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The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.

In the last few years several studies have investigated the neural mechanisms underlying spatial orientation in Drosophila melanogaster. Convergent results suggest that this mechanism is associated with specific neural circuits located within the Central Complex (CC). Furthermore such circuits appear to be associated with visual attention, specifically with selective attention processes implicated in the control of action. Our aim was to understand how wild-type flies react to the abrupt appearance of a visual distractor during an ongoing locomotor action. Thus, we adapted the well-known ‘Buridan paradigm’, used to study walking behaviour in flies, so we could specifically address the mechanisms involved in action selection. We found that flies tended to react in one of two ways when confronted with a visual distractor during ongoing locomotion. Flies either: (i) committed to a new path situated midway between the original target and the distractor, consistent with a novelty effect; or (ii) remained on the original trajectory with a slight deviation in direction of the distractor. We believe that these results provide the first indication of how flies react, from the motor point of view, in a bi-stable context requiring the presence of selection-for-action mechanisms. Some considerations on the neural circuits underlying such behavioural responses are advanced.

Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality. Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.

INTRODUCTION The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. METHODS We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) . RESULTS We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. CONCLUSION PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, we modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, we determined that the glutamate transporter EAAT1 mediated the regulatory functions of TDP-43 in the glia and demonstrated that genetic or pharmacological compensations of EAAT1 activity were sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS.

Scorpions are among the oldest venomous living organisms and the family Buthidae is the largest and most medically relevant one. Scorpion venoms include many toxic peptides, but recently, a metalloprotease from Tityus serrulatus called antarease was reported to be capable of cleaving VAMP2, a protein involved in the neuroparalytic syndromes of tetanus and botulism. We have produced antarease and an inactive metalloprotease mutant in a recombinant form and analyzed their enzymatic activity on recombinant VAMP2 in vitro and on mammalian and insect neuromuscular junction. The purified recombinant antarease paralyzed the neuromuscular junctions of mice and of Drosophila melanogaster whilst the mutant was inactive. We were unable to demonstrate any cleavage of VAMP2 under conditions which leads to VAMP proteolysis by botulinum neurotoxin type B. Antarease caused a reduced release probability, mainly due to defects upstream of the synaptic vesicles fusion process. Paired pulse experiments indicate that antarease might proteolytically inactivate a voltage-gated calcium channel.

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo . Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro . These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.

Organisms are known to be equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. In this study, the effects of extreme changes in oxygen availability and atmospheric pressure on physiological phenotypes of Drosophila melanogaster were investigated to explore adaptation mechanisms. The changes in citrate synthase activity (CSA), lifespan, and behavioral function in different atmospheric conditions were evaluated. In the CAS test, hyperoxia significantly increased CSA; both hypoxia and hyperbaric conditions caused a significant decrease in CSA. In the survivorship test, all changed atmospheric conditions caused a significant reduction in lifespan. The lifespan reduced more after hypoxia exposure than after hyperbaria exposure. In behavioral function test, when mechanical agitation was conducted, bang-sensitive flies showed a stereotypical sequence of initial muscle spasm, paralysis, and recovery. The percentage of individuals that displayed paralysis or seizure was measured on the following day and after 2 weeks from each exposure. The majority of flies showed seizure behavior 15 days after exposure, especially after 3 h of exposure. The percentage of individuals that did not undergo paralysis or seizure and was able to move in the vial, was also tested. The number of flies that moved and raised the higher level of the vial decreased after exposure. Animal's speed decreased significantly 15 days after exposure to extreme environmental conditions. In summary, the alteration of oxygen availability and atmospheric pressure may lead to significant changes in mitochondria mass, lifespan, and behavioral function in D. melanogaster.

Summary Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P1′ position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein–protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane.

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (β-Bungarotoxin, β-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body.

