A new protein curbs the hypertrophic effect of myostatin inhibition, adding remarkable endurance to motor performance in mice

Current efforts to improve muscle performance are focused on muscle trophism via inhibition of the myostatin pathway: however they have been unsuccessful in the clinic to date. In this study, a novel protein has been created by combining the soluble activin receptor, a strong myostatin inhibitor, to the C-terminal agrin nLG3 domain (ActR-Fc-nLG3) involved in the development and maintenance of neuromuscular junctions. Both domains are connected via the constant region of an Igg1 monoclonal antibody. Surprisingly, young male mice treated with ActR-Fc-nLG3 showed a remarkably increased endurance in the rotarod test, significantly longer than the single domain compounds ActR-Fc and Fc-nLG3 treated animals. This increase in endurance was accompanied by only a moderate increase in body weights and wet muscle weights of ActR-Fc-nLG3 treated animals and were lower than expected. The myostatin inhibitor ActR-Fc induced, as expected, a highly significant increase in body and muscle weights compared to control animals and ActR-Fc-nLG3 treated animals. Moreover, the prolonged endurance effect was not observed when ActR-Fc and Fc-nLG3 were dosed simultaneously as a mixture and the body and muscle weights of these animals were very similar to ActR-Fc treated animals, indicating that both domains need to be on one molecule. Muscle morphology induced by ActR-Fc-nLG3 did not appear to be changed however, close examination of the neuromuscular junction showed significantly increased acetylcholine receptor surface area for ActR-Fc-nLG3 treated animals compared to controls. This result is consistent with published observations that endurance training in rats increased acetylcholine receptor quantity at neuromuscular junctions and provide evidence that improving nerve-muscle interaction could be an important factor for sustaining long term muscle activity

Identification and quantification of dolichol and dolichoic acid in neuromelanin from substantia nigra of the human brain.

Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14–20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units

Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders

Broad spectrum and prolonged efficacy of dimiracetam in models of neuropathic pain

Dimiracetam, a bicyclic 2-pyrrolidinone derivative originally developed as cognition enhancer, is a member of the nootropic family for which anecdotal efficacy in models of neuropathic pain has been reported. Its antineuropathic activity was evaluated in established models of neuropathic pain induced by nerve injury, chemotherapy or MIA-induced osteoarthritis. Acutely, dimiracetam was very effective in models of antiretroviral drug induced painful neuropathy, oxaliplatin-induced hyperalgesia and in the MIA-osteoarthritis. Chronic dimiracetam dosing in the MIA and ART- induced models completely reverted hyperalgesia back to the level of healthy controls. Once reached, the maximal effect was maintained despite dose diminution and increased inter-dose interval. The effect of the last dose outlasted dimiracetam half-life longer than 12 times. In synaptosomal preparations, dimiracetam counteracted the NMDA-induced release of glutamate with highest potency in the spinal cord, possibly via NMDA receptor isoforms containing pH-sensitive GluN1 and GluN2A subunits. Dimiracetam appears to be a promising and safe treatment for neuropathic pain conditions for which there are very limited therapeutic options. \ua9 2014 Elsevier Ltd. All rights reserved

NMJ morphology of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Confocal images of the NMJs in <i>soleus</i> muscle of P30 Control or SARCO mice, treated or not treated with NT-1654 as indicated. The postsynaptic AChRs were stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was stained with anti-neurofilament and synaptophysin antibodies (NF-Syn; green). The postsynapses were highly fragmented, partially or completely lost in <i>soleus</i> muscle of SARCO mice. The postsynapses of NT-1654 treated SARCO mice resembled those in Control mice, showing pretzel like structures and much less fragmentation. Scale bar: 50 µm. <b>B:</b> The postsynapses in Control mice showed fragmentation class 0 (no fragmentation, for illustration see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088739#pone.0088739.s003" target="_blank">figure S3</a>). The postsynapses of NT-1654 treated SARCO mice were mostly classified into class 1, whereas the postsynapse of SARCO mice showed class 1-4 with a peak at class 3. <b>C:</b> Many NMJs of SARCO mice have terminal nerve sprouting, which was dramatically reduced in the NT1654 treated SARCO mice. Data present mean ± standard deviation, n  =  2 mice, 100 NMJs were counted in each mouse.</p

Fiber type distribution of <i>m. soleus</i> of NT-1654 and vehicle treated SARCO mice.

<p><b>A:</b> Consecutive muscle cross sections of <i>soleus</i> stained with myosine heavy chain (MHC) specific antibodies or cytochrome C oxidase (COX) staining as indicated. Control and treated SARCO mice show clearly separated type I and II fibers. SARCO mice have a significantly increased amount of hybrid fibers (indicated with asterisks). Cytochrome C staining of <i>soleus</i> sections shows a massive reduction of reactivity in SARCO mice which is reverted back to WT levels in treated SARCO mice. <b>B:</b> Quantitative analysis of muscle fibers of <i>soleus.</i> Bars represent mean values ± standard deviation. Control and treated SARCO mice have almost no hybrid fibers and are indistinguishable from each other. Compared to treated ones, SARCO mice have significantly increased hybrid fibers. Type I fibers are significantly decreased. The total fiber number in SARCO mice is also significantly decreased compared to treated animals. Number of animals: wt  =  3; SARCO treated  =  5, SARCO  =  3. * p<0.05; ** p<0.01. Scale bar: 100 µm.</p

NMJ recovery after sciatic nerve crush.

<p><b>A:</b> Confocal images of the neuromuscular junctions (NMJs) in <i>soleus</i> muscle of Thy1-YFP mice after 14 days sciatic nerve crushing, treated with NT-1654 or PBS (Control) as indicated. The postsynaptic AChR was stained with Alexa-555 conjugate α-bungarotoxin (red) and the presynapse was visualized by the transgenic expression of YFP in motor neurons (green). The NMJs in <i>soleus</i> muscle are fully re-innervated after 14 days sciatic nerve crushing. The NMJs of NT-1654 treated mice showed significantly less nerve sprouting than those treated with PBS (Control). The number of nerve sprouting in each NMJ was counted and shown in <b>B.</b> NMJs in the NT-1654 treated mice have significantly fewer events of nerve sprouting and significantly fewer number of nerve sprouts than those of Control mice. Data present mean ± standard error, *: p<0.05 (two ways t-test), 100 NMJs were counted in each mouse, n  =  3 mice, scale bar: 50 µm.</p