The Experts below are selected from a list of 1602 Experts worldwide ranked by ideXlab platform
Nicoletta Landsberger - One of the best experts on this subject based on the ideXlab platform.
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Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.
International journal of molecular sciences, 2019Co-Authors: Dario Balestra, Nicoletta Landsberger, Domenico Giorgio, Matteo Bizzotto, Maria Fazzari, Bruria Ben Zeev, Mirko Pinotti, Angelisa FrascaAbstract:Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5′ donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.
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Aminoglycoside drugs induce efficient read-through of CDKL5 nonsense mutations, slightly restoring its kinase activity.
RNA biology, 2019Co-Authors: Maria Fazzari, Angelisa Frasca, Francesco Bifari, Nicoletta LandsbergerAbstract:The X-linked CDKL5 gene codes for a kinase whose mutations have been associated with a suite of neurodevelopmental disorders generally characterized by early-onset epileptic encephalopathy and severe intellectual disability. The impact of these mutations on CDKL5 functions and brain development remain mainly unknown, although the importance of maintaining the catalytic activity is generally recognized. Since no cure exists for CDKL5 disorders, the demand for innovative therapies is a real emergency. The recent discovery that CDKL5 is dosage sensitive poses concerns on conventional protein and gene augmentative therapies. Thus, RNA-based therapeutic approaches might be preferred. We studied the efficacy of read-through therapy on CDKL5 premature termination codons (PTCs) that correspond roughly to 15% of all mutations. Our results provide the first demonstration that all tested CDKL5 nonsense mutations are efficiently suppressed by aminoglycoside drugs. The functional characterization of the restored full-length CDKL5 reveals that read-through proteins fully recover their subcellular localization, but only partially rescue their catalytic activity. Since read-through can cause amino acid substitution, CDKL5 patients carrying the PTC outside the catalytic domain might benefit more from a nonsense suppression therapy. Eventually, we demonstrate that non-aminoglycoside drugs, such as Ataluren (PTC124) and GJ072, are unable to induce read-through activity on CDKL5 PTCs. Although these drugs might be more effective in vivo, these results question the validity of the Ataluren phase 2 clinical trial that is currently ongoing on CDKL5 patients.
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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, 2019Co-Authors: Daniele Vigli, Laura Rusconi, Elena Amendola, Cornelius Gross, Paolo La Montanara, Daniela Valenti, Livia Cosentino, Enza Lacivita, Marcello Leopoldo, Nicoletta LandsbergerAbstract:Abstract 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-HT7R) 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-HT7R.
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CDKL5 localizes at the centrosome and midbody and is required for faithful cell division
Scientific reports, 2017Co-Authors: Isabella Barbiero, Marco Tramarin, Chetan Chandola, Maria Fazzari, Davide Valente, Fiorenza Magi, Anna Bergo, Laura Monteonofrio, Silvia Soddu, Nicoletta LandsbergerAbstract: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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The neurosteroid pregnenolone reverts microtubule derangement induced by the loss of a functional CDKL5-IQGAP1 complex
Human molecular genetics, 2017Co-Authors: Isabella Barbiero, Diana Peroni, Marco Tramarin, Chetan Chandola, Laura Rusconi, Nicoletta Landsberger, Charlotte Kilstrup-nielsenAbstract: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.
Charlotte Kilstrup-nielsen - One of the best experts on this subject based on the ideXlab platform.
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The green tea polyphenol epigallocatechin-3-gallate (EGCG) restores CDKL5-dependent synaptic defects in vitro and in vivo.
