Publications by authors named "Charlotte Kilstrup-Nielsen"

34 Publications

Therapeutic potential of pregnenolone and pregnenolone methyl ether on depressive and CDKL5 deficiency disorders: Focus on microtubule targeting.

J Neuroendocrinol 2021 Aug 19:e13033. Epub 2021 Aug 19.

Department of Biotechnology and Life Sciences, (DBSV), Centre of NeuroScience, University of Insubria, Busto Arsizio, Italy.

Pregnenolone methyl-ether (PME) is a synthetic derivative of the endogenous neuroactive steroid pregnenolone (PREG), which is an important modulator of several brain functions. In addition to being the precursor of steroids, PREG acts directly on various targets including microtubules (MTs), the functioning of which is fundamental for the development and homeostasis of nervous system. The coordination of MT dynamics is supported by a plethora of MT-associated proteins (MAPs) and by a specific MT code that is defined by the post-translational modifications of tubulin. Defects associated with MAPs or tubulin post-translational modifications are linked to different neurological pathologies including mood and neurodevelopmental disorders. In this review, we describe the beneficial effect of PME in major depressive disorders (MDDs) and in CDKL5 deficiency disorder (CDD), two pathologies that are joint by defective MT dynamics. Growing evidence indeed suggests that PME, as well as PREG, is able to positively affect the MT-binding of MAP2 and the plus-end tracking protein CLIP170 that are both found to be deregulated in the above mentioned pathologies. Furthermore, PME influences the state of MT acetylation, the deregulation of which is often associated with neurological abnormalities including MDDs. By contrast to PREG, PME is not metabolised into other downstream molecules with specific biological properties, an aspect that makes this compound more suitable for therapeutic strategies. Thus, through the analysis of MDDs and CDD, this work focuses attention on the possible use of PME for neuronal pathologies associated with MT defects.
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http://dx.doi.org/10.1111/jne.13033DOI Listing
August 2021

MECP2 mutations affect ciliogenesis: a novel perspective for Rett syndrome and related disorders.

EMBO Mol Med 2020 06 8;12(6):e10270. Epub 2020 May 8.

Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy.

Mutations in MECP2 cause several neurological disorders of which Rett syndrome (RTT) represents the best-defined condition. Although mainly working as a transcriptional repressor, MeCP2 is a multifunctional protein revealing several activities, the involvement of which in RTT remains obscure. Besides being mainly localized in the nucleus, MeCP2 associates with the centrosome, an organelle from which primary cilia originate. Primary cilia function as "sensory antennae" protruding from most cells, and a link between primary cilia and mental illness has recently been reported. We herein demonstrate that MeCP2 deficiency affects ciliogenesis in cultured cells, including neurons and RTT fibroblasts, and in the mouse brain. Consequently, the cilium-related Sonic Hedgehog pathway, which is essential for brain development and functioning, is impaired. Microtubule instability participates in these phenotypes that can be rescued by HDAC6 inhibition together with the recovery of RTT-related neuronal defects. Our data indicate defects of primary cilium as a novel pathogenic mechanism that by contributing to the clinical features of RTT might impact on proper cerebellum/brain development and functioning, thus providing a novel therapeutic target.
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http://dx.doi.org/10.15252/emmm.201910270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278541PMC
June 2020

Microtubules: A Key to Understand and Correct Neuronal Defects in CDKL5 Deficiency Disorder?

Int J Mol Sci 2019 Aug 21;20(17). Epub 2019 Aug 21.

Department of Biotechnology and Life Sciences, (DBSV), University of Insubria, Via Manara 7, 21052 Busto Arsizio (VA), Italy.

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked gene that encodes a serine/threonine kinase. CDD is characterised by the early onset of seizures and impaired cognitive and motor skills. Loss of CDKL5 in vitro and in vivo affects neuronal morphology at early and late stages of maturation, suggesting a link between CDKL5 and the neuronal cytoskeleton. Recently, various microtubule (MT)-binding proteins have been identified as interactors of CDKL5, indicating that its roles converge on regulating MT functioning. MTs are dynamic structures that are important for neuronal morphology, migration and polarity. The delicate control of MT dynamics is fundamental for proper neuronal functions, as evidenced by the fact that aberrant MT dynamics are involved in various neurological disorders. In this review, we highlight the link between CDKL5 and MTs, discussing how CDKL5 deficiency may lead to deranged neuronal functions through aberrant MT dynamics. Finally, we discuss whether the regulation of MT dynamics through microtubule-targeting agents may represent a novel strategy for future pharmacological approaches in the CDD field.
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http://dx.doi.org/10.3390/ijms20174075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747382PMC
August 2019

Cannabidivarin completely rescues cognitive deficits and delays neurological and motor defects in male mutant mice.