Organisms are known to be equipped with an adaptive plasticity as the phenotype of traits in response to the imposed environmental challenges as they grow and develop. In this study, the effects of extreme changes in oxygen availability and atmospheric pressure on physiological phenotypes of Drosophila melanogaster were investigated to explore adaptation mechanisms. The changes in citrate synthase activity (CSA), lifespan, and behavioral function in different atmospheric conditions were evaluated. In the CAS test, hyperoxia significantly increased CSA; both hypoxia and hyperbaric conditions caused a significant decrease in CSA. In the survivorship test, all changed atmospheric conditions caused a significant reduction in lifespan. The lifespan reduced more after hypoxia exposure than after hyperbaria exposure. In behavioral function test, when mechanical agitation was conducted, bang-sensitive flies showed a stereotypical sequence of initial muscle spasm, paralysis, and recovery. The percentage of individuals that displayed paralysis or seizure was measured on the following day and after 2 weeks from each exposure. The majority of flies showed seizure behavior 15 days after exposure, especially after 3 h of exposure. The percentage of individuals that did not undergo paralysis or seizure and was able to move in the vial, was also tested. The number of flies that moved and raised the higher level of the vial decreased after exposure. Animal's speed decreased significantly 15 days after exposure to extreme environmental conditions. In summary, the alteration of oxygen availability and atmospheric pressure may lead to significant changes in mitochondria mass, lifespan, and behavioral function in D. 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.

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.

The Drosophila Rel transcription factor Dorsal and its inhibitor Cactus participate in a signal transduction pathway involved in several biologic processes, including embryonic pattern formation, immunity, and muscle development. In contrast with embryonic muscle, where Dorsal is reportedly absent, this protein and Cactus accumulates in the neuromuscular junctions in the muscle of both larvae and adults. The phenotype of homozygous dorsal mutant larvae suggested that Dorsal and Cactus maybe necessary for normal function and maintenance of the neuromuscular system. Here we investigate if these proteins can respond to synaptic activity. Using larval body wall preparations and antibodies specific for Dorsal or Cactus we show that the amount of these proteins at the neuromuscular junction is substantially decreased after electrical stimulation of the nerves or incubation in glutamate, the principal transmitter in this type of synapse. The specificity of the response was tested with a glutamate receptor antagonist (argiotoxin 636). Because the effect can be reproduced using a calcium ionophore (ionomycin treatment) as well as blocked by the inhibition of the muscle ryanodine receptor (tetracaine treatment), the involvement of calcium in this process seems likely. We also observed that the inhibition of the calcium dependent protein phosphatase calcineurin prevents the effect of glutamate on the fluorescence for Dorsal and Cactus, suggesting its participation in a signal transduction cascade that may activate Dorsal in the muscle independently of Toll. Our results are consistent with a novel function of the Rel factor Dorsal in a molecular pathway turned on by neural activity and/or contractile activity. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 525–536, 2003

Collagen VI is an extracellular matrix protein with broad distribution in several tissues. Although Col6a1 is expressed by Schwann cells, the role of collagen VI in the peripheral nervous system (PNS) is yet unknown. Here we show that Schwann cells, but not axons, contribute to collagen VI deposition in peripheral nerves. By using Col6a1-null mice, in which collagen VI deposition is compromised, we demonstrate that lack of collagen VI leads to increased myelin thickness (P

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.

The SNARE proteins VAMP/synaptobrevin, SNAP-25 and Syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine, derived from experiments performed with botulinum neurotoxins, which led to the identification of one arginine in SNAP-25 and one aspartate in Syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions among SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and Syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

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.

Homer represents a new and diversified family of proteins made up of several isoforms. The presence of Homer isoforms, referable to 1b/c and 2a/b, was investigated in fast- and slow-twitch skeletal muscles from both rat and mouse. Homer 1b/c was identical irrespective of the muscle, and Homer 2a/b was instead characteristic of the slow-twitch phenotype. Transition in Homer isoform composition was studied in two established experimental models of atrophy, i.e., denervation and disuse of slow-twitch skeletal muscles of the rat. No change of Homer 1b/c was observed up to 14 days after denervation, whereas Homer 2a/b was found to be significantly decreased at 7 and 14 days after denervation by 70 and 90%, respectively, and in parallel to reduction of muscle mass; 3 days after denervation, relative mRNA was reduced by 90% and remained low thereafter. Seven-day hindlimb suspension decreased Homer 2a/b protein by 70%. Reconstitution of Homer 2 complement by in vivo transfection of denervated soleus allowed partial rescue of the atrophic phenotype, as far as muscle mass, muscle fiber size, and ubiquitinazion are concerned. The counteracting effects of exogenous Homer 2 were mediated by downregulation of MuRF1, Atrogin, and Myogenin, i.e., all genes known to be upregulated at the onset of atrophy. On the other hand, slow-to-fast transition of denervated soleus, another landmark of denervation atrophy, was not rescued by Homer 2 replacement. The present data show that 1) downregulation of Homer 2 is an early event of atrophy, and 2) Homer 2 participates in the control of ubiquitinization and ensuing proteolysis via transcriptional downregulation of MuRF1, Atrogin, and Myogenin. Homers are key players of skeletal muscle plasticity, and Homer 2 is required for trophic homeostasis of slow-twitch skeletal muscles.

