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Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABAA receptor antagonist picrotoxin had no positive effects on LTP in Cdkl5 KO mice, the selective GABAB receptor antagonist CGP55845 restored LTP magnitude, suggesting that exaggerated GABAB receptor-mediated inhibition contributes to LTP impairment in mutants. Moreover, acute in vivo treatment with CGP55845 increased the number of PSD95 positive puncta as well as density and maturation of dendritic spines in PRC, and restored NOR memory in Cdkl5 KO mice. The present data show the efficacy of limiting excessive GABAB receptor-mediated signaling in improving synaptic plasticity and cognition in CDD mice.

Microglia, the immune cells of the central nervous system (CNS), have recently taken center stage in research due to their role in CNS diseases. An abnormal immune response during critical windows of development and consequent abnormal production of neuro-inflammatory mediators has an impact on the function and structure of the brain and contributes to the pathogenesis of several neurodevelopmental disorders such as Autism Spectrum Disorders. CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disease caused by de novo mutations in the X-linked CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, autistic features, motor impairment, visual deficits, and early-onset epilepsy. To date, little is known about the etiology of CDD and no therapies are available. Recently, a major cytokine dysregulation proportional to clinical severity, inflammatory status, and redox imbalance was evidenced in plasma from CDD patients, suggesting a subclinical chronic inflammatory status in children affected by this pathology. However, it is still unknown whether such an inflammatory state may even be mirrored at the cerebral level and whether it can contribute to the pathophysiology of CDD. Cdkl5 knockout (KO) mouse models, recently created to investigate the role of CDKL5 in the etiology of CDD, recapitulate various features of the disorder. Previous studies have shown impaired neuronal maturation and survival in the hippocampus of Cdkl5 KO mice, but the knowledge of the substrates underlying these alterations is still limited. Interestingly, we found increased microglial activation in the brain of a mouse model of CDD, the Cdkl5 KO mouse. We found alterations of microglial cell morphology and number, increased levels of AIF-1 and proinflammatory cytokines, and increased STAT3 signaling in the brain of Cdkl5 KO mice. Remarkably, treatment with Luteolin (a natural anti-inflammatory flavonoid) recovers microglia alterations as well as impaired neuronal survival and maturation in Cdkl5 KO mice, and prevents increased NMDA-induced cell death in the hippocampus. Our results suggest that neuro-inflammatory processes contribute to the pathogenesis of CDD and imply the potential usefulness of Luteolin as a treatment option in CDD patients.

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3β or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3β/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3β and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3β/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.

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.

BackgroundCDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental disorder characterized by early-onset epilepsy, intellectual disability, and autistic features, is caused by mutations in the CDKL5 gene. Evidence in animal models of CDD showed that absence of CDKL5 negatively affects neuronal survival, as well as neuronal maturation and dendritic outgrowth; however, knowledge of the substrates underlying these alterations is still limited. Neuro-inflammatory processes are known to contribute to neuronal dysfunction and death. Recent evidence shows a subclinical chronic inflammatory status in plasma from CDD patients. However, to date, it is unknown whether a similar inflammatory status is present in the brain of CDD patients and, if so, whether this plays a causative or exacerbating role in the pathophysiology of CDD. MethodsWe evaluated microglia activation using AIF-1 immunofluorescence, proinflammatory cytokine expression and signaling in the brain of a mouse model of CDD, the Cdkl5 KO mouse, which is characterized by an impaired survival of hippocampal neurons that worsens with age. Hippocampal neuron survival was determined by DCX, NeuN and cleaved caspase-3 immunostaining in Cdkl5 KO mice treated with luteolin (10 mg/kg), a natural anti-inflammatory flavonoid. Since hippocampal neurons of Cdkl5 KO mice exhibit increased susceptibility to excitotoxic stress, we evaluated neuronal survival in Cdkl5 KO mice injected with NMDA (60 mg/kg) after a 7-day treatment with luteolin. ResultsWe found increased microglial activation in the brain of the Cdkl5 KO mouse. We found alterations in microglial cell morphology and number, increased levels of AIF-1 and proinflammatory cytokines, and activation of STAT3 signaling. Remarkably, treatment with luteolin recovers microglia alterations as well as neuronal survival and maturation in Cdkl5 KO mice, and prevents the increase in NMDA-induced cell death in the hippocampus. ConclusionsOur results suggest that neuro-inflammatory processes contribute to the pathogenesis of CDD and imply the potential usefulness of luteolin as a treatment option in CDD patients.

