MicroRNA signatures predict dysregulated vitamin D receptor and calcium pathways status in limb girdle muscle dystrophies (LGMD) 2A/2B.

miRNA expression profile and predicted pathways involved in selected limb-girdle muscular dystrophy (LGMD)2A/2B patients were investigated. A total of 187 miRNAs were dysregulated in all patients, with six miRNAs showing opposite regulation in LGMD2A versus LGMD2B patients. Silico analysis evidence: (1) a cluster of the dysregulated miRNAs resulted primarily involved in inflammation and calcium metabolism, and (2) two genes predicted as controlled by calcium-assigned miRNAs (Vitamin D Receptor gene and Guanine Nucleotide Binding protein beta polypeptide 1gene) showed an evident upregulation in LGMD2B patients, in accordance with miRNA levels. Our data support alterations in calcium pathway status in LGMD 2A/B, suggesting myofibre calcium imbalance as a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd.

Expression of transglutaminase 2 does not differentiate focal myositis from generalized inflammatory myopathies.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), sporadic inclusion-body myositis (s-IBM) and focal myositis (FM) are a heterogeneous group of autoimmune disorders of skeletal muscle. An increased transglutaminase 2 (TG2) expression has been found in DM, PM and s-IBM. The aim of our study was to investigate TG2 expression in FM in comparison with other IIM. MATERIALS AND METHODS: We re-examined tissue material we have gathered in the course of our previous studies on IIM, investigating muscle expression of TG2 in patients with FM in comparison with DM, PM and s-IBM using immunocytochemistry and real-time RT-PCR. RESULTS: Immunocytochemistry revealed an increased TG2 signal in endomysial vessels, in atrophic and degenerating/regenerating muscle fibres in PM, DM, s-IBM and FM; in s-IBM, some vacuoles were immunostained too. Real-time RT-PCR study confirmed a significantly increased expression of TG2 in all IIM muscles examined. CONCLUSIONS: Our study demonstrates the presence of TG2 in FM muscles. The study suggests that TG2 expression does not represent a distinctive marker to differentiate FM from generalized IIM. TG2 over-expression in inflamed skeletal muscle does not seem have a pathogenetic role in such a disease, but it could represent a way to contain the inflammatory process.

Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells.

The stimulation of NMDA receptor increased [3H]GABA release from preloaded cultured retina cells. This effect appears to be mediated by NO production, since addition of L-NA reduces NMDA-evoked [3H]GABA release. Spermine/NO complex, an NO donor, mimics the effect produced by NMDA. The addition of zaprinast, a phosphodiesterase inhibitor, as well as 8-Br-cGMP enhances the NMDA-evoked [3H]GABA release. These results agree with the existence in chick retina cells of NO/cGMP pathways and support a role for NO in NMDA-evoked events. The activation of this receptor complex through maturative stages of the retina together with the NO-mediated increase in GABA release may account for NMDA differentiative effect in culturing retina cells.

NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells.

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells. These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure. These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.

Preliminary report: Amino acid profile in platelets of diabetic patients.

Platelet levels of 19 amino acids were measured in 20 outpatients with type 1 (age [mean +/- SE], 35.5 +/- 2.0 years) and 27 with type 2 (age, 58.4 +/- 1.4 years) diabetes, and 20 young (age 33.7 +/- 1.3 years) and 20 older (age 57.4 +/- 1.5 years) healthy volunteers. Platelet levels of most amino acids tended to be lower in patients with type 1 diabetes than in healthy controls. In particular, asparagine, glycine, taurine, alanine, valine, cysteine, leucine, phenylalanine, and lysine levels, expressed as nmol/10(8) platelets, were significantly lower. Only taurine significantly decreased in patients with type 2 diabetes, whereas threonine, alanine, and isoleucine increased.

Botulinum toxin in upper limb spasticity: study of reciprocal inhibition between forearm muscles.