J Psychopharmacol 2019 07 14;33(7):894-907. Epub 2019 May 14.

1 Department of Biotechnology and Life Sciences (DBSV), University of Insubria, Varese, Italy.

Background: Recent evidence suggests that 2-week treatment with the non-psychotomimetic cannabinoid cannabidivarin (CBDV) could be beneficial towards neurological and social deficits in early symptomatic mutant mice, a model of Rett syndrome (RTT).

Aim: The aim of this study was to provide further insights into the efficacy of CBDV in -null mice using a lifelong treatment schedule (from 4 to 9 weeks of age) to evaluate its effect on recognition memory and neurological defects in both early and advanced stages of the phenotype progression.

Methods: CBDV 0.2, 2, 20 and 200 mg/kg/day was administered to -null mice from 4 to 9 weeks of age. Cognitive and neurological defects were monitored during the whole treatment schedule. Biochemical analyses were carried out in brain lysates from 9-week-old wild-type and knockout mice to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) levels as well as components of the endocannabinoid system.

Results: CBDV rescues recognition memory deficits in mutant mice and delays the appearance of neurological defects. At the biochemical level, it normalizes BDNF/IGF1 levels and the defective PI3K/AKT/mTOR pathway in mutant mice at an advanced stage of the disease. deletion upregulates CB1 and CB2 receptor levels in the brain and these changes are restored after CBDV treatment.

Conclusions: CBDV administration exerts an enduring rescue of memory deficits in mutant mice, an effect that is associated with the normalization of BDNF, IGF-1 and rpS6 phosphorylation levels as well as CB1 and CB2 receptor expression. CBDV delays neurological defects but this effect is only transient.
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http://dx.doi.org/10.1177/0269881119844184DOI Listing
July 2019

A Novel Mecp2 Knock-in Model Displays Similar Behavioral Traits But Distinct Molecular Features Compared to the Mecp2-Null Mouse Implying Precision Medicine for the Treatment of Rett Syndrome.

Mol Neurobiol 2019 Jul 6;56(7):4838-4854. Epub 2018 Nov 6.

Neuroscience Division, IRCCS San Raffaele Scientific Institute, 20132, Milan, Italy.

MeCP2 is a fundamental protein associated with several neurological disorders, including Rett syndrome. It is considered a multifunctional factor with a prominent role in regulating chromatin structure; however, a full comprehension of the consequences of its deficiency is still lacking. Here, we characterize a novel mouse model of Mecp2 bearing the human mutation Y120D, which is localized in the methyl-binding domain. As most models of Mecp2, the Mecp2 mouse develops a severe Rett-like phenotype. This mutation alters the interaction of the protein with chromatin, but surprisingly, it also impairs its association with corepressors independently on the involved interacting domains. These features, which become overt mainly in the mature brain, cause a more accessible and transcriptionally active chromatin structure; conversely, in the Mecp2-null brain, we find a less accessible and transcriptionally inactive chromatin. By demonstrating that different MECP2 mutations can produce concordant neurological phenotypes but discordant molecular features, we highlight the importance of considering personalized approaches for the treatment of Rett syndrome.
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http://dx.doi.org/10.1007/s12035-018-1412-2DOI Listing
July 2019

Rescue of prepulse inhibition deficit and brain mitochondrial dysfunction by pharmacological stimulation of the central serotonin receptor 7 in a mouse model of CDKL5 Deficiency Disorder.

Neuropharmacology 2019 01 13;144:104-114. Epub 2018 Oct 13.

