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.
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 P2Y 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.
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.
1 Searching for the structural requirements improving the potency and the stereoselectivity of Na + channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid a-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers.2 Their ability to block Na + currents, elicited from 7100 to 720 mV at 0.3 Hz (tonic block) and 2 ± 10 Hz (use-dependent block) frequencies, was investigated in vitro on single ®bres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method.3 The a-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2-and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. 4 Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. 5 Antimyotonic activity, evaluated as the shortening of the time of righting re¯exes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more eective for R-To5 than for RTocainide. 6 Thus, constraining the chiral centre of tocainide in a-proline cycle leads to more potent and stereoselective use-dependent Na + channel blockers with improved therapeutic potential.
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