To establish whether botulinum A toxin (BTX-A) acts on modifying reciprocal inhibition between forearm muscles in spasticity, 20 patients with post-stroke upper limb spasticity lasting for more than 1 year were studied. Clinical examination, physiotherapeutic evaluation, standardized video-tape assessment and electrophysiological testing (flexor carpi radialis muscle M and H responses with study of reciprocal inhibition) were performed at baseline and 2 weeks, 1, 2, 3, 4 months after BTX-A treatment. BTX-A induced a significant decrease of tone and an improvement of motility and functional status, with a significant decrease of the M wave and the H reflex. The reduction in both inhibitory phases of reciprocal inhibition did not change after BTX-A treatment differently from that reported in upper limb dystonia. These findings indicate that the efficacy of BTX-A in upper limb spasticity is mainly due to peripheral effects.

Glutamate-induced increases in transglutaminase activity in primary cultures of astroglial cells.

Glutamate exposure of astroglial cells caused ligand-gated channel receptor activation, associated with excitotoxic cell response. We investigated the effects of 24 h glutamate exposure on transglutaminase in astrocytes primary cultures at 7, 14, and 21 days in vitro (DIV). Increases in enzyme activity were observed as a function of cell differentiation stage in glutamate-treated cultures. These effects were significantly reduced when GYKI 52466, an AMPA/KA receptors inhibitor, was added to the culture medium prior to incubation with glutamate. Microscopy observation on transglutaminase-mediated, fluorescent dansylcadaverine incorporation in living cells was consistent with these results. Western blotting analysis with monoclonal antibody showed that glutamate also up-regulated tissue transglutaminase expression, which reached the highest values in 14 DIV cultures. Confocal laser scanning microscopy analysis of immunostained astroglial cells showed a mainly cytoplasmic localisation of the enzyme both in control and treated cultures; nevertheless, counterstaining with the nuclear dye acridine orange demonstrated the presence of tissue transglutaminase also into the nucleus of glutamate-exposed and 21 DIV cells. The increases in enzyme expression and localisation in the nucleus of glutamate-treated astroglial cells may be part of biochemical alterations induced by excitotoxic stimulus.

Electrophysiological study of neuromuscular system involvement in mitochondrial cytopathy.

OBJECTIVES: To define the neuromuscular involvement in 'mitochondrial' patients with clinical evidence of a neuromuscular disorder, and to evaluate if the proposed electrophysiological protocol was suitable to reveal a subclinical neuropathy or myopathy in 'mitochondrial' patients with no clinical sign of a neuromuscular disturbance. METHODS: Quantitative concentric needle electromyography (CNEMG), single fiber electromyography (SFEMG) and nerve conduction studies (NCS) were performed in 33 patients with mitochondrial cytopathies. Lastly, we studied 9 clinically unaffected relatives. RESULTS: NCS were abnormal in 18% of patients, with CNEMG and SFEMG in 58% of cases, but there was not a complete overlapping of the positivity of the different techniques. No asymptomatic relatives showed abnormalities of the electrophysiological studies. CONCLUSIONS: Electrophysiological findings did not correlate with any specific biochemical or genetic defect, but were consistent with clinical diagnosis in almost all of the patients with clinical signs of myopathy and/or neuropathy. Increase of both SFEMG jitter and fiber density was significantly tied to a neuropathic process. CNEMG and SFEMG were altered in about 30% of subjects without clinical signs of myopathy or neuropathy and were therefore able to reveal a subclinical involvement of neuromuscular system in some patients who had external ophthalmoplegia or retinitis only.

Extra-muscle involvement in dystrophinopathies: an electroretinography and evoked potential study.

