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

Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time-lapse video and microarray

The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video timelapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of focal adhesion contacts. Analysis of markers revealed that mesenchymal stem cells constitutively expressed multi-lineage traits, including several pertaining to the neural one. However, the applied ‘neural induction’ protocol neither significantly modulated the expression of such markers, nor induced de novo translation of other neural-specific proteins. Similarly, global expression profiling of over 21,000 genes demonstrated that gene transcription was poorly affected. Most strikingly, we found that the set of genes whose expression was altered by the inductive treatment did not match those sets of genes differentially expressed when comparing untreated mesenchymal stem cells and immature neural tissues. Conversely, by comparing these gene expression profiles with that obtained from comparisons between the same cells and an unrelated nonneural organ, such as liver, we found that the adopted neural induction protocol was no more effective in redirecting human mesenchymal stem cells toward a neural phenotype than toward an endodermal hepatic pathway. Key words: Bone-marrow-derived stem cells, Neurogenesis, Transdifferentiation Summar

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

Disruption of the Cellular Regulation of CDKL5 Might be Relevant for Rett Syndrome

Mutations in the human X-linked cyclin dependent kinase like 5 (CDKL5) gene have recently been identified in some Rett patients with the Hanefeld variant as well as in girls with mental retardation associated with early seizures. We have previously shown that CDKL5 works in a pathway common with that of MeCP2, the main cause of classic Rett Syndrome (RTT). In fact, the two proteins associate and the kinase is able to mediate the phosphorylation of MeCP2 in vitro. This suggests that mutations in CDKL5 cause RTT in part because important MeCP2 functions are impaired. Furthermore, CDKL5 might play a secondary role in RTT by acting as a modifier gene thereby influencing disease severity in patients with mutations in MECP2. Even though our results do suggest a common molecular pathway belonging to CDKL5 and MeCP2, we still have to reveal in which brain areas and when the two factors are communicating. Furthermore, we have to get insight into the functional relevance of the identified interaction. Our immunohistochemistry and western blot experiments show that in adult brain the two proteins have overlapping expression patterns. However, whereas MeCP2 levels appear rather uniform through late embryogenesis and postnatal stages as well as in the different brain areas, CDKL5 levels appear to be subject to a dynamic modulation. In particular, the CDKL5 protein is virtually absent in the mouse embryo brain and is strongly induced in early post-natal stages. Furthermore, whereas MeCP2 is confined to the nuclear compartment, CDKL5 is found in both the nuclear and cytoplasmic compartments indicating a possible role in transmitting signals between these two compartments. Interestingly, the subcellular distribution of CDKL5 varies in the different areas of the adult mouse brain as well as during development. Experiments in cell cultures have allowed us to show that CDKL5 shuttles between the nucleus and the cytoplasm and that an active nuclear export mechanism depending on the C-terminal tail of the protein is responsible for the cytoplasmic localization. The relevance of this regulation seems to be demonstrated by the fact that all the late RTT truncating mutations identified so far lead to an abnormal accumulation of the kinase into the nucleus

Disruption of the Cellular Regulation of CDKL5 Might be Relevant for Rett Syndrome

Mutations in the human X-linked cyclin dependent kinase like 5 (CDKL5) gene have recently been identified in some Rett patients with the Hanefeld variant as well as in girls with mental retardation associated with early seizures. We have previously shown that CDKL5 works in a pathway common with that of MeCP2, the main cause of classic Rett Syndrome (RTT). In fact, the two proteins associate and the kinase is able to mediate the phosphorylation of MeCP2 in vitro. This suggests that mutations in CDKL5 cause RTT in part because important MeCP2 functions are impaired. Furthermore, CDKL5 might play a secondary role in RTT by acting as a modifier gene thereby influencing disease severity in patients with mutations in MECP2. Even though our results do suggest a common molecular pathway belonging to CDKL5 and MeCP2, we still have to reveal in which brain areas and when the two factors are communicating. Furthermore, we have to get insight into the functional relevance of the identified interaction. Our immunohistochemistry and western blot experiments show that in adult brain the two proteins have overlapping expression patterns. However, whereas MeCP2 levels appear rather uniform through late embryogenesis and postnatal stages as well as in the different brain areas, CDKL5 levels appear to be subject to a dynamic modulation. In particular, the CDKL5 protein is virtually absent in the mouse embryo brain and is strongly induced in early post-natal stages. Furthermore, whereas MeCP2 is confined to the nuclear compartment, CDKL5 is found in both the nuclear and cytoplasmic compartments indicating a possible role in transmitting signals between these two compartments. Interestingly, the subcellular distribution of CDKL5 varies in the different areas of the adult mouse brain as well as during development. Experiments in cell cultures have allowed us to show that CDKL5 shuttles between the nucleus and the cytoplasm and that an active nuclear export mechanism depending on the C-terminal tail of the protein is responsible for the cytoplasmic localization. The relevance of this regulation seems to be demonstrated by the fact that all the late RTT truncating mutations identified so far lead to an abnormal accumulation of the kinase into the nucleus

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail,”

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment

Disruption of the Cellular Regulation of CDKL5 Might be Relevant for Rett Syndrome

Mutations in the human X-linked cyclin dependent kinase like 5 (CDKL5) gene have recently been identified in some Rett patients with the Hanefeld variant as well as in girls with mental retardation associated with early seizures. We have previously shown that CDKL5 works in a pathway common with that of MeCP2, the main cause of classic Rett Syndrome (RTT). In fact, the two proteins associate and the kinase is able to mediate the phosphorylation of MeCP2 in vitro. This suggests that mutations in CDKL5 cause RTT in part because important MeCP2 functions are impaired. Furthermore, CDKL5 might play a secondary role in RTT by acting as a modifier gene thereby influencing disease severity in patients with mutations in MECP2. Even though our results do suggest a common molecular pathway belonging to CDKL5 and MeCP2, we still have to reveal in which brain areas and when the two factors are communicating. Furthermore, we have to get insight into the functional relevance of the identified interaction. Our immunohistochemistry and western blot experiments show that in adult brain the two proteins have overlapping expression patterns. However, whereas MeCP2 levels appear rather uniform through late embryogenesis and postnatal stages as well as in the different brain areas, CDKL5 levels appear to be subject to a dynamic modulation. In particular, the CDKL5 protein is virtually absent in the mouse embryo brain and is strongly induced in early post-natal stages. Furthermore, whereas MeCP2 is confined to the nuclear compartment, CDKL5 is found in both the nuclear and cytoplasmic compartments indicating a possible role in transmitting signals between these two compartments. Interestingly, the subcellular distribution of CDKL5 varies in the different areas of the adult mouse brain as well as during development. Experiments in cell cultures have allowed us to show that CDKL5 shuttles between the nucleus and the cytoplasm and that an active nuclear export mechanism depending on the C-terminal tail of the protein is responsible for the cytoplasmic localization. The relevance of this regulation seems to be demonstrated by the fact that all the late RTT truncating mutations identified so far lead to an abnormal accumulation of the kinase into the nucleus