Center for Behavioral Sciences and Mental Health, Istituto Superiore di Sanità, 00161 Rome, Italy. Electronic address:

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause CDKL5 Deficiency Disorder (CDD), a rare neurodevelopmental syndrome characterized by severe behavioural and physiological symptoms. No cure is available for CDD. CDKL5 is a kinase that is abundantly expressed in the brain and plays a critical role in neurodevelopmental processes, such as neuronal morphogenesis and plasticity. This study provides the first characterization of the neurobehavioural phenotype of 1 year old Cdkl5-null mice and demonstrates that stimulation of the serotonin receptor 7 (5-HTR) with the agonist molecule LP-211 (0.25 mg/kg once/day for 7 days) partially rescues the abnormal phenotype and brain molecular alterations in Cdkl5-null male mice. In particular, LP-211 treatment completely normalizes the prepulse inhibition defects observed in Cdkl5-null mice and, at a molecular level, restores the abnormal cortical phosphorylation of rpS6, a downstream target of mTOR and S6 kinase, which plays a direct role in regulating protein synthesis. Moreover, we demonstrate for the first time that mitochondria show prominent functional abnormalities in Cdkl5-null mouse brains that can be restored by pharmacological stimulation of brain 5-HTR.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.018DOI Listing
January 2019

The antidepressant tianeptine reverts synaptic AMPA receptor defects caused by deficiency of CDKL5.

Hum Mol Genet 2018 06;27(12):2052-2063

Department of Biotechnology and Life Sciences and Center of Neuroscience, University of Insubria, 21052 Busto Arsizio, Italy.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a complex neurological disorder, characterized by infantile seizures, impairment of cognitive and motor skills and autistic features. Loss of Cdkl5 in mice affects dendritic spine maturation and dynamics but the underlying molecular mechanisms are still far from fully understood. Here we show that Cdkl5 deficiency in primary hippocampal neurons leads to deranged expression of the alpha-amino-3-hydroxy-5-methyl-4-iso-xazole propionic acid receptors (AMPA-R). In particular, a dramatic reduction of expression of the GluA2 subunit occurs concomitantly with its hyper-phosphorylation on Serine 880 and increased ubiquitination. Consequently, Cdkl5 silencing skews the composition of membrane-inserted AMPA-Rs towards the GluA2-lacking calcium-permeable form. Such derangement is likely to contribute, at least in part, to the altered synaptic functions and cognitive impairment linked to loss of Cdkl5. Importantly, we find that tianeptine, a cognitive enhancer and antidepressant drug, known to recruit and stabilise AMPA-Rs at the synaptic sites, can normalise the expression of membrane inserted AMPA-Rs as well as the number of PSD-95 clusters, suggesting its therapeutic potential for patients with mutations in CDKL5.
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http://dx.doi.org/10.1093/hmg/ddy108DOI Listing
June 2018

CDKL5 localizes at the centrosome and midbody and is required for faithful cell division.

Sci Rep 2017 07 24;7(1):6228. Epub 2017 Jul 24.

Department of Biotechnology and Life Sciences, University of Insubria, 21052, Busto Arsizio, Italy.

The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.
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http://dx.doi.org/10.1038/s41598-017-05875-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524905PMC
July 2017

The neurosteroid pregnenolone reverts microtubule derangement induced by the loss of a functional CDKL5-IQGAP1 complex.

Hum Mol Genet 2017 09;26(18):3520-3530

Department of Biotechnology and Life Sciences and Center of Neuroscience, University of Insubria, 21052 Busto Arsizio, Italy.

CDKL5 is a protein kinase that plays a key role for neuronal functions as testified by the onset of complex neuronal dysfunctions in patients with genetic lesions in CDKL5. Here we identify a novel interactor of CDKL5, IQGAP1, a fundamental regulator of cell migration and polarity. In accordance with a functional role of this interaction, depletion of CDKL5 impairs cell migration and impedes the localization of IQGAP1 at the leading edge. Moreover, we demonstrate that CDKL5 is required for IQGAP1 to form a functional complex with its effectors, Rac1 and the microtubule plus end tracking protein CLIP170. These defects eventually impact on the microtubule association of CLIP170, thus deranging their dynamics. CLIP170 is a cellular target of the neurosteroid pregnenolone; by blocking CLIP170 in its active conformation, pregnenolone is capable of restoring the microtubule association of CLIP170 in CDKL5 deficient cells and rescuing morphological defects in neurons devoid of CDKL5. These findings provide novel insights into CDKL5 functions and pave the way for target-specific therapeutic strategies for individuals affected with CDKL5-disorder.
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http://dx.doi.org/10.1093/hmg/ddx237DOI Listing
September 2017

Brain phosphorylation of MeCP2 at serine 164 is developmentally regulated and globally alters its chromatin association.