Neurobiology of disease, 2020Co-Authors: L. Trovò, Elisabetta Ciani, Isabella Barbiero, Marco Tramarin, Laura Rusconi, C. Fuchs, R. De Rosa, Charlotte Kilstrup-nielsenAbstract:CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder that is characterised by early-onset seizures, intellectual disability, gross motor impairment, and autistic-like features. CDD is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene that encodes a serine/threonine kinase with a predominant expression in the brain. Loss of CDKL5 causes neurodevelopmental alterations in vitro and in vivo, including defective dendritic arborisation and spine maturation, which most likely underlie the cognitive defects and autistic features present in humans and mice. Here, we show that treatment with epigallatocathechin-3-gallate (EGCG), the major polyphenol of green tea, can restore defects in dendritic and synaptic development of primary CDKL5 knockout (KO) neurons. Furthermore, defective synaptic maturation in the hippocampi and cortices of adult CDKL5-KO mice can be rescued through the intraperitoneal administration of EGCG, which is however not sufficient to normalise behavioural CDKL5-dependent deficits. EGCG is a pleiotropic compound with numerous cellular targets, including the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) that is selectively inhibited by EGCG. DYRK1A controls dendritic development and spine formation and its deregulation has been implicated in neurodevelopmental and degenerative diseases. Treatment with another DYRK1A inhibitor, harmine, was capable of correcting neuronal CDKL5-dependent defects; moreover, DYRK1A levels were upregulated in primary CDKL5-KO neurons in concomitance with increased phosphorylation of Tau, a well-accepted DYRK1A substrate. Altogether, our results indicate that DYRK1A deregulation may contribute, at least in part, to the neurodevelopmental alterations caused by CDKL5 deficiency.
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Microtubules: A Key to Understand and Correct Neuronal Defects in CDKL5 Deficiency Disorder?
International journal of molecular sciences, 2019Co-Authors: Isabella Barbiero, Roberta De Rosa, Charlotte Kilstrup-nielsenAbstract:CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked CDKL5 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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The neurosteroid pregnenolone reverts microtubule derangement induced by the loss of a functional CDKL5-IQGAP1 complex
Human molecular genetics, 2017Co-Authors: Isabella Barbiero, Diana Peroni, Marco Tramarin, Chetan Chandola, Laura Rusconi, Nicoletta Landsberger, Charlotte Kilstrup-nielsenAbstract: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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Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.
PloS one, 2016Co-Authors: Ralph D. Hector, Nicoletta Landsberger, Charlotte Kilstrup-nielsen, Owen Dando, Peter C. Kind, Mark E.s. Bailey, Stuart CobbAbstract: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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CDKL5 and Shootin1 Interact and Concur in Regulating Neuronal Polarization
PloS one, 2016Co-Authors: Mohammad Sarfaraz Nawaz, Laura Rusconi, Nicoletta Landsberger, Sara Ricciardi, Paolo La Montanara, Elisa Giarda, Francesco Bedogni, Dalila Ciceri, Tiziana Alberio, Charlotte Kilstrup-nielsenAbstract: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.
Elisabetta Ciani - One of the best experts on this subject based on the ideXlab platform.
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The green tea polyphenol epigallocatechin-3-gallate (EGCG) restores CDKL5-dependent synaptic defects in vitro and in vivo.
Neurobiology of disease, 2020Co-Authors: L. Trovò, Elisabetta Ciani, Isabella Barbiero, Marco Tramarin, Laura Rusconi, C. Fuchs, R. De Rosa, Charlotte Kilstrup-nielsenAbstract:CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder that is characterised by early-onset seizures, intellectual disability, gross motor impairment, and autistic-like features. CDD is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene that encodes a serine/threonine kinase with a predominant expression in the brain. Loss of CDKL5 causes neurodevelopmental alterations in vitro and in vivo, including defective dendritic arborisation and spine maturation, which most likely underlie the cognitive defects and autistic features present in humans and mice. Here, we show that treatment with epigallatocathechin-3-gallate (EGCG), the major polyphenol of green tea, can restore defects in dendritic and synaptic development of primary CDKL5 knockout (KO) neurons. Furthermore, defective synaptic maturation in the hippocampi and cortices of adult CDKL5-KO mice can be rescued through the intraperitoneal administration of EGCG, which is however not sufficient to normalise behavioural CDKL5-dependent deficits. EGCG is a pleiotropic compound with numerous cellular targets, including the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) that is selectively inhibited by EGCG. DYRK1A controls dendritic development and spine formation and its deregulation has been implicated in neurodevelopmental and degenerative diseases. Treatment with another DYRK1A inhibitor, harmine, was capable of correcting neuronal CDKL5-dependent defects; moreover, DYRK1A levels were upregulated in primary CDKL5-KO neurons in concomitance with increased phosphorylation of Tau, a well-accepted DYRK1A substrate. Altogether, our results indicate that DYRK1A deregulation may contribute, at least in part, to the neurodevelopmental alterations caused by CDKL5 deficiency.
