Gregory J. Pelka

Mutations of CDKL5 Cause a Severe Neurodevelopmental Disorder with Infantile Spasms and Mental Retardation

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused, in most classic cases, by mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). A large degree of phenotypic variation has been observed in patients with RTT, both those with and without MECP2 mutations. We describe a family consisting of a proband with a phenotype that showed considerable overlap with that of RTT, her identical twin sister with autistic disorder and mild-to-moderate intellectual disability, and a brother with profound intellectual disability and seizures. No pathogenic MECP2 mutations were found in this family, and the Xq28 region that contains the MECP2 gene was not shared by the affected siblings. Three other candidate regions were identified by microsatellite mapping, including 10.3 Mb at Xp22.31-pter between Xpter and DXS1135, 19.7 Mb at Xp22.12-p22.11 between DXS1135 and DXS1214, and 16.4 Mb at Xq21.33 between DXS1196 and DXS1191. The ARX and CDKL5 genes, both of which are located within the Xp22 region, were sequenced in the affected family members, and a deletion of nucleotide 183 of the coding sequence (c.183delT) was identified in CDKL5 in the affected family members. In a screen of 44 RTT cases, a single splice-site mutation, IVS13-1G-->A, was identified in a girl with a severe phenotype overlapping RTT. In the mouse brain, Cdkl5 expression overlaps--but is not identical to--that of Mecp2, and its expression is unaffected by the loss of Mecp2. These findings confirm CDKL5 as another locus associated with epilepsy and X-linked mental retardation. These results also suggest that mutations in CDKL5 can lead to a clinical phenotype that overlaps RTT. However, it remains to be determined whether CDKL5 mutations are more prevalent in specific clinical subgroups of RTT or in other clinical presentations.

Environmental enrichment ameliorates a motor coordination deficit in a mouse model of Rett syndrome –Mecp2 gene dosage effects and BDNF expression

Rett syndrome, commonly associated with mutations of the methyl CpG‐binding protein 2 (MECP2) gene, is characterised by an apparently normal early postnatal development followed by deterioration of acquired cognitive and motor coordination skills in early childhood. To evaluate whether environmental factors may influence the disease outcome of Rett syndrome, we tested the effect of environmental enrichment from 4 weeks of age on the behavioural competence of mutant mice harboring a Mecp2 tm1Tam‐null allele. Our findings show that enrichment improves motor coordination in heterozygous Mecp2 +/− females but not Mecp2 −/y males. Standard‐housed Mecp2 +/− mice had an initial motor coordination deficit on the accelerating rotarod, which improved with training then deteriorated in subsequent weeks. Enrichment resulted in a significant reduction in this coordination deficit in Mecp2 +/− mice, returning the performance to wild‐type levels. Brain‐derived neurotrophic factor (BDNF) protein levels were 75 and 85% of wild‐type controls in standard‐housed and environmentally enriched Mecp2 +/− cerebellum, respectively. Mecp2 −/y mice showed identical deficits of cerebellar BDNF (67% of wild‐type controls) irrespective of their housing environment. Our findings demonstrate a positive impact of environmental enrichment in a Rett syndrome model; this impact may be dependent on the existence of one functional copy of Mecp2.

Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Application of lacZ transgenic mice to cell lineage studies.