Mutations in the EFHC1 gene have been linked to juvenile myoclonic epilepsy. To understand EFHC1 function in vivo, we generated knockout Drosophila for the fly homolog Defhc1.1. We found that the neuromuscular junction synapse of Defhc1.1 mutants displays an increased number of satellite boutons resulting in increased spontaneous neurotransmitter release. Defhc1.1 binds to microtubules in vitro and overlaps in vivo with axonal and synaptic microtubules. Elimination of Defhc1.1 from synaptic terminals reduces the number of microtubule loops, suggesting that Defhc1.1 is a negative regulator of microtubule dynamics. In fact, pharmacological treatment of Defhc1.1 mutants with vinblastine, an inhibitor of microtubule dynamics, suppresses the satellite bouton phenotype. Furthermore, Defhc1.1 mutants display overgrowth of the dendritic arbor and Defhc1.1 overexpression reduces dendrite elaboration. These results suggest that Defhc1.1 functions as an inhibitor of neurite growth by finely tuning the microtubule cytoskeleton dynamics and that EFHC1-dependent juvenile myoclonic epilepsy may result from augmented spontaneous neurotransmitter release due to overgrowth of neuronal processes.

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 neuromuscular system of Drosophila melanogaster has been studied for many years for its relative simplicity and because of the genetic and molecular versatilities. Three main types of striated muscles are present in this dipteran: fibrillar muscles, tubular muscles and supercontractile muscles. The visceral muscles in adult flies and the body wall segmental muscles in embryos and larvae belong to the group of supercontractile muscles. Larval body wall muscles have been the object of detailed studies as a model for neuromuscular junction function but have received much less attention with respect to their mechanical properties and to the control of contraction. In this review we wish to assess available information on the physiology of the Drosophila larval muscular system. Our aim is to establish whether this system has the requisites to be considered a good model in which to perform a functional characterization of Drosophila genes, with a known muscular expression, as well as Drosophila homologs of human genes, the dysfunction of which, is known to be associated with human hereditary muscle pathologies.

In humans, mutations in ZASP (the gene for Z-band alternatively spliced PDZ-motif protein) are associated with dilated cardiomyopathy and left ventricular non-compaction. In particular, mutations in or around the Zasp motif seem to be related to myofibrillar myopathy. Thus, “zaspopathies” include symptoms such as Z-line disgregation, proximal and distal muscle weakness, cardiomyopathies, and peripheral neuropathies. In order to understand the role of ZASP in muscle structure and function, we have performed a molecular characterization of the Drosophila ortholog of human ZASP and a functional analysis following the post-transcriptional silencing of the Drosophila gene. Transcriptional analysis of dzasp has revealed six additional exons, with respect to the known 16, and multiple splice variants. We have produced transgenic lines harboring constructs that, through the use of the UAS/Gal4 binary system, have enabled us to drive dsRNA interference of dzasp in a tissue-specific manner. Knockdown individuals show locomotor defects associated with alterations of muscle structure and ultrastructure, consistent with a role of dzasp specifically in the maintenance of muscular integrity.

Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin "hinge" region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial "melting" of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was approximately 5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus, alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed.

The study of the effect of snake presynaptic neurotoxins with phospholipase A2 activity on nerve terminals has recently unveiled the inhibitory action of a lysophosphatidylcholine (LysoPC)/fatty acid mixture. We report here that these neurotoxins have no activity on Drosophila melanogaster nerve terminals. However, a 1:1 mixture of LysoPC and oleic acid induces an early increase, followed by an inhibition of both evoked and spontaneous neurotransmitter release. This effect is also induced by LysoPC alone. The present findings provide an indirect evidence that the lipid hemifusion-to-pore transition is a key event in neuroexocytosis in Drosophila. Moreover, these findings substantiate the use of LysoPC as a general agonist of membrane fusion at nerve terminals. This novel tool could contribute to the unraveling of the molecular steps involved in neuroexocytosis, particularly in Drosophila, where it is straightforward to combine it with electrophysiology and genetics.

