Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients

Unconventional Transcriptional Response to Environmental Enrichment in a Mouse Model of Rett Syndrome

Background: Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) and one of the leading causes of mental retardation in females. RTT is characterized by psychomotor retardation, purposeless hand movements, autistic-like behavior and abnormal gait. We studied the effects of environmental enrichment (EE) on the phenotypic manifestations of a RTT mouse model that lacks MeCP2 (Mecp2 2/y). Principal Findings: We found that EE delayed and attenuated some neurological alterations presented by Mecp2 2/y mice and prevented the development of motor discoordination and anxiety-related abnormalities. To define the molecular correlate of this beneficial effect of EE, we analyzed the expression of several synaptic marker genes whose expression is increased by EE in several mouse models. Conclusions/Significance: We found that EE induced downregulation of several synaptic markers, suggesting that th

Daily Rhythmic Behaviors and Thermoregulatory Patterns Are Disrupted in Adult Female MeCP2-Deficient Mice

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome, severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature, gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2 deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities

Characterization of the 55-Residue Protein Encoded by the 9S E1A mRNA of Species C Adenovirus

Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A

Generation of Mice Deficient in both KLF3/BKLF and KLF8 Reveals a Genetic Interaction and a Role for These Factors in Embryonic Globin Gene Silencing

Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin xpression during development

On the growth and welfare effects of monopolistic distortions

Endogenous growth, Competition, Deregulation, Poverty trap, Trade liberalization, F15, F43, O31, O34, O41,

Downstream targets of methyl CpG binding protein 2 and their abnormal expression in the frontal cortex of the human Rett syndrome brain

Background: The Rett Syndrome (RTT) brain displays regional histopathology and volumetric reduction, with frontal cortex showing such abnormalities, whereas the occipital cortex is relatively less affected. Results: Using microarrays and quantitative PCR, the mRNA expression profiles of these two neuroanatomical regions were compared in postmortem brain tissue from RTT patients and normal controls. A subset of genes was differentially expressed in the frontal cortex of RTT brains, some of which are known to be associated with neurological disorders (clusterin and cytochrome c oxidase subunit 1) or are involved in synaptic vesicle cycling (dynamin 1). RNAi-mediated knockdown of MeCP2 in vitro, followed by further expression analysis demonstrated that the same direction of abnormal expression was recapitulated with MeCP2 knockdown, which for cytochrome c oxidase subunit 1 was associated with a functional respiratory chain defect. Chromatin immunoprecipitation (ChIP) analysis showed that MeCP2 associated with the promoter regions of some of these genes suggesting that loss of MeCP2 function may be responsible for their overexpression. Conclusions: This study has shed more light on the subset of aberrantly expressed genes that result from MECP2 mutations. The mitochondrion has long been implicated in the pathogenesis of RTT, however it has not been at the forefront of RTT research interest since the discovery of MECP2 mutations. The functional consequence of the underexpression of cytochrome c oxidase subunit 1 indicates that this is an area that should be revisited.National Health and Medical Research Council (Australia) (NHMRC) (project grant 185202)National Health and Medical Research Council (Australia) (NHMRC) (project grant 346603