Loss of CDKL5 Causes Synaptic GABAergic Defects That Can Be Restored with the Neuroactive Steroid Pregnenolone-Methyl-Ether

: CDKL5 deficiency disorder (CDD) is an X-linked neurodevelopmental disorder characterised by early-onset drug-resistant epilepsy and impaired cognitive and motor skills. CDD is caused by mutations in cyclin-dependent kinase-like 5 (CDKL5), which plays a well-known role in regulating excitatory neurotransmission, while its effect on neuronal inhibition has been poorly investigated. We explored the potential role of CDKL5 in the inhibitory compartment in Cdkl5-KO male mice and primary hippocampal neurons and found that CDKL5 interacts with gephyrin and collybistin, two crucial organisers of the inhibitory postsynaptic sites. Through molecular and electrophysiological approaches, we demonstrated that CDKL5 loss causes a reduced number of gephyrin puncta and surface exposed γ2 subunit-containing GABAA receptors, impacting the frequency of miniature inhibitory postsynaptic currents, which we ascribe to a postsynaptic function of CDKL5. In line with previous data showing that CDKL5 loss impacts microtubule (MT) dynamics, we showed that treatment with pregnenolone-methyl-ether (PME), which promotes MT dynamics, rescues the above defects. The impact of CDKL5 deficiency on inhibitory neurotransmission might explain the presence of drug-resistant epilepsy and cognitive defects in CDD patients. Moreover, our results may pave the way for drug-based therapies that could bypass the need for CDKL5 and provide effective therapeutic strategies for CDD patients

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders

Molecular and functional characterization of CDKL5, a novel X-linked kinase, mainly involved in female mental retardation

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5; OMIM300203) have recently been identified in patients with infantile spasms and mental retardation that had previously been diagnosed with atypical Rett Syndrome or X-linked Infantile Spasms. Although CDKL5 appears to play a critical role for proper brain function, it remains largely uncharacterized. The general aim of this project is to initiate the comprehension of the cellular and molecular defects caused by mutations in CDKL5

DNA methylation in the control of gene silencing during development and human disorders (DisChrom)

\u201cChromatin diseases\u201d (CD) are genetic diseases resulting from mutations in components of chromatin and/or in enzymes that modify DNA methylation and histone modifications. This alters chromatin status at specific genes and/or genomic regions, thereby causing drastic effects on gene expression. CD are ideal model system since their nature of monogenic disease, making them more \u201cmolecularly controlled\u201d with respect to the multifactorial nature of cancer. Furthermore, the factors involved in remodeling the chromatin structure are potential drug targets in the treatment of human diseases, making the investigation of epigenetic factors and therapies a priority for research. The DisChrom ITN aims to study, at different levels, the molecular mechanisms underlying four chromatin diseases, the potential for therapeutic intervention and the development of new technologies to dissect the epigenetic abnormalites associated with these conditions. Our interest will be focused on four diseases: i) Rett syndrome ii) alpha thalassemia with mental retardation (ATRX) sindrome iii) facioscapulohumeral muscular dystrophy (FSHD) iv) Immunodeficiency, Centromeric region instability, Facial anomalies (ICF) syndrome

Molecular mechanisms underlying the regulation of PBX subcellular localization

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Molecular genetics of Rett syndrome: when epigenetic signals go unrecognized

Rett syndrome (RTT) is an X-linked progressive neurodevelopmental disorder causing mental retardation mainly in girls. Affected patients are characterized by a period of normal development followed by a rapid regression phase that leaves them with a profound mental handicap. Mutations in the MECP2 gene, coding for a transcriptional repressor involved in chromatin remodeling, are the primary cause of Rett syndrome but are also found in patients affected by learning disability, neonatal encephalopathy, autism and mental retardation therefore making RTT paradigmatic for the study of autism spectrum disorders. Recent experiments indicate that MeCP2-deficient neurons are not permanently damaged, since Mecp2 reactivation leads to robust abrogation of advanced neurological defects in both young and adult animals. Although these results do not provide immediate therapeutic strategies for RTT, they establish the principle of reversibility of RTT and related disorders suggesting the necessity to develop therapeutical approaches. In this communication we will summarize the fundamental discoveries of the last 9 years relevant to MeCP2 related neurological disorder and we will discuss the new challenges inspired by these discoveries. In particular, we will underline the importance of the identification of the critical factors that function downstream of MeCP2 as well as of the modifier genes that subdue the disease symptoms. As Huda Zhogbi wrote \u201cRTT story started in the clinic, but today has inspired many exciting basic science studies in neurobiology and epigenetics. It is anticipated that the next chapter in this story will involve translating some of the discoveries back to the clinic to benefit patients with RTT and patients with related neurological disorders\u201d

PBX1 nuclear export is regulated independently of PBX–MEINOX interaction by PKA phosphorylation of the PBC-B domain

The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. We investigated the processes controlling PBX1 nuclear export. Here we show that in the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation in duces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. Our results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain

Molecular characterization of CDKL5, a novel kinase involved in Rett syndrome and infantile spasms

