Molecular characterization of CDKL5 in the nervous system and in the pathogenesis of rett syndrome.

Rett syndrome (RS) is an X-linked neurodevelopmental disorder that represents the second cause of mental retardation in females. Symptoms manifest after a period of apparently normal development and are characterized by stereotypic hand movements, mental retardation, epileptic crisis, hyperventilation, constipation and cardiovascular abnormalities. The vast majority of patients affected by the classical form of RS carries mutations in the methyl-CpG binding protein 2 (MeCP2), a ubiquitous protein that, binding to methylated promoters, represses the transcription of downstream genes. Moreover, according to the fact that RS is exclusively a neurological disease, MeCP2 has been recently shown to dynamically modulate the transcription of specific neuronal genes, such as Bdnf. Importantly, the phosphorylation of MeCP2 has been demonstrated to be required for Bdnf transcription upon neuronal activity. Besides the classical form of RS, also a number of variants have been reported: some of them cause a milder clinical picture than the classical form, while others display a more severe phenotype. Among the latter, the Hanefeld variant characterized by the absence of an initial asyntomatic period and by the early onset of pharmacologically untreatable seizures. Several patients affected by this variant have been found to carry mutations in the cyclin dependent kinase like 5 (CDKL5), a serine/threonine kinase made up of an N-terminal catalytic domain and a long and uncharacterized C-terminal tail. Previous publications demonstrated that CDKL5 and MeCP2 interact physically and that the kinase is able to autophosphorylate and to mediate the phosphorylation of recombinant MeCP2 in vitro. The aim of this work was to elucidate the impact of RS causing mutations in CDKL5 on its physiological functions as well as its expression pattern in adult and developing brain. The kinase activity, subcellular localization, and protein stability of different RS causing missense or truncating mutations have been analyzed in comparison to the wild type protein. The Δ781 mutant, lacking the last 250 aa of the protein, revealed to be catalytically hypermorphic: in fact, both the auto- and the heterophosphorylation activity of this mutant were increased if compared to the wild type protein. Moreover, when overexpressed in cells, it accumulated exclusively in the nucleus, loosing the capability to shuttle between the two main cellular compartments, as the wt protein does. Also its half-life was altered, showing a reduced degradation rate when compared to the wt kinase. In parallel, the analysis of other truncated mutants showed an altered subcellular localization and proteasomal degradation when the very C-terminal tail of the protein was missing. These results clearly suggest a pivotal role played by the last 200 aa of CDKL5 in the modulation of its catalytic activity, the regulation of its nucleo-cytoplasmic shuttling and its degradation. The concomitant analysis of two disease causing missense mutations of CDKL5, C152F and R175S, revealed that point mutations affecting the catalytic domain of the protein strongly impair its functions. In fact, conversely to the truncated mutants, the R175S and the C152F derivatives showed to be hypomorphic or catalytically inactive, respectively. According to the hypothesis that the catalytic activity of the protein is requested for CDKL5 to enter the nucleus, the hypomorphic and the kinase dead artificial derivative, K42R, were retained in the cytosolic compartment. All together, these results indicate that altered subcellular localization and expression levels of CDKL5 might be the origin of a pathological state. In parallel, also the expression pattern of endogenous murine protein in a physiological context was analysed. The western blot experiments performed on adult mouse tissues revealed that CDKL5 expression is heterogeneous: this kinase was, in fact, virtually absent in some brain districts (hypothalamus and cerebellum), while it was well expressed in others (cortex, hippocampus, striatum and thalamus). Also its subcellular localization differed depending on the brain region: in the majority of the analysed areas, its cytosolic and nuclear fraction were almost equally represented, while in cerebellum and striatum the amount of cytosolic protein widely exceeded the nuclear one. These data suggest that in mammalian brain CDKL5 expression and localization are tightly regulated both at a regional and at a cellular level. The analysis of the same features in new born mice showed the importance of CDKL5 in the delicate process of postnatal development. In fact, the expression level of this protein was strongly upregulated in the first days of postnatal life, decreasing, then, to lower levels in the adulthood. Concomitantly, the nuclear fraction of the kinase gradually increased during postnatal development, being almost absent in the first days of life and reaching the 40% of total protein in adult mouse brain. Finally, the involvement of CDKL5 in the process of neuronal maturation and differentiation is also suggested by the experiments performed in Neuro-2a cells with the shRNA tool: the long term silencing of the kinase, in fact, prevented neurite extension upon induction of differentiation. Taken together, the results showed in this work indicate that the RS pathological state may be correlated to an altered capability of the mutated protein (i) to phosphorylate its substrates (itself or other targets), (ii) to be properly localized within the cell, and (iii) to be readily degraded by the proteasome. Moreover, the study performed in the developing mouse brain and in Neuro-2a cells has suggested a role of the kinase in postnatal development and neuronal differentiation. Further work will be aimed to investigate more in detail this interesting issue.