Denervation deeply affects muscle structure and function, the alterations being different in slow and fast muscles. Because the effects of denervation on fast muscles are still controversial, and high-throughput studies on gene expression in denervated muscles are lacking, we studied gene expression during atrophy progression following denervation in mouse tibialis anterior (TA). The sciatic nerve was cut close to trochanter in adult CD1 mice. One, three, seven, and fourteen days after denervation, animals were killed and TA muscles were dissected out and utilized for physiological experiments and gene expression studies. Target cDNAs from TA muscles were hybridized on a dedicated cDNA microarray of muscle genes. Seventy-one genes were found differentially expressed. Microarray results were validated, and the expression of relevant genes not probed on our array was monitored by real-time quantitative PCR (RQ-PCR). Nuclear- and mitochondrial-encoded genes implicated in energy metabolism were consistently downregulated. Among genes implicated in muscle contraction (myofibrillar and sarcoplasmic reticulum), genes typical of fast fibers were downregulated, whereas those typical of slow fibers were upregulated. Electrophoresis and Western blot showed less pronounced changes in myofibrillar protein expression, partially confirming changes in gene expression. Isometric tension of skinned fibers was little affected by denervation, whereas calcium sensitivity decreased. Functional studies in mouse extensor digitorum longus muscle showed prolongation in twitch time parameters and shift to the left in force-frequency curves after denervation. We conclude that, if studied at the mRNA level, fast muscles appear not less responsive than slow muscles to the interruption of neural stimulation.

Homer represents a new and diversified family of proteins that includes several isoforms, Homer 1, 2, and 3; some of these isoforms have been reported to be present in striated muscles. In this study, the presence of Homer isoforms 1a, 1b/c/d, 2b, and 3 was thoroughly investigated in rat skeletal muscles under resting conditions. Transition in Homer isoforms compositon was studied under experimental conditions of short-term and long-term adaptation, e.g., fatigue and regeneration, respectively. First, we show that Homer 1a was constitutively expressed and was transiently upregulated during regeneration. In C2C12 cell cultures, Homer 1a was also upregulated during formation of myotubes. No change of Homer 1a was observed in fatigue. Second, Homer 1b/c/d and Homer 2b were positively and linearly related to muscle mass change during regeneration, and third, Homer 3 was not detectable under resting conditions but was transiently expressed during regeneration although with a temporal pattern distinct from that of Homer 1a. Thus a switch in Homer isoforms is associated to muscle differentiation and regeneration. Homers may play a role not only in signal transduction of skeletal muscle, in particular regulation of Ca2+ release from sarcoplasmic reticulum (Ward CW, Feng W, Tu J, Pessah IN, Worley PF, and Schneider MF. Homer protein increases activation of Ca2+ sparks in permeabilized skeletal muscle. J Biol Chem 279: 5781–5787, 2004), but also in adaptation.

Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila. KD was produced by post-transcriptional silencing employing a transgene encoding a dsRNA fragment of the Drosophila homolog of human Surf1, activated by the UAS transcriptional activator. Two alternative drivers, Actin5C–GAL4 or elav–GAL4, were used to induce silencing ubiquitously or in the CNS, respectively. Actin5C–GAL4 KD produced 100% egg-to-adult lethality. Most individuals died as larvae, which were sluggish and small. The few larvae reaching the pupal stage died as early imagos. Electron microscopy of larval muscles showed severely altered mitochondria. elav–GAL4-driven KD individuals developed to adulthood, although cephalic sections revealed low COX-specific activity. Behavioral and electrophysiological abnormalities were detected, including reduced photoresponsiveness in KD larvae using either driver, reduced locomotor speed in Actin5C–GAL4 KD larvae, and impaired optomotor response as well as abnormal electroretinograms in elav–GAL4 KD flies. These results indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, we employed a caki null mutant in the model organism Drosophila melanogaster. By means of electrophysiological methods, we analyzed, in adult flies, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system. We found that in caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ was increased, the response of the giant fiber pathway to continuous stimulation was impaired, and electroretinographic responses to single and continuous repetitive stimuli were altered and optomotor behavior was abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function.