Dystrophin is present in various tissues other than skeletal and cardiac muscles, including the central nervous system (CNS) and the outer plexiform layer of the retina. Therefore lack of dystrophin might be related to mental retardation or to changes in electrophysiological tests exploring retina and CNS. We performed electroretinography, VEPs, BAEPs, SEPs and MEPs in 18 patients with Duchenne muscular dystrophy (DMD), 18 with Becker muscular dystrophy (BMD) and 12 obligate carriers. We observed a marked reduction of the b-wave amplitude in the scotopic ERG, mainly in DMD patients. Oscillatory potentials were altered in all groups, even in carriers, suggesting that dystrophin may be also involved in retinal circulation. VEPs changes confirmed the role of dystrophin in visual function. The other evoked potentials were altered only in a small percentage of subjects but changes of different tests did not overlap in individual subjects. Neurophysiological abnormalities did not correlate with type, site and size of alteration in the dystrophin gene.

Onset of hypothyroidism with polymyositis-like clinical features in elderly patients.

Impaired muscle function may be a predominant aspect of hypothyroidism and is virtually present in all patients with overt thyroid failure. Less common is the onset of hypothyroidism with clinical features mimicking a polymyositis. We have observed 3 patients, whose age ranged 63-68 years, presenting with muscle aches, cramps, proximal weakness and stiffness. Two patients had dysphagia. Serum creatine kinase (CK) and electromyography (EMC) were altered in two patients. Muscle biopsy showed type II atrophy, sporadic type I and type II grouping, "core-like" areas, and some myopathic changes such as central nuclei and muscle necrosis. No inflammatory changes were present. Immunohistochemistry of several muscle cytoskeletal proteins revealed increased desmin in "corelike" areas. Detection of serum thyroid hormone levels revealed very low triiodo-L-thyronine (T3) and thyroxine (T4), whereas thyroid-stimulating hormone (TSH) was greatly increased as well as serum anti-thyroglobulin, anti-peroxidase and anti-microsome antibodies. The patients were diagnosed having a hypothyroid myopathy due to Hashimoto thyroiditis. L-thyroxine treatment normalized clinical and hormone levels, but serum antibodies remained elevated. Muscle biopsy was fundamental to establish the correct diagnosis in our patients. Presence of over-expression of desmin in cores, as described in target lesions in neurogenic diseases, may suggest a nerve-mediated pathogenesis of hypothyroid myopathy.

RAGE-NF-kappaB pathway activation in response to oxidative stress in facioscapulohumeral muscular dystrophy.

OBJECTIVES: An increased expression of adenine nucleotide translocator (ANT1), found in facioscapulohumeral muscular dystrophy (FSHD), is known to lead to a decrease in nuclear factor-kappaB (NF-kappaB) DNA binding and to sensitize muscle cells to oxidative stress and apoptosis. Receptor for advanced glycation end products (RAGE) mediated by NF-kappaB activation is involved in proinflammatory pathomechanism and in muscle fiber regeneration in inflammatory myopathies and in limb girdle muscular dystrophy. Oxidative stress can stimulate RAGE- NF-kappaB pathway. Our purpose was to verify if oxidative stress may induce RAGE- NF-kappaB pathway activation in FSHD, contributing to the pathogenesis of such a disease. MATERIALS AND METHODS: On muscle samples of eight patients with FSHD, eight patients with Duchenne muscular dystrophy and eight normal controls the following studies were carried out: immunocytochemistry for activated NF-kappaB; electrophoretic mobility shift assay of NF-kappaB DNA binding activity; Western blot studies of RAGE and ANT1; hydrogen peroxide (HP), peroxidase and glutathione peroxidase (GPx) assays. RESULTS: An increased RAGE and ANT1 expression in FSHD with moderate increase of NF-kappaB DNA binding activity was found together with an increased production of HP and a reduced activity of peroxidase and GPx. CONCLUSIONS: Our data confirm that response to oxidative stress and ANT1 increased activity are early events in FSHD muscle. The study also reveals that the RAGE- NF-kappaB pathway, induced by oxidative stress, is activated independently of the presence of a clear histochemical evidence of muscle damage in FSHD.

Neuroprotective effects of lamotrigine and remacemide on excitotoxicity induced by glutamate agonists in isolated chick retina.

