An agonist of CXCR4 induces a rapid recovery from the neurotoxic effects of Vipera ammodytes and Vipera aspis venoms.

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA2 neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.

Mechanical and electrophysiological properties of the sarcolemma of muscle fibers in two murine models of muscle dystrophy: col6a1-/- and mdx.

This study aimed to analyse the sarcolemma of Col6a1-/- fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1-/- and increased in mdx fibers. In the membrane of Col6a1-/- fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.

The inhibitor kappaB-ortholog Cactus is necessary for normal neuromuscular function in Drosophila melanogaster.

The Drosophila inhibitor-kappaB ortholog Cactus acts as an inhibitor of the Rel-transcription factors Dorsal and Dif. In blastoderm cells and immune competent cells, Cactus inhibits Dorsal and Dif by preventing their nuclear localization. Cactus, Dorsal and Dif are also expressed in somatic muscles, where Cactus and Dorsal, but not Dif, are enriched at the neuromuscular junction. Mutations in dorsal cause neuromuscular defects and mislocalization of Cactus. Here, we investigated whether mutations in cactus affect the neuromuscular system and subcellular localization of Dorsal and Dif. Using locomotion assays, as well as physiological and immunochemical methods, we found that wild type Cactus is necessary for the normal function of the larval neuromuscular system. The phenotype comprises i) altered bouton numbers and impaired neurotransmitter release in the neuromuscular junctions in the abdominal segments, ii) muscular weakness and iii) poor locomotion performance, probably reflecting a general neuromuscular impairment. Interestingly, in cactus mutants the subcellular localization of Dorsal and Dif in muscle is not affected, whereas cactus protein is not detected in the nucleus. This suggests, together with the similarities between the phenotypes induced by cactus and dorsal mutations, that in larval muscles the function of Cactus might be cooperation to the transcriptional activity of Rel proteins more than their cytoplasmic retention. The similarities with inhibitor-kappaB/nuclear factor kappaB interactions and muscle pathology in mammals point to Drosophila as a suitable experimental system to clarify the complex interactions of these proteins in muscle postembryonic development and activity.

Comparative analysis of the nonA region in Drosophila identifies a highly diverged 5' gene that may constrain nonA promoter evolution.

A genomic fragment from Drosophila virilis that contained all the no-on-transientA (nonA) coding information, plus several kilobases of upstream material, was identified. Comparisons of nonA sequences and the gene nonA-like in D. melanogaster, a processed duplication of nonA, suggest that it arose before the split between D. melanogaster and D. virilis. In both species, another gene that lies <350 bp upstream from the nonA transcription starts, and that probably corresponds to the lethal gene l(1)i19, was identified. This gene encodes a protein that shows similarities to GPI1, which is required for the biosynthesis of glycosylphosphatidylinositol (GPI), a component for anchoring eukaryotic proteins to membranes, and so we have named it dGpi1. The molecular evolution of nonA and dGpi1 sequences show remarkable differences, with the latter revealing a level of amino acid divergence that is as high as that of transformer and with extremely low levels of codon bias. Nevertheless, in D. melanogaster hosts, the D. virilis fragment rescues the lethality associated with a mutation of l(1)i19e, as well as the viability and visual defects produced by deletion of nonA(-). The presence of dGpi1 sequences so close to nonA appears to have constrained the evolution of the nonA promoter.

Molecular Dissection of the 5' Region of no-on-transientA of Drosophila melanogaster Reveals cis-Regulation by Adjacent dGpi1 Sequences.

The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies approximately 350 bp downstream of the dGpi1 gene. Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries approximately 2 kb of 5' regulatory material and that encodes most of the coding sequence of dGpi1. We have analyzed this 5' region by making a series of deleted fragments, fusing them to yeast GAL4 sequences, and driving UAS-nonA expression in a mutant nonA background. Regions that both silence and enhance developmental tissue-specific expression of nonA and that are necessary for generating optomotor visual responses are identified. Some of these overlap the dGpi1 sequences, revealing cis-regulation by neighboring gene sequences. The largest 5' fragment was unable to rescue the normal electroretinogram (ERG) consistently, and no rescue at all was observed for the courtship song phenotype. We suggest that sequences within the nonA introns that were missing in the UAS-nonA cDNA may carry enhancer elements for these two phenotypes. Finally, we speculate on the striking observation that some of the cis-regulatory regions of nonA appear to be embedded within the coding regions of dGpi1.

Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.

Giant neuron pathway neurophysiological activity in per(0) mutants of Drosophila melanogaster.