Sci Rep 2016 06 21;6:28295. Epub 2016 Jun 21.

San Raffaele Rett Research Unit, San Raffaele Scientific Institute, Milan, Italy.

MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA.
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http://dx.doi.org/10.1038/srep28295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915018PMC
June 2016

Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

PLoS One 2016 17;11(6):e0157758. Epub 2016 Jun 17.

Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912119PMC
July 2017

MeCP2 Related Studies Benefit from the Use of CD1 as Genetic Background.

PLoS One 2016 20;11(4):e0153473. Epub 2016 Apr 20.

San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.

MECP2 mutations cause a number of neurological disorders of which Rett syndrome (RTT) represents the most thoroughly analysed condition. Many Mecp2 mouse models have been generated through the years; their validity is demonstrated by the presence of a broad spectrum of phenotypes largely mimicking those manifested by RTT patients. These mouse models, between which the C57BL/6 Mecp2tm1.1Bird strain probably represents the most used, enabled to disclose much of the roles of Mecp2. However, small litters with little viability and poor maternal care hamper the maintenance of the colony, thus limiting research on such animals. For this reason, past studies often used Mecp2 mouse models on mixed genetic backgrounds, thus opening questions on whether modifier genes could be responsible for at least part of the described effects. To verify this possibility, and facilitate the maintenance of the Mecp2 colony, we transferred the Mecp2tm1.1Bird allele on the stronger CD1 background. The CD1 strain is easier to maintain and largely recapitulates the phenotypes already described in Mecp2-null mice. We believe that this mouse model will foster the research on RTT.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153473PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838291PMC
August 2016

CDKL5 and Shootin1 Interact and Concur in Regulating Neuronal Polarization.

PLoS One 2016 5;11(2):e0148634. Epub 2016 Feb 5.

Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of Biotechnology and Life Sciences, Centre of Neuroscience, University of Insubria, 21052 Busto Arsizio, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148634PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746202PMC
July 2016

MeCP2 Affects Skeletal Muscle Growth and Morphology through Non Cell-Autonomous Mechanisms.

PLoS One 2015 22;10(6):e0130183. Epub 2015 Jun 22.

San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Insitute, Milan, Italy; Department of Medical Biotechnology and Translational Medicine, University of Milan, Segrate Italy.

Rett syndrome (RTT) is an autism spectrum disorder mainly caused by mutations in the X-linked MECP2 gene and affecting roughly 1 out of 10.000 born girls. Symptoms range in severity and include stereotypical movement, lack of spoken language, seizures, ataxia and severe intellectual disability. Notably, muscle tone is generally abnormal in RTT girls and women and the Mecp2-null mouse model constitutively reflects this disease feature. We hypothesized that MeCP2 in muscle might physiologically contribute to its development and/or homeostasis, and conversely its defects in RTT might alter the tissue integrity or function. We show here that a disorganized architecture, with hypotrophic fibres and tissue fibrosis, characterizes skeletal muscles retrieved from Mecp2-null mice. Alterations of the IGF-1/Akt/mTOR pathway accompany the muscle phenotype. A conditional mouse model selectively depleted of Mecp2 in skeletal muscles is characterized by healthy muscles that are morphologically and molecularly indistinguishable from those of wild-type mice raising the possibility that hypotonia in RTT is mainly, if not exclusively, mediated by non-cell autonomous effects. Our results suggest that defects in paracrine/endocrine signaling and, in particular, in the GH/IGF axis appear as the major cause of the observed muscular defects. Remarkably, this is the first study describing the selective deletion of Mecp2 outside the brain. Similar future studies will permit to unambiguously define the direct impact of MeCP2 on tissue dysfunctions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476581PMC
April 2016

Defects During Mecp2 Null Embryonic Cortex Development Precede the Onset of Overt Neurological Symptoms.