Sphingomyelin derivatives exert various second messenger actions in numerous tissues. Sphingosine (SPH) and sphingosine 1-phosphate (S1P) are two major sphingomyelin derivatives present at high levels in blood. The aim of the present work was to investigate whether S1P and SPH exert relevant actions in mouse skeletal muscle contractility and fatigue. Exogenous S1P and SPH administration caused a significant reduction of tension decline during fatigue of extensor digitorum longus muscle. Final tension after the fatiguing protocol was 40% higher than in untreated muscle. Interestingly, N, N-dimethylsphingosine, an inhibitor of SPH kinase (SK), abolished the effect of supplemented SPH but not that of S1P, suggesting that SPH acts through its conversion to S1P. Moreover, SPH was not effective in Ca2+-free solutions, in agreement with the hypothesis that SPH action is dependent on its conversion to S1P by the Ca2+-requiring enzyme SK. In contrast to SPH, S1P produced its positive effects on fatigue in Ca2+-free conditions, indicating that S1P action does not require Ca2+entry and most likely is receptor mediated. The effects of S1P could be ascribed in part to its ability to prevent the reduction (−20 mV) of action potential amplitude caused by fatigue. In conclusion, these results indicate that extracellular S1P has protective effects during the development of muscle fatigue and that the extracellular conversion of SPH to S1P may represent a rheostat mechanism to protect skeletal muscle from possible cytotoxic actions of SPH.

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 μM dantrolene did not affect fatigue development, whereas 20 μM 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 μM 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.

In the present study we aimed to determine the functional properties and the myosin heavy chain (MHC) isoform composition of single chemically skinned fibres from the vocal muscle of four adult men (age: 55-67 years). Single fibres, dissected from the bioptic samples, were chemically skinned and isometric tension (P0) and maximal shortening velocity (V0) were measured at pCa 4.6. MHC and myosin light chain (MLC) composition of fibre segments and MHC distribution of the biopsy samples were analysed by SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) and densitometry. Four MHC isoforms (1, 2A, 2X and a fourth isoform, provisionally called L) and five MLC isoforms (MLC1s, MLC1f, MLC3f, MLC2f, MLC2s) were identified. The major findings of this study were: (1) fast MHC isoforms (in particular MHC-2A) and fast fibres were predominant, (2) one-third of the fibres were mixed or hybrid, i.e. expressed more than one MHC isoform, (3) V0 and P0 values were determined by the MHC isoform composition and mixed fibres showed functional properties which were intermediate between pure fibres; MHC-L was associated with V0 values similar to those of MHC-2A, (4) compared with limb muscles, V0 values of laryngeal fibres were similar to those of limb muscle fibres containing the same MHC isoform whereas P0 values were lower for slow and fast 2X fibres and similar for fibres expressing MHC-2A.

Skeletal muscle type 2B fibers normally receive a moderate level of motoneuron discharge. As a consequence, we hypothesize that type 2B fiber properties should be less sensitive to the absence of the nerve. Therefore, we have investigated the response of sarcoplasmic reticulum and myofibrillar proteins of type 2B fibers isolated from rat extensor digitorum longus muscle after denervation (2 and 7 days). Single fibers were identified by SDS-PAGE of myosin heavy chain isoforms. Electrophysiological and isometric contractile properties of the whole muscle were also analyzed. The pCa-tension relationship of type 2B single fibers was shifted to the left at 2 days and to right at 7 days after denervation, with significant differences in the Hill coefficients and pCa threshold values in 2- vs. 7-day-denervated fibers. The sarcoplasmic reticulum Ca2+uptake capacity and rate significantly decreased after 2 days of denervation, whereas both increased at 7 days. Caffeine sensitivity of sarcoplasmic reticulum Ca2+release was transitory and markedly increased in 2-day-denervated fibers. Our results indicate that type 2B fiber functional properties are highly sensitive to the interruption of nerve supply. Moreover, most of 2-day-denervated changes were reverted at 7 days.

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

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.

The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies ∼350 bp downstream of the dGpi1 gene. Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries ∼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.

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.

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.