First insights on the signaling pathways related to CDKL5 regulation and on its possible involvement in synaptic plasticity

Rett syndrome (RTT) is an X-linked form of mental retardation that occurs sporadically once every 10,000-15,000 female births. After a period of normal development (6-18 months), the patients show a rapid regression of acquired speech and motor skills and the development of several symptoms including mental retardation, seizures, intermittent hyperventilation and stereotypic hand movements. This condition is mainly stable and signs of progressive neurodegeneration are absent. Almost 80% of Rett cases are associated with mutations in the MECP2 (methyl CpG binding protein 2) gene. MeCP2 is a nuclear protein that binds methylated DNA and recruits histone deacetylases and co-repressor complexes to suppress transcription. It belongs to the MBD family of proteins involved in the epigenetic regulation of gene-expression. Recently, mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients characterized by a subset of Rett clinical phenotypes and generally suffering of infantile spasms and severe mental retardation. The product of CDKL5 is a serine/threonine kinase that belongs to the CMGC family; the exact functions exerted by this kinase and its regulatory mechanisms remain mainly unknown. CDKL5 is present in the nucleus and in the cytosol of neurons and its expression shows a continued increase during development; accordingly, CDKL5 is a critical regulator of neuronal morphogenesis, neurite growth and dendritic arborization. In the cytosol, CDKL5 phosphorylates NGL-1 (Netrin-G1 Ligand 1), a regulator of early synapse formation and maturation. In the nucleus, CDKL5 binds and phosphorylates in vitro MeCP2. Furthermore, in the nucleus CDKL5 colocalizes with nuclear speckles and is probably involved in the regulation of mRNA splicing. Recently our group has demonstrated that the expression levels and the subcellular distribution of CDKL5 are modified by neuronal activation. In particular, a glutamate bath induces in cultured hippocampal neurons the rapid exit of the kinase from the nucleus and its proteasome-dependent degradation. The significance of this response remains to be elucidated. Furthermore, BDNF induces in rat cortical cultures, a rapid phosphorylation of CDKL5. The main aim of this work was to study how neuronal depolarization or activation by BDNF affects Cdkl5 regulation, in terms of gene transcription, post translation modifications of its final protein product and the involved signaling pathway(s). We found that, both in primary murine neuronal cultures and cortical slices, depolarization affects the expression of the gene, both at the transcriptional and post-transcriptional levels, together with its phosphorylation state. The response is affected by the maturation stage of the treated neurons and the involved signaling pathways have been characterized. We speculate that the observed regulation of Cdkl5 during neuronal depolarization could be related to a role of the kinase in neuronal activation. Electrophysiological approaches will be required to confirm the involvement of CDKL5 in the regulation of neuronal activity; furthermore, the identification of novel interactors of Cdkl5 should help in understanding its physiological functions in the central nervous system and the pathological consequences of a malfunctioning CDKL5

Cyclin-dependent-like kinase 5 is required for pain signaling in human sensory neurons and mouse models

Cyclin-dependent-like kinase 5 (Cdkl5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognised anamnestic deficiency in pain perception. Consistent with a role in nociception, we discovered that Cdkl5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in iPS-derived human nociceptors. CDKL5 deficient mice display defective epidermal innervation and conditional deletion of Cdkl5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, Cdkl5 interacts with CaMKIIα to control outgrowth as well as TRPV1-dependent signaling, which are disrupted in both Cdkl5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for Cdkl5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder

What We Know and Would Like to Know about CDKL5 and Its Involvement in Epileptic Encephalopathy

In the last few years, the X-linked serine/threonine kinase cyclin-dependent kinase-like 5 (CDKL5) has been associated with early-onset epileptic encephalopathies characterized by the manifestation of intractable epilepsy within the first weeks of life, severe developmental delay, profound hypotonia, and often the presence of some Rett-syndrome-like features. The association of CDKL5 with neurodevelopmental disorders and its high expression levels in the maturing brain underscore the importance of this kinase for proper brain development. However, our present knowledge of CDKL5 functions is still rather limited. The picture that emerges from the molecular and cellular studies suggests that CDKL5 functions are important for regulating both neuronal morphology through cytoplasmic signaling pathways and activity-dependent gene expression in the nuclear compartment. This paper surveys the current state of CDKL5 research with emphasis on the clinical symptoms associated with mutations in CDKL5, the different mechanisms regulating its functions, and the connected molecular pathways. Finally, based on the available data we speculate that CDKL5 might play a role in neuronal plasticity and we adduce and discuss some possible arguments supporting this hypothesis