Cereb Cortex 2016 06 15;26(6):2517-2529. Epub 2015 May 15.

San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.

MeCP2 is associated with several neurological disorders; of which, Rett syndrome undoubtedly represents the most frequent. Its molecular roles, however, are still unclear, and data from animal models often describe adult, symptomatic stages, while MeCP2 functions during embryonic development remain elusive. We describe the pattern and timing of Mecp2 expression in the embryonic neocortex highlighting its low but consistent expression in virtually all cells and show the unexpected occurrence of transcriptional defects in the Mecp2 null samples at a stage largely preceding the onset of overt symptoms. Through the deregulated expression of ionic channels and glutamatergic receptors, the lack of Mecp2 during early neuronal maturation leads to the reduction in the neuronal responsiveness to stimuli. We suggest that such features concur to morphological alterations that begin affecting Mecp2 null neurons around the perinatal age and become evident later in adulthood. We indicate MeCP2 as a key modulator of the transcriptional mechanisms regulating cerebral cortex development. Neurological phenotypes of MECP2 patients could thus be the cumulative result of different adverse events that are already present at stages when no obvious signs of the pathology are evident and are worsened by later impairments affecting the central nervous system during maturation and maintenance of its functionality.
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http://dx.doi.org/10.1093/cercor/bhv078DOI Listing
June 2016

Synaptic synthesis, dephosphorylation, and degradation: a novel paradigm for an activity-dependent neuronal control of CDKL5.

J Biol Chem 2015 Feb 2;290(7):4512-27. Epub 2015 Jan 2.

From the Department of Theoretical and Applied Sciences, Section of Biomedical Research; University of Insubria, 21052 Busto Arsizio, Italy and the San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy

Mutations in the X-linked CDKL5 (cyclin-dependent kinase-like 5) gene have been associated with several forms of neurodevelopmental disorders, including atypical Rett syndrome, autism spectrum disorders, and early infantile epileptic encephalopathy. Accordingly, loss of CDKL5 in mice results in autistic-like features and impaired neuronal communication. Although the biological functions of CDKL5 remain largely unknown, recent pieces of evidence suggest that CDKL5 is involved in neuronal plasticity. Herein, we show that, at all stages of development, neuronal depolarization induces a rapid increase in CDKL5 levels, mostly mediated by extrasomatic synthesis. In young neurons, this induction is prolonged, whereas in more mature neurons, NMDA receptor stimulation induces a protein phosphatase 1-dependent dephosphorylation of CDKL5 that is mandatory for its proteasome-dependent degradation. As a corollary, neuronal activity leads to a prolonged induction of CDKL5 levels in immature neurons but to a short lasting increase of the kinase in mature neurons. Recent results demonstrate that many genes associated with autism spectrum disorders are crucial components of the activity-dependent signaling networks regulating the composition, shape, and strength of the synapse. Thus, we speculate that CDKL5 deficiency disrupts activity-dependent signaling and the consequent synapse development, maturation, and refinement.
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http://dx.doi.org/10.1074/jbc.M114.589762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326854PMC
February 2015

Methyl-CpG binding protein 2 (MeCP2) localizes at the centrosome and is required for proper mitotic spindle organization.

J Biol Chem 2015 Feb 19;290(6):3223-37. Epub 2014 Dec 19.

From the Department of Theoretical and Applied Sciences, Section of Biomedical Research, University of Insubria, 21052 Busto Arsizio, Italy, the San Raffaele Rett Research Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy

