Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.

MuSK levels differ between adult skeletal muscles and influence postsynaptic plasticity.

Muscle-specific tyrosine kinase (MuSK) is involved in the formation and maintenance of the neuromuscular junction (NMJ), and is necessary for NMJ integrity. As muscle involvement is strikingly selective in pathological conditions in which MuSK is targeted, including congenital myasthenic syndrome with MuSK mutation and MuSK antibody-seropositive myasthenia gravis, we hypothesized that the postsynaptic response to MuSK-agrin signalling differs between adult muscles. Transcript levels of postsynaptic proteins were compared between different muscles in wild-type adult mice. MuSK expression was high in the soleus and sternomastoid muscles and low in the extensor digitorum longus (EDL) and omohyoid muscles. The acetylcholine receptor (AChR) α subunit followed a similar expression pattern, whereas expression of Dok-7, Lrp4 and rapsyn was comparable between the muscles. We subsequently examined muscles in mice that overexpressed a miniaturized form of neural agrin or MuSK. In these transgenic mice, the soleus and sternomastoid muscles responded with formation of ectopic AChR clusters, whereas such clusters were almost absent in the EDL and omohyoid muscles. Electroporation of Dok-7 revealed its important role as an activator of MuSK in AChR cluster formation in adult muscles. Together, our findings indicate for the first time that adult skeletal muscles harbour different endogenous levels of MuSK and that these levels determine the ability to form ectopic AChR clusters upon overexpression of agrin or MuSK. We believe that these findings are important for our understanding of adult muscle plasticity and the selective muscle involvement in neuromuscular disorders in which MuSK is diminished.

Minor sarcoplasmic reticulum membrane components that modulate excitation-contraction coupling in striated muscles.

In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca(2+) stored in the sarcoplasmic reticulum by a process called excitation-contraction coupling. Excitation-contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation-contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca(2+) release channel, and Ca(2+) storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation-contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation-contraction coupling is important in order to understand how mutations in genes involved in Ca(2+) signalling cause neuromuscular disorders.

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice.

Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures.

Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death.

Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling.

The effect of drugs of abuse on NMDAR1 receptor expression in the rat limbic system.

An increasing body of evidence points to the role of N-methyl-D-aspartate (NMDA) receptors in the limbic system in the mechanism of drug dependence. We studied the influence of acute and repeated morphine (20 mg/kg i.p. or increasing dose for 10 days) and cocaine (3x20 mg/kg i.p. per day at hourly intervals, for 1 or 5 days) administration on the expression of glutamate NMDA receptor subunit 1 (NMDAR1) in the central and basolateral nuclei of the rat amygdala and hippocampal formation. Acute or chronic morphine and cocaine administration increased NMDAR1 mRNA level in the central and basolateral nuclei of the amygdala; morphine did so 3 h after the last dose and 48 h after withdrawal, cocaine 3 h after acute and last chronic dose. Morphine did not change the NMDAR1 mRNA level in the hippocampal formation, but chronic cocaine did decrease it in the dentate gyrus only. Our study suggests a possible link between the expression of NMDAR1 and changes in limbic system neuronal activity and behaviour after administration of morphine and cocaine. In summary, the present study demonstrated that morphine and cocaine influenced the expression of NMDAR1 in the structure of the limbic system which could be involved in dependence phenomena.

Formalin-induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonists in mice.

The effects of adenosine analogues on pain have been shown to depend on the subtype receptor involved as well as on the nociceptive stimuli and on the route of administration. In the first experiment of the present study intraperitoneal administration of the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) (0.015, 0.03, 0.09, 0.15, 0.21, 0.3 mg/kg) induced dose-dependent analgesia to formalin pain in both phases characterizing the test. The A(2a) receptor agonist 2-[p-2-(carbonyl-ethyl)-phenyethylamino]-5'-N-ethylcarboxaminoadenosine (CGS21680) (0.025, 0.05, 0.1, 0.15 mg/kg) significantly affected behavioral responses to formalin only during the early phase. In the second experiment the interaction between adenosine and the opioid system was investigated through both behavioral and neurochemical studies. The opioid antagonist naltrexone (0.1 mg/kg) did not affect the antinociception induced by CPA (0.21 mg/kg) and CGS21680 (0.05 mg/kg). Autoradiographic studies showed that formalin administration significantly modified mu-opioid receptor density in the superficial laminae of the spinal cord and in the paracentral thalamic nucleus, contralateral to the side of formalin injection. CPA and CGS21680 counteracted these effects induced by formalin. In conclusion the present study confirms and extends the role of A(1) and A(2a) adenosine receptors in the modulation of inflammatory pain and their interaction with the mu-opioid system, and suggests further investigation of these purinergic receptors from a therapeutic perspective.

Knockdown of spinal opioid receptors by antisense targeting beta-arrestin reduces morphine tolerance and allodynia in rat.

The development of morphine tolerance and sciatic nerve injury-induced allodynia after functional knockdown of spinal opioid receptors using antisense oligonucleotides targeting beta-arrestin was investigated. Ineffectiveness of morphine in neuropathic pain suggests an implication of the same mechanism in these two processes. The development of morphine tolerance (10 microg intrathecally (i.th.), every 12 h) was significantly inhibited in rats, which received i.th. beta-arrestin antisenses (2 nM). Acute and chronic (6 days) i.th. administration of antisenses antagonized the allodynia in the rat model of neuropathic pain. Our results demonstrated that i.th. administration of beta-arrestin antisenses delayed development of tolerance to morphine and nerve injury-induced cold allodynia, which suggest that both of the investigated phenomena may be mediated by a similar mechanism, e.g. receptor desensitization. Moreover, the antisense oligonucleotides targeting beta-arrestin may constitute a new approach to the therapy of neuropathic pain.

Effect of cocaine and amphetamine on biosynthesis of proenkephalin and prodynorphin in some regions of the rat limbic system.

A vast body of evidence points to the role of the limbic system in the mechanism of drug dependence. Opioid peptides localized in the limbic system may play a role in central effects of substances of abuse. The goal of the present study was to investigate the influence of acutely and chronically administered drugs of abuse, cocaine and amphetamine on biosynthesis of prodynorphin and proenkephalin in the rat amygdala, the structure involved in the mechanism of drug addiction. Acute injection of cocaine (20 mg/kg ip every hour for 3 h) or amphetamine (2.5 mg/kg) did not changed or decreased the level of proenkephalin mRNA in the central nucleus of the amygdala. In contrast, the level of prodynorphin mRNA was significantly increased in this structure after cocaine. Repeated cocaine administration (20 mg/kg ip every hour for 3 h, for 5 days) had no effect on the proenkephalin and prodynorphin mRNA in the central nucleus of the amygdala. Chronic amphetamine (2.5 mg/kg twice daily for 5 days) administration decreased proenkephalin and increased prodynorphin mRNA level in the central nucleus of the amygdala (at 24 and 48 h). Moreover, significant increase in prodynorphin mRNA level was observed in the hippocampal dentate gyrus after acute (cocaine) and chronic (cocaine, amphetamine) administration of the psychostimulants. The observed adaptive changes in the activity of two opioid systems in two structures of the limbic system, central nucleus of amygdala and hippocampus, may contribute to the neurochemical mechanism of drug addiction after psychostimulants. These studies also indicate that the changes in opioid gene expression in the central nucleus of the amygdala are not parallel to those observed in the nucleus accumbens after cocaine and amphetamine, which suggests that peptidergic systems in the structures of extended amygdala might be regulated by different neurochemical mechanisms after psychostymulant administration.