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Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder.
Human molecular genetics, 2019Co-Authors: Leonardo Lupori, Claudia Fuchs, Raffaele Mazziotti, Elisabetta Ciani, Elena Putignano, Giulia Sagona, Antonia Stefanov, Debora Napoli, Enrica Strettoi, Tommaso PizzorussoAbstract:CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD.
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CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling.
Brain pathology (Zurich Switzerland), 2019Co-Authors: Claudia Fuchs, Stefania Trazzi, Giorgio Medici, Laura Gennaccaro, Elisa Ren, Giuseppe Galvani, Chiara Berteotti, Manuela Loi, Elisabetta CianiAbstract:CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF-β action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-β1 treatment normalized defective neuronal survival and maturation in CDKL5 knockout (KO) neurons. Moreover, we demonstrate that CDKL5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-β1 prevents increased NMDA-induced cell death in hippocampal neurons from CDKL5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of CDKL5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.
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Treatment with the GSK3-beta inhibitor Tideglusib improves hippocampal development and memory performance in juvenile, but not adult, CDKL5 knockout mice.
The European journal of neuroscience, 2018Co-Authors: Claudia Fuchs, Stefania Trazzi, Roberto Rimondini, Laura Gennaccaro, Norma Fustini, Elisabetta CianiAbstract:Cyclin-dependent kinase-like 5 (CDKL5) disorder is a severe neurodevelopmental disorder characterized by early-onset epileptic seizures, severe developmental delay, and intellectual disability. To date, no effective pharmacological treatments are available to improve the neurological phenotype that is due to mutations in the CDKL5 gene. Murine models of CDKL5 disorder have recently been generated, making the preclinical testing of pharmacological interventions possible. Using a CDKL5 knockout (KO) mouse model, we recently demonstrated that deficiency of CDKL5 causes defects in postnatal hippocampal development and hippocampus-dependent learning and memory. These defects were accompanied by an increased activity of GSK3β, an important inhibitory regulator of many neuronal functions. Pharmacological inhibition of GSK3β activity was able to recover hippocampal defects and cognitive performance in juvenile CDKL5 KO mice, suggesting that GSK3β inhibitors might be a potential therapeutic option for CDKL5 disorder. As GSK3β inhibitors have been shown to have differential medication responses in young people and adults, this study was designed to examine whether GSK3β is a possible therapeutic target both in juvenile and in adult CDKL5 patients. We found that treatment with the GSK3β inhibitor Tideglusib during the juvenile period improved hippocampal development and hippocampus-dependent behaviors in CDKL5 KO mice, while treatment later on in adulthood had no positive effects. These results suggest that pharmacological interventions aimed at normalizing impaired GSK3β activity might have different age-dependent outcomes in CDKL5 disorder. This is of utmost importance in the development of therapeutic approaches in CDKL5 patients and in the design of rational clinical trials.
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CDKL5 deficiency entails sleep apneas in mice.
