactory system. In the adult rat, crush lesion of the sciatic nerve results in a rapid expression of B-50/GAP-43 mRNA followed by synthesis of B-50/GAP-43 protein in dorsal root ganglia and transport of the protein into the newly formed sprouts. Presumably, the neurotrophic effect of melanocortins on peripheral nerve repair is not brought about by enhancement of B-50/GAP-43 synthesis per se. Bulbectomy (central) or Triton X-100 lesioning (peripheral) of the olfactory epithelium results in a differential expression of B-50/GAP-43 and the olfactory marker protein characterizing two stages in the regeneration of this epithelium. Evidence that the degree of phosphorylation may co-determine the role of B-50/GAP-43 in neurite outgrowth is discussed.

A congenital myasthenic disorder with paucity of secondary synaptic clefts: deficiency and altered distribution of acetylcholine receptors.

Congenital myasthenia (CM) constitutes a heterogeneous group of disorders with different underlying defects. The authors investigated a case of CM, presenting with congenital contractures. Endplate studies in the first year of life showed a developmental disorder of postsynaptic membranes. Clinical follow-up demonstrated a beneficial effect of pyridostigmine, resulting in normal motor development. Results of a second biopsy at age 4 are reported in this paper. Microelectrode study showed small Mepp amplitudes, which returned to nearly normal in the presence of neostigmine. In the electronmicroscope the postsynaptic membranes showed a paucity of infoldings, as in the first biopsy. These membranes showed only scanty, patchy enhancement with two different methods for localization of AChR. The extrajunctional membranes showed evidence of local presence of AChR. Our results show a developmental disorder of postsynaptic membranes with a deficiency and altered distribution of AChRs.

Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts.

Adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate the initial sprouting response in the crushed rat sciatic nerve. In this report a detailed analysis of the neurotrophic action of Org.2766 [a degradation resistant ACTH(4-9) analog] and alpha-MSH is described. Org.2766 treatment results in enhanced numbers of outgrowing sprouts in the damaged nerve. The growth velocity of the sprouts is not affected. The peptide effect is dose-dependent. A single peptide injection administered immediately following the crush stimulates the formation of sprouts significantly. Continued high blood levels of Org.2766 are probably not critical for the neurotrophic effect of these peptides, since a more moderate dosing protocol (injections given every 48 hr) was more effective than more frequent injections (injections given every 12 hr). The present results further the understanding of the mode of action of ACTH/alpha-MSH-like peptides and underscore the necessity to test a wide range of doses and injection protocols to avoid false negative results in clinical work being planned to start in the near future.

Effects of ciliary neurotrophic factor on retrograde cell reaction after facial nerve crush in young adult rats.

Locally applied ciliary neurotrophic factor (CNTF) has a powerful effect on retrograde axonal reaction following facial nerve crush in neonatal rats. We examined whether it also exerts a strong effect on retrograde axonal reaction in young adult rats when administered subcutaneously. The dose was 1 mg/kg body weight, three times a week, similar to that used in a previous experiment in which CNTF was reported to have an effect. We studied changes in the morphology of the motor nerve cell bodies, in the number of perineuronal microglial cells and in the expression of five proteins. It appeared that CNTF prevented swelling of the facial motoneuron cell bodies but it did not influence the swelling of the nucleus nor the shift of the nucleus towards the periphery. In saline-treated rats, facial nerve crush resulted from day two to day six in a marked increase in the number of perineuronal glial cells. This increase was neither diminished nor augmented by CNTF. Following facial nerve crush, the immunoreactivity of the proteins C3bi, GFAP, B-50 and CGRP increased in the glial cells and motoneuron cell bodies, whilst the immunoreactivity of synaptophysin at the membrane of the motoneuron cell bodies decreased. CNTF had no obvious effect on these changes. It was concluded that in young adult rats under the present conditions, CNTF had only a small effect on a specific aspect of the retrograde cell reaction. The small effects might be explained by the minor availability of CNTF to the motoneuron cell bodies. The gain in body weight of rats treated with CNTF was less than that of saline-treated rats.