Acetyl-L-Carnitine Improves Behavior and Dendritic Morphology in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a devastating neurodevelopmental disorder affecting 1 in 10,000 girls. Approximately 90% of cases are caused by spontaneous mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Girls with RTT suffer from severe motor, respiratory, cognitive and social abnomalities attributed to early deficits in synaptic connectivity which manifest in the adult as a myriad of physiological and anatomical abnormalities including, but not limited to, dimished dendritic complexity. Supplementation with acetyl-L-carnitine (ALC), an acetyl group donor, ameliorates motor and cognitive deficits in other disease models through a variety of mechanisms including altering patterns of histone acetylation resulting in changes in gene expression, and stimulating biosynthetic pathways such as acetylcholine. We hypothesized ALC treatment during critical periods in cortical development would promote normal synaptic maturation, and continuing treatment would improve behavioral deficits in the Mecp2^1lox mouse model of RTT. In this study, wildtype and Mecp2^1lox mutant mice received daily injections of ALC from birth until death (postnatal day 47). General health, motor, respiratory, and cognitive functions were assessed at several time points during symptom progression. ALC improved weight gain, grip strength, activity levels, prevented metabolic abnormalities and modestly improved cognitive function in Mecp2 null mice early in the course of treatment, but did not significantly improve motor or cognitive functions assessed later in life. ALC treatment from birth was associated with an almost complete rescue of hippocampal dendritic morphology abnormalities with no discernable side effects in the mutant mice. Therefore, ALC appears to be a promising therapeutic approach to treating early RTT symptoms and may be useful in combination with other therapies.     

Gait Analysis in a Mecp2 Knockout Mouse Model of Rett Syndrome Reveals Early-Onset and Progressive Motor Deficits

Rett syndrome (RTT) is a genetic disorder characterized by a range of features including cognitive impairment, gait abnormalities and a reduction in purposeful hand skills. Mice harbouring knockout mutations in the Mecp2 gene display many RTT-like characteristics and are central to efforts to find novel therapies for the disorder. As hand stereotypies and gait abnormalities constitute major diagnostic criteria in RTT, it is clear that motor and gait-related phenotypes will be of importance in assessing preclinical therapeutic outcomes. We therefore aimed to assess gait properties over the prodromal phase in a functional knockout mouse model of RTT. In male Mecp2 knockout mice, we observed alterations in stride, coordination and balance parameters at 4 weeks of age, before the onset of other overt phenotypic changes as revealed by observational scoring. These data suggest that gait measures may be used as a robust and early marker of MeCP2-dysfunction in future preclinical therapeutic studies.     

Histone H3 and H4 gene deletions in Saccharomyces cerevisiae

The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes. Strains with deletions of each of these loci were constructed by gene replacement techniques. Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable. In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally. However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression. The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid. The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay.     

Characterization of the MeCP2R168X Knockin Mouse Model for Rett Syndrome

Rett syndrome, one of the most common causes of mental retardation in females, is caused by mutations in the X chromosomal gene MECP2. Mice deficient for MeCP2 recapitulate some of the symptoms seen in patients with Rett syndrome. It has been shown that reactivation of silent MECP2 alleles can reverse some of the symptoms in these mice. We have generated a knockin mouse model for translational research that carries the most common nonsense mutation in Rett syndrome, R168X. In this article we describe the phenotype of this mouse model. In male MeCP2^R168X mice life span was reduced to 12–14 weeks and bodyweight was significantly lower than in wild type littermates. First symptoms including tremor, hind limb clasping and inactivity occurred at age 27 days. At age 6 weeks nest building, rotarod, open-field and elevated plus maze experiments showed impaired motor performance, reduced activity and decreased anxiety-like behavior. Plethysmography at the same time showed apneas and irregular breathing with reduced frequency. Female MeCP2^R168X mice showed no significant abnormalities except decreased performance on the rotarod at age 9 months. In conclusion we show that the male MeCP2^R168X mice have a phenotype similar to that seen in MECP2 knockout mouse models and are therefore well suited for translational research. The female mice, however, have a much milder and less constant phenotype making such research with this mouse model more challenging.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.     

Histone H3K4 demethylases are essential in development and differentiation.

Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. The methylation of Lys4 of histone H3 seems to be of particular significance. It is associated with active regions of the genome, and in Drosophila it is catalyzed by trithorax-group proteins that have become paradigms of developmental regulators at the level of chromatin. Like other histone methylation events, H3K4 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics. However, the described demethylases had no strictly assigned biological functions and the identity of the histone demethylases that would contribute to the epigenetic changes specifying certain biological processes was unknown. Recently, several groups presented evidence that a family of 4 JmjC domain proteins results in the global changes of histone demethylation, and in elegant studies using model organisms, they demonstrated the importance of this family of histone demethylases in cell fate determination. All 4 proteins possess the demethylase activity specific to H3K4 and belong to the poorly described JARID1 protein family.     

小鼠不同细胞间组蛋白修饰变化对MafA基因转录表达的影响

目的通过比较不同细胞类型之间MafA基因转录起始区的组蛋白修饰差异,探讨组蛋白修饰对MafA基因转录表达的作用。方法采用染色质免疫共沉淀-实时定量PCR法检测小鼠胰岛素瘤β细胞（NIT-1）、NIH小鼠成纤维细胞（NIH3T3）及小鼠胚胎干细胞（mES）三者中的MafA和MLH1基因转录起始区组蛋白修饰（H3K4m3、H3K9m3和H3乙酰化）的状况。同时采用实时定量RT-PCR检测上述三种细胞各基因mRNA表达水平。分析基因的H3K4m3、H3K9m3和H3乙酰化修饰与基因表达之间的相互关系。结果 （1）以mES细胞为参照,NIT-1细胞MafA基因的转录起始区的H3K4m3修饰水平明显增高（P〈0.05）,H3K9m3修饰水平明显降低（P〈0.05）;NIH 3T3细胞MafA基因的转录起始区的H3K9m3修饰水平明显增高（P〈0.05）,H3K4m3修饰水平明显降低（P〈0.05）;（2）MafA基因的仅在NIT-1细胞表达,其表达与H3K4m3修饰存在直线相关（相关系数0.995）;与H3K9m3修饰存在直线负相关（相关系数-0.751）;（3）管家基因MLH1的表达与所检测组蛋白修饰无相关性。结论 H3K9m3与H3K4m3修饰能相互协调,共同调控MafA基因的表达,对胚胎干细胞向β细胞分化具有重要的意义。     

Histone H4 Promotes Prothrombin Autoactivation

Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation. The effect is abrogated by mutation of the catalytic Ser-525 and requires the presence of the Gla domain. Fluorescence titrations document direct binding of histone H4 to prothrombin with an affinity in the low nm range. Stopped flow data and luminescence resonance energy transfer measurements indicate that the binding mechanism obeys conformational selection. Among the two conformations of prothrombin, collapsed and fully extended, histone H4 binds selectively to the collapsed form and induces a transition toward a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases by 13 Å. These findings confirm the molecular plasticity of prothrombin emerged from recent structural studies and suggest that different conformations of the inter-kringle linker domain determine the functional behavior of prothrombin. The results also broaden our mechanistic understanding of the prothrombotic phenotype observed during cellular damage due to the release of histones in the blood stream. Prothrombin autoactivation induced by histone H4 emerges as a mechanism of pathophysiological relevance through which thrombin is generated independently of activation of the coagulation cascade.