Mutations in MECP2 cause a broad spectrum of neuropsychiatric disorders of which Rett syndrome represents the best defined condition. Both neuronal and non-neuronal functions of the methyl-binding protein underlie the related pathologies. Nowadays MeCP2 is recognized as a multifunctional protein that modulates its activity depending on its protein partners and posttranslational modifications. However, we are still missing a comprehensive understanding of all MeCP2 functions and their involvement in the related pathologies. The study of human mutations often offers the possibility of clarifying the functions of a protein. Therefore, we decided to characterize a novel MeCP2 phospho-isoform (Tyr-120) whose relevance was suggested by a Rett syndrome patient carrying a Y120D substitution possibly mimicking a constitutively phosphorylated state. Unexpectedly, we found MeCP2 and its Tyr-120 phospho-isoform enriched at the centrosome both in dividing and postmitotic cells. The molecular and functional connection of MeCP2 to the centrosome was further reinforced through cellular and biochemical approaches. We show that, similar to many centrosomal proteins, MeCP2 deficiency causes aberrant spindle geometry, prolonged mitosis, and defects in microtubule nucleation. Collectively, our data indicate a novel function of MeCP2 that might reconcile previous data regarding the role of MeCP2 in cell growth and cytoskeleton stability and that might be relevant to understand some aspects of MeCP2-related conditions. Furthermore, they link the Tyr-120 residue and its phosphorylation to cell division, prompting future studies on the relevance of Tyr-120 for cortical development.
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http://dx.doi.org/10.1074/jbc.M114.608125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318997PMC
February 2015

MeCP2 post-translational modifications: a mechanism to control its involvement in synaptic plasticity and homeostasis?

Front Cell Neurosci 2014 13;8:236. Epub 2014 Aug 13.

Division of Neuroscience, San Raffaele Rett Research Center, San Raffaele Scientific Institute Milan, Italy ; Section of Biomedical Research, Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of Theoretic and Applied Sciences, University of Insubria Busto Arsizio, Italy.

Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.
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http://dx.doi.org/10.3389/fncel.2014.00236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131190PMC
August 2014

Rett syndrome and the urge of novel approaches to study MeCP2 functions and mechanisms of action.

Neurosci Biobehav Rev 2014 Oct 2;46 Pt 2:187-201. Epub 2014 Mar 2.

San Raffaele Rett Research Center, Division of Neuroscience, San Raffaele Scientific Institute, Milan 20132, Italy; Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of Theoretical and Applied Sciences, Division of Biomedical Research, University of Insubria, Busto Arsizio 21052, Italy. Electronic address:

Rett syndrome (RTT) is a devastating genetic disorder that worldwide represents the most common genetic cause of severe intellectual disability in females. Most cases are caused by mutations in the X-linked MECP2 gene. Several recent studies have demonstrated that RTT mimicking animal models do not develop an irreversible condition and phenotypic rescue is possible. However, no cure for RTT has been identified so far, and patients are only given symptomatic and supportive treatments. The development of clinical applications imposes a more comprehensive knowledge of MeCP2 functional role(s) and their relevance for RTT pathobiology. Herein, we thoroughly survey the knowledge about MeCP2 structure and functions, highlighting the necessity of identifying more functional domains and the value of molecular genetics. Given that, in our opinion, RTT ultimately is generated by perturbations in gene transcription and so far no genes/pathways have been consistently linked to a dysfunctional MeCP2, we have used higher-level bioinformatic analyses to identify commonly deregulated mechanisms in MeCP2-defective samples. In this review we present our results and discuss the possible value of the utilized approach.
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http://dx.doi.org/10.1016/j.neubiorev.2014.01.011DOI Listing
October 2014

CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons.

Nat Cell Biol 2012 Sep 26;14(9):911-23. Epub 2012 Aug 26.

Stem Cell and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.
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http://dx.doi.org/10.1038/ncb2566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485419PMC
September 2012

What we know and would like to know about CDKL5 and its involvement in epileptic encephalopathy.

Neural Plast 2012 17;2012:728267. Epub 2012 Jun 17.

Theoretical and Applied Sciences, Division of Biomedical Research, University of Insubria, 21052 Busto Arsizio, Italy.

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.
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http://dx.doi.org/10.1155/2012/728267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3385648PMC
April 2013

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

J Biol Chem 2011 Oct 5;286(42):36550-8. Epub 2011 Aug 5.

Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of Biomedical, Informatics and Communication Sciences, University of Insubria, Busto Arsizio 21052, Italy.

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.
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http://dx.doi.org/10.1074/jbc.M111.235630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196071PMC
October 2011

A novel transcript of cyclin-dependent kinase-like 5 (CDKL5) has an alternative C-terminus and is the predominant transcript in brain.

Hum Genet 2012 Feb 12;131(2):187-200. Epub 2011 Jul 12.

NSW Centre for Rett Syndrome Research, The Children's Hospital at Westmead, Sydney, Australia.