In Drosophila melanogaster, the clock gene period (per) has a clearly defined role in the molecular machinery involved in generating free-running circadian rhythms. per mutations also influence rhythms in the Drosophila love song and in the ultradian timescale. The relationship between these two phenomena has so far escaped satisfactory explanation. Here we analyzed the neurophysiological activity of the giant fiber neural pathway in per(0) flies. Under constant light, and at relatively low stimulation frequencies (1-2 Hz), per(01) flies habituate significantly earlier than they do under 12 h light-dark cycles. The results suggest an involvement of per in phenomena of short-term neural plasticity.

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres.

The Ca2+ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca2+ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca2+ capacity and the rate of Ca2+ uptake decreased from the control values of 0.384 +/- 0.030 micromol (mg fibre protein)-1 and 19.8 +/- 1.9 nmol min-1 (mg fibre protein)-1, respectively, to 0.210 +/- 0.016 micromol (mg fibre protein)-1 and 13.5 +/- 0.9 nmol min-1 (mg fibre protein)-1; the calculated amount of Ca2+ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 micromol (mg fibre protein)-1. In the 7-day denervated muscle, the SR Ca2+ capacity and the rate of Ca2+ uptake increased to 0.517 +/- 0.06 micromol (mg fibre protein)-1 and 21.6 +/- 2.3 nmol min-1 (mg fibre protein)-1, respectively; the calculated amount of Ca2+ released increased to 0.217 micromol (mg fibre protein)-1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca2+ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation.

Slow-to-fast transformation of denervated soleus muscle of the rat, in the presence of an antifibrillatory drug.

The myofibrillar changes of rat denervated soleus muscle were studied in the presence and in the absence of an antifibrillatory drug. After bilateral sciaticotomy, a concentrated solution of procainamide hydrochloride was steadily released, by way of a miniosmotic pump, in the space between the soleus and the gastrocnemius muscles of one leg. Fibrillation activity of soleus muscles was checked electromyografically at 3- to 5-day intervals. On the 21st day following denervation the muscles were excised, stained for adenosine triphosphatase activity and analysed for myosin heavy chain (MHC) isoforms. In the denervated-procainamide-treated muscles fibrillation was consistently (-75% on average) depressed in comparison to the contralateral denervated muscles. Type 1 (slow) fibres and MHC isoform were also significantly reduced, to the advantage of type 2A (fast) fibres and MHC isoform. The results support the view that denervation inactivity, like other kinds of muscle inactivity, favours the expression of fast type myofibrillar isoforms, and that this effect is counteracted, at least partially, by the spontaneous activity of the denervated muscle.

Nerve control of type 2A MHC isoform expression in regenerating slow skeletal muscle.

Bupivacaine-induced regeneration was studied in rat soleus muscle under several conditions, with the focus on type 2A and type 1 myosin heavy chain (MHC) isoform expression. In denervated muscles, type 1 was absent, whereas type 2A was widely expressed, a pattern of regeneration which appeared to be independent of fibrillation activity of the muscle. Both type 1 and type 2A isoforms were absent in muscles regenerated during tetrodotoxin (TTX) block of impulse conduction in the sciatic nerve, but type 2A was still present when the TTX block was associated with the vinblastine block of axoplasmic flow; vinblastine block alone caused the coexpression of type 1 and type 2A isoforms in the majority of fibers. These results suggest that axoplasmic flow carries some chemical factor that inhibits 2A MHC isoform expression. The results are also of clinical interest, contributing to the understanding of factors controlling muscle differentiation and adaptation.

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles.

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.

Lack of type 1 and type 2A myosin heavy chain isoforms in rat slow muscle regenerating during chronic nerve block.

The degeneration-regeneration process was induced by bupivacaine injection in innervated, denervated, and nerve-blocked rat soleus muscles. Nerve block was obtained by superfusion of the sciatic nerve with tetrodotoxin (TTX). Two weeks after bupivacaine injection, immunohistochemical and electrophoretical analyses showed the presence of type 1 myosin heavy chain (MHC) only in innervated regenerated muscles, type 2A in innervated and denervated, but not in TTX-paralyzed muscles, and type 2X under all experimental conditions. The presence of type 1 MHC in the innervated, and its absence in both denervated and TTX-paralyzed muscles were also verified immunohistochemically 1 week after bupivacaine injection. It is concluded that the nerve impulses play a determinant role in the expression of 1 and 2A MHC isoforms in the innervated regenerating muscle. The possible causes of the absence of the type 2A MHC isoform in the TTX-paralyzed muscles are discussed.