Rett Syndrome (RTT) is an X-linked neurological disorder and represents the second cause of mental retardation in females. Mutations in the methyl-CpG binding protein (MeCP2) gene cause the majority of RTT cases. Recently, mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene have been found in some RTT patients with the Hanefeld variant. Pathogenic mutations in CDKL5 were also found in females with early signs of developmental delay and epileptic seizure onset, further reinforcing the importance of this gene in mental retardation and epilepsy. We are characterizing the role of CDKL5 in the nervous system thereby clarifying the molecular mechanisms involved in disease onset. We have previously shown that CDKL5 and MeCP2 function in a common pathway; in fact, they associate and the kinase is able to mediate the phosphorylation of MeCP2. This suggests that CDKL5 might also play an indirect role in RTT acting as a modifier gene that, by regulating MeCP2 functions, is able to influence disease severity in patients with mutations in MeCP2. Here we will show that both CDKL5 expression and its subcellular localization are highly modulated during embryogenesis and post-natal development. In addition, in adult mouse, CDKL5 protein level and its cytoplasmic/nuclear fraction are tightly regulated in the different brain areas. Moreover, we will present data demonstrating that CDKL5 shuttles between the nucleus and the cytoplasm and that an active nuclear export mechanism is involved in regulating its localization. Our analysis suggests that the C-terminal tail of the kinase is responsible for the cytoplasmic localization. Importantly, we will show that a number of RTT truncating mutations, found in this region, are mislocalized in the nucleus. We believe that this analysis will contribute in drawing a phenotype-genotype correlation in patients with mutations in CDKL5 and in understanding the role of CDKL5 as an MeCP2 modifier gene

Molecular characterization of CDKL5, a novel kinase involved in Rett syndrome and infantile spasms

Rett Syndrome (RTT) is an X-linked neurological disorder affecting mainly females. In the classical form, patients have normal period of development of 6-18 months where after they display developmental arrest and a progressive regression leading to the loss of speech and purposeful movements with the appearance of a severe mental retardation. Atypical forms with differences in disease onset and severity exist. Mutations in the methyl-CpG binding protein (MECP2) gene, located on Xq28, cause the majority of RTT cases but have been found in less than half of atypical RTT patients. Recently, mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene, on Xp22, have been found in some RTT patients with the Hanefeld variant, characterized by the onset of seizures in the very first months of life. Furthermore, mutations in CDKL5 have been found in girls with infantile spasms and mental retardation, suggesting an important role of this gene for neuronal function. A main interest of our laboratory is to characterize the role of CDKL5 in the nervous system thereby clarifying the molecular mechanisms involved in disease onset. With this communication we will present our results showing that CDKL5 and MeCP2 function in a common pathway in accordance with the fact that mutations in the two genes cause a similar phenotype. In fact, besides sharing an overlapping expression pattern correlating with neuronal maturation and synaptogenesis, CDKL5 and MeCP2 associate and in vitro the kinase is able to mediate the phosphorylation of the methyl-binding protein. Furthermore, we will show our unpublished data indicating that CDKL5 catalytic activities are subject to different levels of regulation mediated by its long C-terminal tail. First of all, the C-terminus influences negatively the catalytic activity of the kinase and, moreover, an active nuclear export mediated by the tail is involved in regulating the subcellular localization of CDKL5. Finally, the turn over of the kinase seems to be regulated and disease causing mutations might affect it. It is important to keep in mind that besides missense mutations in the N-terminal catalytic domain a number of truncating mutations are found in the C-terminal tail; with the functional analysis of these mutated derivatives we try to provide a molecular explanation to their contribution to Rett Syndrome. We believe that this analysis will contribute in drawing a phenotype-genotype correlation

Characterization of HIPK2 that, by Associating with MeCP2, Might Function as a Modifier Gene in Rett Syndrome.

Mutations in the methyl CpG-binding protein 2 (MECP2) gene, located on Xq28, are responsible for almost all cases of classic RTT. Conversely, less than half of the patients with one of the variant forms of RTT carry mutations in MECP2. It seems, thus, that other genes are involved in causing RTT; moreover the fact that there are patients with milder phenotypes in spite of severe mutations argues that modifier genes might restrict the clinical outcome by regulating MeCP2 functions. To search for MeCP2 interacting proteins possibly involved in RTT we performed a yeast two-hybrid screening and identified among the positive clones HIPK2 (homeodomain interacting protein kinase 2) that belongs to a family of Ser/Thr kinases originally identified as corepressors for homeodomain transcription factors. HIPK2 has a clear role in regulating cell growth and genotoxic stress-induced apoptosis. Furthermore, its involvement in the nervous system is indicated by the neuronal defects of null mice that partially overlap those observed in Mecp2 ko mice. Since important MeCP2 functions in the nervous system are regulated by its phosphorylation we found it interesting to analyze the functional role of its interaction with HIPK2. We have thus confirmed that the two proteins associate in vitro and in vivo and phosphorylation assays have shown that MeCP2 is significantly phosphorylated by HIPK2 in vitro. Importantly, these assays have also allowed us to establish that Ser80 within the MBD of MeCP2 is a specific target of HIPK2. Functional assays have shown that ectopic MeCP2 causes an increase in cell death and an additive effect of the two proteins in inducing apoptosis in cultured cells was observed. Importantly, the role of MeCP2 in inducing apoptosis together with HIPK2 is lost when Ser80 is mutated or a kinase dead derivative of HIPK2 is used. Presently we are analyzing whether MeCP2 is a target of the kinase also in vivo and the role of the interaction for the nervous system. In favor of the hypothesis that the two proteins work in a common molecular pathway we have shown by immunohistochemistry experiments that the expression pattern of MeCP2 and HIPK2 in the brain of adult mice is highly similar. We therefore believe that these studies are relevant for understanding whether this novel MeCP2 interactor acts as a modifier gene influencing disease severity in RTT patients with mutations in MeCP2