Journal of sleep research, 2017Co-Authors: Viviana Lo Martire, Stefano Bastianini, Rocchina Viggiano, Chiara Berteotti, Elisabetta Ciani, Sara Alvente, Alessandro Silvani, Alice Valli, Giovanna ZoccoliAbstract:A recently discovered neurodevelopmental disorder caused by the mutation of the cyclin-dependent kinase-like 5 gene (CDKL5) entails complex autistic-like behaviours similar to Rett syndrome, but its impact upon physiological functions remains largely unexplored. Sleep-disordered breathing is common and potentially life-threatening in patients with Rett syndrome; however, evidence is limited in children with CDKL5 disorder, and is lacking altogether in adults. The aim of this study was to test whether the breathing pattern during sleep differs between adult CDKL5 knockout (CDKL5-KO) and wild-type (WT) mice. Using whole-body plethysmography, sleep and breathing were recorded non-invasively for 8 h during the light period. Sleep apneas occurred more frequently in CDKL5-KO than in WT mice. A receiver operating characteristic (ROC) analysis discriminated CDKL5-KO significantly from WT mice based on sleep apnea occurrence. These data demonstrate that sleep apneas are a core feature of CDKL5 disorder and a respiratory biomarker of CDKL5 deficiency in mice, and suggest that sleep-disordered breathing should be evaluated routinely in CDKL5 patients.
Claudia Fuchs - One of the best experts on this subject based on the ideXlab platform.
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Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder.
Human molecular genetics, 2019Co-Authors: Leonardo Lupori, Claudia Fuchs, Raffaele Mazziotti, Elisabetta Ciani, Elena Putignano, Giulia Sagona, Antonia Stefanov, Debora Napoli, Enrica Strettoi, Tommaso PizzorussoAbstract:CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD.
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CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling.
Brain pathology (Zurich Switzerland), 2019Co-Authors: Claudia Fuchs, Stefania Trazzi, Giorgio Medici, Laura Gennaccaro, Elisa Ren, Giuseppe Galvani, Chiara Berteotti, Manuela Loi, Elisabetta CianiAbstract:CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene, a member of a highly conserved family of serine-threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF-β action, is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF-β1 treatment normalized defective neuronal survival and maturation in CDKL5 knockout (KO) neurons. Moreover, we demonstrate that CDKL5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF-β1 prevents increased NMDA-induced cell death in hippocampal neurons from CDKL5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of CDKL5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.
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Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.
Neural plasticity, 2018Co-Authors: Claudia Fuchs, Stefania Trazzi, Stefano Bastianini, Giorgio Medici, Viviana Lo Martire, Laura Gennaccaro, Simone Bettini, Elisa Ren, Giovanna Zoccoli, Roberto RimondiniAbstract:CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female CDKL5 knockout (CDKL5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous CDKL5 +/- mice. We found that CDKL5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, CDKL5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female CDKL5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.
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CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder
Human Molecular Genetics, 2018Co-Authors: Stefania Trazzi, Marianna De Franceschi, Claudia Fuchs, Stefano Bastianini, Rocchina Viggiano, Leonardo Lupori, Raffaele Mazziotti, Giorgio Medici, Viviana Lo Martire, Roberto RimondiniAbstract:: Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in CDKL5-null mice. Notably, systemically administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.
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Treatment with the GSK3-beta inhibitor Tideglusib improves hippocampal development and memory performance in juvenile, but not adult, CDKL5 knockout mice.
The European journal of neuroscience, 2018Co-Authors: Claudia Fuchs, Stefania Trazzi, Roberto Rimondini, Laura Gennaccaro, Norma Fustini, Elisabetta CianiAbstract:Cyclin-dependent kinase-like 5 (CDKL5) disorder is a severe neurodevelopmental disorder characterized by early-onset epileptic seizures, severe developmental delay, and intellectual disability. To date, no effective pharmacological treatments are available to improve the neurological phenotype that is due to mutations in the CDKL5 gene. Murine models of CDKL5 disorder have recently been generated, making the preclinical testing of pharmacological interventions possible. Using a CDKL5 knockout (KO) mouse model, we recently demonstrated that deficiency of CDKL5 causes defects in postnatal hippocampal development and hippocampus-dependent learning and memory. These defects were accompanied by an increased activity of GSK3β, an important inhibitory regulator of many neuronal functions. Pharmacological inhibition of GSK3β activity was able to recover hippocampal defects and cognitive performance in juvenile CDKL5 KO mice, suggesting that GSK3β inhibitors might be a potential therapeutic option for CDKL5 disorder. As GSK3β inhibitors have been shown to have differential medication responses in young people and adults, this study was designed to examine whether GSK3β is a possible therapeutic target both in juvenile and in adult CDKL5 patients. We found that treatment with the GSK3β inhibitor Tideglusib during the juvenile period improved hippocampal development and hippocampus-dependent behaviors in CDKL5 KO mice, while treatment later on in adulthood had no positive effects. These results suggest that pharmacological interventions aimed at normalizing impaired GSK3β activity might have different age-dependent outcomes in CDKL5 disorder. This is of utmost importance in the development of therapeutic approaches in CDKL5 patients and in the design of rational clinical trials.