What We Know and Would Like to Know about CDKL5 and Its Involvement in Epileptic Encephalopathy

In the last few years, the X-linked serine/threonine kinase cyclin-dependent kinase-like 5 (CDKL5) has been associated with early-onset epileptic encephalopathies characterized by the manifestation of intractable epilepsy within the first weeks of life, severe developmental delay, profound hypotonia, and often the presence of some Rett-syndrome-like features. The association of CDKL5 with neurodevelopmental disorders and its high expression levels in the maturing brain underscore the importance of this kinase for proper brain development. However, our present knowledge of CDKL5 functions is still rather limited. The picture that emerges from the molecular and cellular studies suggests that CDKL5 functions are important for regulating both neuronal morphology through cytoplasmic signaling pathways and activity-dependent gene expression in the nuclear compartment. This paper surveys the current state of CDKL5 research with emphasis on the clinical symptoms associated with mutations in CDKL5, the different mechanisms regulating its functions, and the connected molecular pathways. Finally, based on the available data we speculate that CDKL5 might play a role in neuronal plasticity and we adduce and discuss some possible arguments supporting this hypothesis

CDKL5 and shootin1 interact and concur in regulating neuronal polarization

In the last years, the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with epileptic encephalopathies characterized by the early onset of intractable epilepsy, severe developmental delay, autistic features, and often the development of Rett syndrome-like features. Still, the role of CDKL5 in neuronal functions is not fully understood. By way of a yeast two hybrid screening we identified the interaction of CDKL5 with shootin1, a brain specific protein acting as a determinant of axon formation during neuronal polarization. We found evidence that CDKL5 is involved, at least in part, in regulating neuronal polarization through its interaction with shootin1. Indeed, the two proteins interact in vivo and both are localized in the distal tip of outgrowing axons. By using primary hippocampal neurons as model system we find that adequate CDKL5 levels are required for axon specification. In fact, a significant number of neurons overexpressing CDKL5 is characterized by supernumerary axons, while the silencing of CDKL5 disrupts neuronal polarization. Interestingly, shootin1 phosphorylation is reduced in neurons silenced for CDKL5 suggesting that the kinase affects, directly or indirectly, the post-translational modification of shootin1. Finally, we find that the capacity of CDKL5 to generate surplus axons is attenuated in neurons with reduced shootin1 levels, in agreement with the notion that two proteins act in a common pathway. Altogether, these results point to a role of CDKL5 in the early steps of neuronal differentiation that can be explained, at least in part, by its association with shootin1

CDKL5 promotes axon formation.

<p>(A) Western blot showing CDKL5 levels in primary hippocampal neurons nucleofected before plating with bicistronic vectors expressing GFP alone or together with CDKL5 or CDKL5-K42R. Cell lysates were prepared at DIV5 and analyzed for CDKL5 levels using Tuj1 as loading control. (B) Representative images showing the localization of endogenous and exogenous CDKL5 in the soma of nucleofected GFP-positive neurons at DIV5. The exposure time of the GFP-expressing neuron (left) was increased to reveal the staining of endogenous CDKL5. (C) Representative images showing hippocampal neurons at DIV5 transfected with vectors expressing GFP together with CDKL5 or the K42R derivative. GFP and CDKL5 signals are in green and red, respectively. The arrows indicate neurons with increased CDKL5 levels. Scale bar: 20 μm. (D) Quantitative analysis of neuronal polarization. Axon specification was analyzed at DIV5 by determining the number of neurons with a single axon (polarized, black bars), multiple axons (dark grey bars) and neurons with no axon (light grey bars). Data are expressed as mean of 4 independent experiments ±SEM; ***p<0,001, **p<0,01, *p<0,05 (n≥100 neurons/condition, Student’s <i>t</i> test). (E) Graph showing the length of the longest axon and dendrite of transfected neurons. Data present neurite length as means ±SEM (n>28 neurons/condition, 4 independent experiments); **p<0,01. (Student’s <i>t</i> test).</p

CDKL5 regulates axon outgrowth through shootin1.