Cell lineage analyses trace the hierarchy of cell types derived from a progenitor population. Critical to these analyses is the ability to track reliably all or defined subsets of the clonal descendants of the progenitor population. This necessitates marking the cells with a heritable and cell autonomous marker. Transgenes encoding molecules that can be visualized directly in situ without compromising cell differentiation, such as the reporter, β-galactosidase encoded by lacZ and chloramphenicol acetyltransferase, encoded by the CAT gene are the most widely used. The lacZ can be readily detected using a sensitive histo-chemical assay such that cells in which the β-galactosidase gene is transcrip-tionally active produce a blue stain in tissue sections or in whole mounts (1). Some lineage studies demand the simultaneous detection of the lacZ product and other tissue-specific proteins or transcripts. In this chapter, we discuss the experimental strategies in which the lacZ transgene can be utilized in the analysis of cell lineages, and we detail assays for detecting β-galactosidase by X-gal histochemistry or immunological localization of the enzyme in combination with mRNA in situ hybridization, immunochemistry, and histochemical procedures, such as alkaline phosphatase staining. 1.1. Choosing the Appropriate Transgenic Animals for Lineage Analysis The expression of the lacZ transgene varies according to the nature of the regulatory elements (promoter and enhancer) that drive gene expression, the number of active copies, and the chromosomal domain where the transgene is localized. The choice of transgene is dictated by the specific questions to be addressed. Several examples of transgenes give ubiquitous nonlineage biased expression. For example, in transgenic mice where the lacZ gene is regulated by the hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase promoter, the transgene is expressed in all lineages at high levels during development (2). Similarly, the ROSA-β-geo transgene produced by site-directed mutagenesis (3) and the lacZ reporter driven by rat β-actin promoter (4) also provide ubiquitous tagging of multiple cell lineages. Ubiquitously expressed transgenes provide the most ideal marker for unbiased cell lineage analyses, provided the lineage progenitors or founder cells to be studied can be isolated as a pure cell population. Integration of the transgene into a specific chromosome may be useful in some cases for tracing cell lineages. Transgenes that are expressed in the same pattern as neighboring genes at the site of integration provide a ready marker for any tissue-specific pattern of expression. For example, transgenes integrated into the X chromosome may behave like the endogenous X-linked gene; however, the fact that the expression pattern is due to a transgene, not an endogenous gene, must be taken into account when interpreting the results (5, 6). In female mice that carry the lacZ transgene on only one X chromosome, X-inactivation during embryonic development generates two cell populations, one that expresses the X-linked transgene and another that does not. Since the status of X-inactivation is heritable, descendants of either population are stable for the transgene expression. Several studies on the lineage relationship of cells in tissues, such as the retina, brain, and tongue, have been performed using the mosaicism of transgene expression generated by random X-chromosome inactivation (7–10). Finally, some transgenes display lineage and stage-specific expression through use of a tissue-specific promoter or as a consequence of their unique integration sites. For example, in Wnt1-lacZ transgenic mice, the transgene is regulated by the Wnt1 3′-cis-acting enhancer element, which directs specific expression to the dorsal part of the neural tube and subsequently in the neural crest cells derived from this region (11). Another example is R197 transgenic mice, in which the lacZ gene is expressed principally in the muscle lineage from early organogenesis stages onward as a consequence of its integration site (12). Provided that the expression of the transgene is tissue specific for a defined period of development, such transgenes offer an excellent tool for tracing the differentiation of cell lineages. A prerequisite for using these mice and any other transgenic lines is to establish the pattern of transmission and ascertain the tissue and temporal specificity of transgene expression.

Alleviating Transcriptional Inhibition of the Norepinephrine Slc6a2 Transporter Gene in Depolarized Neurons

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increased Slc6a2 mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on the Slc6a2 promoter sequence by chromatin immunopurification strategies. Increased Slc6a2 gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on the Slc6a2 promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism of Slc6a2 gene regulation.

Chromatin context and ncRNA highlight targets of MeCP2 in brain

The discovery that Rett syndrome (RTT) is caused by mutation of the methyl-CpG-binding-protein MeCP2 provided a major breakthrough in understanding the neurodevelopmental disorder and accelerated MeCP2 research. However, gene regulation by MeCP2 is complicated. The current consensus for MeCP2 remains as a classical repressor complex, with major emphasis on its role in methylation-dependent binding and repression. However, recent evidence indicates additional regulatory roles, suggesting non-classical mechanisms in gene activation. This has opened the field of MeCP2 research and suggests that the gene targets may not be the usual suspects, that is, dependent only on DNA methylation. Here we examine how chromatin binding and sequence preference may confer MeCP2 functionality, and connect relevant pathways in an active genome. Finding both genomic and proteomic evidence to indicate MeCP2 spliceosome interaction, we consequently discovered broad MeCP2 enrichment of the transcriptome while our focus toward long non-coding RNA (lncRNA) revealed MeCP2 association with RNCR3. Our data may indicate an as-yet-unappreciated role between lncRNA and MeCP2. We hypothesize that ncRNA may mediate chromatin-remodeling events by interacting with MeCP2, thereby conferring changes in gene expression. We consider that these results may suggest new mechanisms of gene regulation conferred by MeCP2 and its interactions upon chromatin structure and gene function.

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

ABSTRACT 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 expression during development.

Affective dysfunction in a mouse model of Rett syndrome: Therapeutic effects of environmental stimulation and physical activity

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X‐linked gene encoding methyl‐CpG‐binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2tm1Tam mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety‐related behavior of the Mecp2+/− mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2+/− mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic–pituitary–adrenal axis dysregulation. EE of Mecp2+/− mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment‐induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

Deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in twins with a Rett syndrome-like phenotype.

Rett syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is primarily caused by variants in MECP2. Variants in other genes such as CDKL5 and FOXG1 are usually associated with individuals who manifest distinct phenotypes that may overlap with RTT. Individuals with phenotypes suggestive of RTT are typically screened for variants in MECP2 and then subsequently the other genes dependent on the specific phenotype. Even with this screening strategy, there are individuals in whom no causative variant can be identified, suggesting that there are other novel genes that contribute to the RTT phenotype. Here we report a de novo deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in identical twins with a RTT-like phenotype. We also demonstrate the reduced expression of Ptpn4 in a Mecp2 null mouse model of RTT, as well as the activation of the PTPN4 promoter by MeCP2. Our findings suggest that PTPN4 should be considered for addition to the growing list of genes that warrant screening in individuals with a RTT-like phenotype.