The X-linked cyclin-dependent kinase-like 5 (CDKL5) gene is an important molecular determinant of early-onset intractable seizures with infantile spasms and Rett syndrome-like phenotype. The gene encodes a kinase that may influence components of molecular pathways associated with MeCP2. In humans there are two previously reported splice variants that differ in the 5' untranslated exons and produce the same 115 kDa protein. Furthermore, very recently, a novel transcript including a novel exon (16b) has been described. By aligning both the human and mouse CDKL5 proteins to the orthologs of other species, we identified a theoretical 107 kDa isoform with an alternative C-terminus that terminates in intron 18. In human brain and all other tissues investigated except the testis, this novel isoform is the major CDKL5 transcript. The detailed characterisation of this novel isoform of CDKL5 reveals functional and subcellular localisation attributes that overlap greatly, but not completely, with that of the previously studied human CDKL5 protein. Considering its predominant expression in the human and mouse brain, we believe that this novel isoform is likely to be of primary pathogenic importance in human diseases associated with CDKL5 deficiency, and suggest that screening of the related intronic sequence should be included in the molecular genetic analyses of patients with a suggestive clinical phenotype.
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http://dx.doi.org/10.1007/s00439-011-1058-xDOI Listing
February 2012

The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis.

Hum Mol Genet 2010 Aug 26;19(16):3114-23. Epub 2010 May 26.

Laboratory of Genetic and Epigenetic Control of Gene Expression, Department of Structural and Functional Biology, University of Insubria, 21052 Busto Arsizio, VA, Italy.

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome.
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http://dx.doi.org/10.1093/hmg/ddq214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908467PMC
August 2010

Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis.

EMBO Rep 2009 Dec 9;10(12):1327-33. Epub 2009 Oct 9.

Department of Experimental Oncology, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Rome 00158, Italy.

Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth.
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http://dx.doi.org/10.1038/embor.2009.217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799202PMC
December 2009

CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery.

Hum Mol Genet 2009 Dec 9;18(23):4590-602. Epub 2009 Sep 9.

Division of Neuroscience, San Raffaele Rett Research Center, San Raffaele Scientific Institute, Milan 20132, Italy.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.
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http://dx.doi.org/10.1093/hmg/ddp426DOI Listing
December 2009

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

J Biol Chem 2008 Oct 13;283(44):30101-11. Epub 2008 Aug 13.

Department of Structural and Functional Biology, University of Insubria, Via Alberto da Giussano 12, 21052 Busto Arsizio (VA), Italy.

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.
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http://dx.doi.org/10.1074/jbc.M804613200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662074PMC
October 2008

Spatio-temporal dynamics and localization of MeCP2 and pathological mutants in living cells.

Epigenetics 2007 Sep 18;2(3):187-97. Epub 2007 Sep 18.

Italian Institute of Technology (IIT), Pisa, Italy.

MECP2 is an X-linked gene coding for a protein functioning as a transcriptional repressor. The protein MeCP2 (Methyl CpG-binding protein) is an abundant component of pericentric heterochromatin and its mutations or duplications are present in around 80% of patients with a neurological disorder known as Rett Syndrome. Although MeCP2 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching in controlled conditions concentration, we demonstrated that most GFP-MeCP2 fusion protein associates strongly and reversibly to pericentric heterocromatin whereas the remaining fraction is bound irreversibly. The mobility of the methyl-binding protein is influenced by the differentiation state of the host cells. Furthermore, residues downstream of the methyl-binding domain are critical for the interaction with chromatin. Whereas the binding is stabilised by the central region it is likely modulated by the most C-terminal region. Importantly, these residues are missing in several disease causing mutations. Our data suggest that alterations in the affinity of MeCP2 for chromatin might contribute to the pathological effects of mutations causing Rett Syndrome.
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http://dx.doi.org/10.4161/epi.2.3.5057DOI Listing
September 2007

Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation.

J Biol Chem 2006 Oct 24;281(42):32048-56. Epub 2006 Aug 24.

Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, 21052 Busto Arsizio (VA), Italy.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions.
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http://dx.doi.org/10.1074/jbc.M606325200DOI Listing
October 2006
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