<p>(A) Immunofluorescence of neurons nucleofected before plating with a bicistronic vector expressing GFP alone or together with CDKL5 and subsequently infected with lentiviral particles expressing shRNAs against shootin1 or LacZ. At DIV5 neurons were stained for GFP, Tau1, and CDKL5 (green, blue, and red, respectively). (B) Quantification of neuronal polarization of GFP-positive neurons with increased CDKL5 expression. Data are expressed as means ±SEM. **p<0,01, *p<0,05. (n≥24 neurons/condition in 3 independent experiments; ANOVA two-way). Scale bar: 20 μm.</p

CDKL5 interacts with shootin1 in vivo.

<p>(A) A yeast two-hybrid screening identified shootin1 as a CDKL5 interacting protein. The C-terminal region of hCDKL5, spanning amino acids 299–1030, was used as bait (upper, thick bar). The diagram below shows shootin1 with its coiled coil domains in black. The clones identified in the screen are indicated as black bars and the minimum CDKL5 interacting region as a black bar. (B) Coimmunoprecipitation of P5-7 brain lysates with anti-CDKL5 (upper, n = 3) or anti-shootin1 (lower, n = 3) antibodies (both rabbit). IgGs were used as negative control. The immunoprecipitates and inputs (5% of the brain lysates) were analyzed by immunoblotting for CDKL5 and shootin1 (using a goat anti-shootin1 antibody). Asterisks indicate the immunoglobulin heavy chains and the open circle an unspecific band detected with anti-CDKL5. (C) Coimmunoprecipitation of HeLa cells overexpressing either Flag-CDKL5 or shootin1 or both proteins together. Whole cell lysates were immunoprecipiated with an anti-Flag resin and inputs (5%) and immunocomplexes analyzed by western blotting as indicated. Asterisk shows an anti-shootin1 reactive protein that copurifies with CDKL5. (n = 3).</p

CDKL5 influences shootin1 phosphorylation in primary cortical neurons.

<p>(A) Total cell extracts of DIV7 cortical neurons were treated with or without lambda phosphatase (λ-PPase) and analyzed by two-dimensional gel electrophoresis and immunoblotting with antibodies against shootin1, β-actin and, as control for the λ-PPase treatment, phopho-ERK1/2. (B) Primary cortical neurons were infected with shLacZ- or shCDKL5#1-expressing viral particles at DIV0 and total cell lysates were prepared at DIV7 and subjected to two-dimensional gel electrophoresis. Shootin1 and NFL were detected by immunoblotting; the single NFL-spot was used as internal control for alignment. Silencing of CDKL5 was confirmed by western blot (right panel). (n = 3).</p

CDKL5 and shootin1 are coexpressed in brains and neurons.

<p>(A) Western blot analysis showing CDKL5 and shootin1 levels in mouse brain at the indicated developmental stages using Tuj1 as loading control. (n = 2) (B) <i>Shootin1</i> is expressed in the cortex, as early as E13, in the cortical plate (cp) and its levels increase ongoing with development (a,b,c,d); <i>Cdkl5</i> (b’,c’,d’) follow the same pattern. Low but detectable levels of <i>shootin1</i> and <i>Cdkl5</i> mRNAs are present in cells migrating out of the ventricular and sub-ventricular zone (vz-svz) towards their final destination in the cortical plate (b,b’). At E18 <i>shootin1</i> and <i>Cdkl5</i> are strongly expressed throughout the whole thickness of the cortex (d,d’). Scale bars: 50 μm: b,b’,c,c’; 100 μm: d,d’; 200 μm: a. (C) Western blot showing CDKL5 and shootin1 levels in cultured primary hippocampal neurons at the indicated stages. A longer exposure of the 18 h time point is shown to the right. (n = 2). (D) Immunofluorescence analysis (left) of hippocampal neurons at stages 2–3 with antibodies against CDKL5 (green) and shootin1 (red). The small panels show the magnification of the minor processes/axons indicated with asterisks. Quantitative profiles showing the fluorescence intensities of shootin1 (red) and CDKL5 (green) from the soma to the distal tip of the neurites/axons indicated with asterisks are shown to the right. Scale bar: 10 μm.</p