The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation.

The neural network of the enteric nervous system (ENS) underlies gastrointestinal functions. However, the molecular mechanisms involved in enteric neuronal connectivity are poorly characterized. Here, we studied the role of semaphorin 5A (Sema5A), previously characterized in the central nervous system, on ENS neuronal connectivity. Sema5A is linked to autism spectrum disorder (ASD), a neurodevelopmental disorder frequently associated with gastrointestinal comorbidities, and potentially associated with ENS impairments. This study investigated in rat enteric neuron cultures and gut explants the role of Sema5A on enteric neuron connectivity and the impact of ASD-associated mutations on Sema5A activity. Our findings demonstrated that Sema5A promoted axonal complexity and reduced functional connectivity in enteric neurons. Strikingly, the ASD-associated mutation S956G in Sema5A strongly affected these activities. This study identifies a critical role of Sema5A in the ENS as a regulator of neuronal connectivity that might be compromised in ASD.

A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.

KEY POINTS: Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron-enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation. ABSTRACT: In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron-enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial-derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y1 receptor- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation.

Quantitative proteomic analysis of dystrophic dog muscle.

Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α). Subsequently, we were able to showed that PGC1-α expression is dramatically reduced in GRMD compared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice.

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

Optimal requirements for high affinity and use-dependent block of skeletal muscle sodium channel by N-benzyl analogs of tocainide-like compounds.

Newly synthesized tocainide analogs were tested for their state-dependent affinity and use-dependent behavior on sodium currents (INa) of adult skeletal muscle fibers by means of the Vaseline-gap voltage clamp method. The drugs had the pharmacophore amino group constrained in position alpha [N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide (To5)] or beta [N-(2,6-dimethylphenyl)pyrrolidine-3-carboxamide (To9)] in a proline-like cycle and/or linked to a lipophilic benzyl moiety as in N-benzyl-tocainide (Benzyl-Toc), 1-benzyl-To5 (Benzyl-To5), and 1-benzyl-To9 (Benzyl-To9). INa were elicited with pulses to -20 mV from different holding potentials (-140, -100, and -70 mV) and stimulation frequencies (2 and 10 Hz). All compounds were voltage-dependent and use-dependent channel blockers. The presence of a proline-like cycle increased the potency; i.e., To5 was 3- and 10-fold more effective than Toc in blocking INa at the holding potential of -140 and -70 mV, respectively. The benzyl group on the amine further enhanced drug effectiveness with the following scale: Benzyl-To9 >/= Benzyl-Toc > Benzyl-To5. At a holding potential of -100 mV and 10-Hz stimulation, Benzyl-To9 blocked INa with a half-maximal concentration of 0.5 microM, being 60 and 400 times more potent than To9 and Toc, respectively. The similar effectiveness of Benzyl-Toc and Benzyl-To9 was paralleled by a similar spatial arrangement by equilibrium geometry modeling. In addition, the latter had a higher pKa value that probably contributed to a slow kinetic during its high use-dependent behavior. Benzyl-To5 had its lowest energy level at a more folded conformation that justifies the less favorable profile among the N-benzylated analogs. The new compounds are the most potent tocainide-like sodium channel blockers so far described and have high therapeutic potentials.

Inhibition of skeletal muscle sodium currents by mexiletine analogues: specific hydrophobic interactions rather than lipophilia per se account for drug therapeutic profile.

In striated fibers, the activity of mexiletine (Mex)-like sodium channel blockers is strongly modulated by the part of the molecule nearby the asymmetric carbon atom. A lipophilic aromatic phenyl group at this levels, as in 2-(2,6-dimethylphenoxy)-1-phenylethanamine (Me4), markedly increases drug potency, while an increased distance between the stereogenic center and the pharmacophore amino group, as in 3-(2,6-dimethylphenoxy)-2-methylpropan-1-amine (Me2), enhances the use-dependent behavior. In order to better evaluate the role of lipophilicity in drug potency in relation to the structural determinants for a specific binding, lipophilic analogs of Me2 and Me4 were synthesized. Compounds 3-[(2,6-dimethylphenyl)thio]-2-methylpropan-1-amine and 2-[(2,6-dimethylphenyl)thio]-1-phenylethanamine were obtained by isosteric substitution of the oxygen atom with sulfur, while the introduction of a chlorine atom in 4- position of the aryloxy ring lead to 3-(4-chloro-2,6-dimethylphenoxy)-2-methylpropan-1-amine and 2-(4-chloro-2,6-dimethylphenoxy)-1-phenylethanamine. The compounds were tested on nearly maximal Na(+) currents elicited with depolarizing steps at 0.3 Hz (tonic block) and 2-10 Hz (use-dependent block) by means of vaseline-gap voltage-clamp method on single frog muscle fibers.The augmented lipophilicity largely increase drug potency in Me2 analogues, the thio and chlorinated compounds being 20- and 10-fold more potent in producing the tonic block, respectively. However, both compounds showed a 2-fold lower use-dependent behavior vs. the high use-dependent Me2. Surprisingly, the same increase in lipophilicity brought about by the same substitutions, in the already high lipophilic and potent Me4 failed to further improve the potency, although both new analogs were more stereoselective than Me4. No correlation was found between logP and potency of all analogs tested. All compounds acted as inactivated channel blockers. In conclusion, lipophilicity differently influences drug profile based on the molecular determinants controlling drug-receptor interaction.

IP(3)-induced tension and IP(3)-receptor expression in rat soleus muscle during postnatal development.

The present study was designed to examine whether changes in Ca(2+) release by inositol-1,4,5-trisphosphate (IP(3)) in 8-, 15-, and 30-day-old rat skeletal muscles could be associated with the expression of IP(3) receptors. Experiments were conducted in slow-twitch muscle in which both IP(3)-induced Ca(2+) release and IP(3)-receptor (IP(3)R) expression have been shown to be larger than in fast-twitch muscle. In saponin-skinned fibers, IP(3) induced transient contractile responses in which the amplitude was dependent on the Ca(2+)-loading period with the maximal IP(3) contracture being at 20 min of loading. The IP(3) tension decreased during postnatal development, was partially inhibited by ryanodine (100 microM), and was blocked by heparin (20-400 microg/ml). Amplification of the DNA sequence encoding for IP(3)R isoforms (using the RT-PCR technique) showed that in slow-twitch muscle, the type 2 isoform is mainly expressed, and its level decreases during postnatal development in parallel with changes in IP(3) responses in immature fibers. IP(3)-induced Ca(2+) release would then have greater participation in excitation-contraction coupling in developing fibers than in mature muscle.