The possible neuroprotective effects of two recently developed antiepileptic compounds, lamotrigine (LTG) and remacemide (REMA), against glutamate agonist-induced excitotoxicity have been investigated in the isolated chick embryo retina model. Retina segments from 15- or 16-day-old embryos were incubated in 1 ml of balanced salt solution, at 25 degrees C for 30 min, in the presence or absence of N-methyl-d-aspartate (NMDA), kainic acid (KA), or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (10 to 200 microM). LTG, REMA, and the active desglycinyl metabolite of REMA (d-REMA) (10-200 microM) were added separately 5 min before glutamate agonists. Retina damage was assessed after 24 h (i) by measuring LDH activity present in the medium, expressed as percentage of total retina LDH activity, and (ii) by histological analysis of retina specimens through scoring for the presence or absence of edema, necrosis, nuclear pyknosis, and cell layer damage. LTG, REMA, and d-REMA reduced LDH release produced by NMDA 58-70% in a dose-dependent manner, with d-REMA being the most potent (EC(50): d-REMA, 25.75 +/- 3.27 microM; REMA, 64.75 +/- 7.75 microM; LTG, 60.50 +/- 6.80 microM; P < 0.001). The drugs had less effect on the LDH release produced by AMPA and KA. Histological analysis confirmed these biochemical results, with all three compounds reducing edema and the number of necrotic and pyknotic nuclei in the ganglion layer. d-REMA provided almost complete protection of the ganglion cell layer against damage produced by NMDA. Combinations of d-REMA and LTG showed additive rather than potentiative effects against NMDA-induced cell injury. The present data provide pharmacological evidence that LTG, REMA, and d-REMA decrease glutamate agonist-induced excitotoxicity in isolated chick retina, findings that might have therapeutic implications for various neurological disorders.

Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina.

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.

Focal hand dystonia in a patient with thoracic outlet syndrome.

A patient affected by thoracic outlet syndrome, with an involvement of the left lower primary trunk due to a rudimentary cervical rib, developed a severe hand dystonia on the same side. The dystonic posture was characterised by a flexion of the wrist with the fingers curled into the palm. Polygraphic recordings performed on the left flexor digitorum superficialis (FDS4) and extensor digitorum superficialis (EDC4) muscles, during a repetitive tapping task of the fourth digit, showed a loss of well formed bursts without a clear silent period along with long duration bursts of cocontraction in antagonistic muscles. The study of reciprocal inhibition between forearm flexor and extensor muscles showed a reduced amount of inhibition in both the disynaptic and the later presynaptic phase of inhibition. The patient underwent an operation with resection of the cervical rib. Twelve hours after the operation the patient experienced a significant improvement of the hand dystonia; the distonia had disappeared completely by two months with a progressive normalisation of reciprocal inhibition.

Electrophysiological studies in mild idiopathic carpal tunnel syndrome.

Many techniques have been reported to improve the diagnosis of carpal tunnel syndrome (CTS), but there is no agreement on the diagnostic yield of these different methods. We used an electrophysiological protocol including the assessment of the orthodromic sensory conduction velocity of the median nerve along the carpal tunnel, comparison of median and ulnar sensory conduction between the ring finger and wrist, short segment incremental median sensory nerve conduction across the carpal tunnel recording from the III digit ('inching test'), the study of the refractory period of transmission (RPT) and calculation of the distoproximal ratio obtained by dividing the nerve conduction velocity in the median nerve between the third digit and the palm and between the palm and wrist in 41 patients with mild CTS (75 symptomatic hands) and in 45 control subjects. The distoproximal ratio calculation was the most sensitive technique (81%), but was also the least specific. The 'inching test', even though less sensitive, had the advantage of localising focal abnormalities of the median nerve along the carpal tunnel. RPT was abnormal in patients with recent symptoms. Combining the different techniques, an overall sensitivity of 92% was reached, 11% higher than the yield of the single best test suggesting that a multimodal approach could be useful. The best procedure for electrodiagnosis of mild CTS was to combine the median/ulnar comparison test with calculation of the disto-proximal ratio.