A low-carbohydrate ketogenic diet remains one of the most effective (but mysterious) treatments for severe pharmacoresistant epilepsy. We have tested for an acute effect of physiological ketone bodies on neuronal firing rates and excitability, to discover possible therapeutic mechanisms of the ketogenic diet. Physiological concentrations of ketone bodies (-hydroxybutyrate or acetoacetate) reduced the spontaneous firing rate of neurons in slices from rat or mouse substantia nigra pars reticulata. This region is thought to act as a "seizure gate," controlling seizure generalization. Consistent with an anticonvulsant role, the ketone body effect is larger for cells that fire more rapidly. The effect of ketone bodies was abolished by eliminating the metabolically sensitive K ATP channels pharmacologically or by gene knock-out. We propose that ketone bodies or glycolytic restriction treat epilepsy by augmenting a natural activity-limiting function served by K ATP channels in neurons.
The Tsx gene resides at the X-inactivation center and is thought to encode a protein expressed in testis, but its function has remained mysterious. Given its proximity to noncoding genes that regulate X-inactivation, here we characterize Tsx and determine its function in mice. We find that Tsx is actually noncoding and the long transcript is expressed robustly in meiotic germ cells, embryonic stem cells, and brain. Targeted deletion of Tsx generates viable offspring and X-inactivation is only mildly affected in embryonic stem cells. However, mutant embryonic stem cells are severely growth-retarded, differentiate poorly, and show elevated cell death. Furthermore, male mice have smaller testes resulting from pachytene-specific apoptosis and a maternal-specific effect results in slightly smaller litters. Intriguingly, male mice lacking Tsx are less fearful and have measurably enhanced hippocampal short-term memory. Combined, our study indicates that Tsx performs general functions in multiple cell types and links the noncoding locus to stem and germ cell development, learning, and behavior in mammals.
The X-chromosome harbors hundreds of disease genes whose associated diseases predominantly affect males. However, a subset, including neurodevelopmental disorders, Rett syndrome (RTT), fragile X syndrome, and CDKL5 syndrome, also affects females. These disorders lack disease-specific treatment. Because female cells carry two X chromosomes, an emerging treatment strategy has been to reawaken the healthy allele on the inactive X (Xi). Here, we focus on methyl-CpG binding protein 2 (MECP2) restoration for RTT and combinatorially target factors in the interactome of Xist, the noncoding RNA responsible for X inactivation. We identify a mixed modality approach combining an Xist antisense oligonucleotide and a small-molecule inhibitor of DNA methylation, which, together, achieve 30,000-fold MECP2 up-regulation from the Xi in cultured cells. Combining a brain-specific genetic ablation with short-term 5-aza-2'-deoxycytidine (Aza) treatment models the synergy in vivo without evident toxicity. The Xi is selectively reactivated. These experiments provide proof of concept for a mixed modality approach for treating X-linked disorders in females.
Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2X cilia ) which is modulated by Na + and by long exposures to ATP. P2X cilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch-clamp technique in the whole-cell configuration. During the initial continuous exposure to extracellular ATP, P2X cilia currents gradually increase in magnitude (priming), yet the permeability to N -methyl-D-glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na + , which readily permeates P2X cilia receptor channels, was found to inhibit the channel extracellular to the electric field. Cation-selective ion channels activated by extracellular ATP (P2X receptor channels) are widely distributed in electrically excitable cells such as neurones, and in non-excitable cells such as immune cells and epithelial cells (North, 2002). Seven subunits of the mammalian P2X receptor family (P2X 1 to P2X 7 ) have been cloned, and all of them can form functional homomeric receptor channels in heterologous expression systems (North, 2002;Jones et al. 2004). Six functional heteromeric receptor channels (P2X 1 /P2X 2 , P2X 1 /P2X 4 , P2X 1 /P2X 5 , P2X 2 /P2X 3 , P2X 2 /P2X 6 and P2X 4 /P2X 6 ) have also been characterized (Torres et al. 1998;Le et al. 1998Le et al. , 1999King et al. 2000;Brown et al. 2002;Nicke et al. 2005). Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that additional hetero-oligomeric assemblies are possible among the different P2X receptor subunits (Torres et al. 1999). The pharmacological and biophysical properties of native P2X receptor channels often deviate from those of heterologously expressed P2X receptor channels. This suggests that native P2X receptor channels might be processed differently or that they are heteromeric channels of yet uncharacterized combinations of P2X subunits.Airway ciliated cells express a P2X receptor channel (P2X cilia ) of unknown composition. This channel is interesting both from a physiological and from a biophysical standpoint because of its modulation by both ATP and Na + . P2X cilia receptor currents gradually increase over tens of seconds during the first exposure to ATP whereas subsequent applications of ATP activate the current significantly faster (Korngreen et al. 1998). This priming phenomenon can be observed for several minutes after the initial application of ATP, and thus ATP can be considered both an agonist and a long-term modulator of the P2X cilia receptor channel. P2X cilia receptor channels are also modulated by Na + (Ma et al. 1999). The effect
Cilia are small organelles protruding from the cell surface that beat synchronously, producing biological transport. Despite intense research for over a century, the mechanisms underlying ciliary beating are still not well understood. Even the nature of the cytosolic molecules required for spontaneous and stimulated beating is debatable. In an effort to resolve fundamental questions related to cilia beating, we developed a method that integrates the whole-cell mode of the patch-clamp technique with ciliary beat frequency measurements on a single cell. This method enables to control the composition of the intracellular solution while the cilia remain intact, thus providing a unique tool to simultaneously investigate the biochemical and physiological mechanism of ciliary beating. Thus far, we investigated whether the spontaneous and stimulated states of cilia beating are controlled by the same intracellular molecular mechanisms. It was found that: (a) MgATP was sufficient to support spontaneous beating. (b) Ca2+ alone or Ca2+-calmodulin at concentrations as high as 1 μM could not alter ciliary beating. (c) In the absence of Ca2+, cyclic nucleotides produced a moderate rise in ciliary beating while in the presence of Ca2+ robust enhancement was observed. These results suggest that the axonemal machinery can function in at least two different modes.
Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by a mutation in the X-linked methyl-CpG-binding protein 2 (MECP2). There is currently no disease-specific treatment, but MECP2 restoration through reactivation of the inactive X (Xi) has been of considerable interest. Progress toward an Xi-reactivation therapy has been hampered by a lack of suitable female mouse models. Because of cellular mosaicism due to random X-chromosome inactivation (XCI), heterozygous females develop only mild RTT. Here, we create an improved female mouse model by introducing a mutation in, the antisense regulator of XCI allelic choice. mice show reduced MECP2 mosaicism and closely phenocopy the severely affected-null males. females demonstrate shortened lifespan, motor weakness, tremors, and gait disturbance. Intriguingly, they also exhibit repetitive behaviors, as is often seen in human RTT, including excessive grooming and biting that result in self-injury. With a allelic series, we vary MECP2 levels in brain and demonstrate a direct, but nonlinear correlation between MECP2 levels and phenotypic improvement. As little as 5-10% MECP2 restoration improves neuromotor function and extends lifespan five- to eightfold. Our study thus guides future pharmacological strategies and suggests that partial MECP2 restoration could have disproportionate therapeutic benefit.
ELISA or Western blot is known as a basic technique to be used for measurement of intracellular proteins, but in some cases, they cannot overcome problems such as normalization between samples or extraneous costs for required commercial kits. In order to address this problem, we developed a rapid and effective method (a hybrid of Western blot and ELISA). We use this new hybrid method to detect and normalize trace protein changes in gene expression intracellularly at a lower cost.
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