Isamu Kameshita - One of the best experts on this subject based on the ideXlab platform.
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Cyclin-Dependent Kinase-Like 5 (CDKL5): Possible Cellular Signalling Targets and Involvement in CDKL5 Deficiency Disorder.
Neural plasticity, 2020Co-Authors: Syouichi Katayama, Noriyuki Sueyoshi, Tetsuya Inazu, Isamu KameshitaAbstract:Cyclin-dependent kinase-like 5 (CDKL5, also known as STK9) is a serine/threonine protein kinase originally identified in 1998 during a transcriptional mapping project of the human X chromosome. Thereafter, a mutation in CDKL5 was reported in individuals with the atypical Rett syndrome, a neurodevelopmental disorder, suggesting that CDKL5 plays an important regulatory role in neuronal function. The disease associated with CDKL5 mutation has recently been recognised as CDKL5 deficiency disorder (CDD) and has been distinguished from the Rett syndrome owing to its symptomatic manifestation. Because CDKL5 mutations identified in patients with CDD cause enzymatic loss of function, CDKL5 catalytic activity is likely strongly associated with the disease. Consequently, the exploration of CDKL5 substrate characteristics and regulatory mechanisms of its catalytic activity are important for identifying therapeutic target molecules and developing new treatment. In this review, we summarise recent findings on the phosphorylation of CDKL5 substrates and the mechanisms of CDKL5 phosphorylation and dephosphorylation. We also discuss the relationship between changes in the phosphorylation signalling pathways and the CDKL5 knockout mouse phenotype and consider future prospects for the treatment of mental and neurological disease associated with CDKL5 mutations.
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subcellular distribution of cyclin dependent kinase like 5 CDKL5 is regulated through phosphorylation by dual specificity tyrosine phosphorylation regulated kinase 1a dyrk1a
Biochemical and Biophysical Research Communications, 2017Co-Authors: Syouichi Katayama, Isamu Kameshita, Naoya Hatano, Yasunori Sugiyama, Noriyuki SueyoshiAbstract:Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization.
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Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5)
Biochemistry, 2015Co-Authors: Syouichi Katayama, Noriyuki Sueyoshi, Isamu KameshitaAbstract:Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase known to be associated with X-linked neurodevelopmental disorders. In a previous study, we identified amphiphysin 1 (Amph1) as a potential substrate for CDKL5 and identified a single phosphorylation site at Ser-293. In this study, we investigated the molecular mechanisms of substrate recognition by CDKL5 using Amph1 as a model substrate. Amph1 served as an efficient CDKL5 substrate, whereas Amph2, a structurally related homologue of Amph1, was not phosphorylated by CDKL5. The sequence around the Amph1 phosphorylation site is RPR293SPSQ, while the corresponding sequence in Amph2 is IPK332SPSQ. To define the amino acid sequence specificity of the substrate, various point mutants of Amph1 and Amph2 were prepared and phosphorylated by CDKL5. Both Amph2(I329R) and Amph1 served as efficient CDKL5 substrates, but Amph1(R290I) did not, indicating that the arginyl residue at the P −3 position is critical for substrate recognition. With regard to pr...
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Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder.
Archives of biochemistry and biophysics, 2013Co-Authors: Mari Sekiguchi, Syouichi Katayama, Noriyuki Sueyoshi, Naoya Hatano, Yasushi Shigeri, Isamu KameshitaAbstract:Abstract Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120 kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.