BackgroundCDKL5 deficiency disorder (CDD), a severe neurodevelopmental disorder characterized by early onset epilepsy, intellectual disability, and autistic features, is caused by mutations in theCDKL5gene. Evidence in animal models of CDD showed that absence of CDKL5 negatively affects neuronal survival, as well as neuronal maturation and dendritic outgrowth; however, knowledge of the substrates underlying these alterations is still limited. Neuroinflammatory processes are known to contribute to neuronal dysfunction and death. Recent evidence shows a subclinical chronic inflammatory status in plasma from CDD patients. However, to date, it is unknown whether a similar inflammatory status is present in the brain of CDD patients and, if so, whether this plays a causative or exacerbating role in the pathophysiology of CDD.MethodsWe evaluated microglia activation using AIF-1 immunofluorescence, proinflammatory cytokine expression, and signaling in the brain of a mouse model of CDD, theCdkl5KO mouse, which is characterized by an impaired survival of hippocampal neurons that worsens with age. Hippocampal neuron survival was determined by DCX, NeuN, and cleaved caspase-3 immunostaining inCdkl5KO mice treated with luteolin (10 mg/kg), a natural anti-inflammatory flavonoid. Since hippocampal neurons ofCdkl5KO mice exhibit increased susceptibility to excitotoxic stress, we evaluated neuronal survival inCdkl5KO mice injected with NMDA (60 mg/kg) after a 7-day treatment with luteolin.ResultsWe found increased microglial activation in the brain of theCdkl5KO mouse. We found alterations in microglial cell morphology and number, increased levels of AIF-1 and proinflammatory cytokines, and activation of STAT3 signaling. Remarkably, treatment with luteolin recovers microglia alterations as well as neuronal survival and maturation inCdkl5KO mice, and prevents the increase in NMDA-induced cell death in the hippocampus.ConclusionsOur results suggest that neuroinflammatory processes contribute to the pathogenesis of CDD and imply the potential usefulness of luteolin as a treatment option in CDD patients.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABA

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Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a severe neurodevelopmental disorder, CDKL5 deficiency disorder (CDD). CDKL5 is fundamental for correct brain development and function, but the molecular mechanisms underlying aberrant neurologic dysfunction in CDD are incompletely understood. Here we show a dysregulation of hippocampal and cortical serotonergic (5-HT) receptor expression in heterozygous Cdkl5 knockout (KO) female mice, suggesting that impaired 5-HT neurotransmission contributes to CDD. We demonstrate that targeting impaired 5-HT signaling via the selective serotonin reuptake inhibitor (SSRI) sertraline rescues CDD-related neurodevelopmental and behavioral defects in heterozygous Cdkl5 KO female mice. In particular, chronic treatment with sertraline normalized locomotion, stereotypic and autistic-like features, and spatial memory in Cdkl5 KO mice. These positive behavioral effects were accompanied by restored neuronal survival, dendritic development and synaptic connectivity. At a molecular level, sertraline increased brain-derived neurotrophic factor (BDNF) expression and restored abnormal phosphorylation levels of tyrosine kinase B (TrkB) and its downstream target the extracellular signal-regulated kinase (ERK1/2). Since sertraline is an FDA-approved drug with an extensive safety and tolerability data package, even for children, our findings suggest that sertraline may improve neurodevelopment in children with CDD. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Mutations in the CDKL5 gene, which encodes a serine/threonine kinase, causes a rare encephalopathy, characterized by early-onset epilepsy and severe intellectual disability, named CDKL5 deficiency disorder (CDD). In vitro and in vivo studies in mouse models of Cdkl5 deficiency have highlighted the role of CDKL5 in brain development and, in particular, in the morphogenesis and synaptic connectivity of hippocampal and cortical neurons. Interestingly, Cdkl5 deficiency in mice increases vulnerability to excitotoxic stress in hippocampal neurons. However, the mechanism by which CDKL5 controls neuronal survival is far from being understood. To investigate further the function of CDKL5 and dissect the molecular mechanisms underlying neuronal survival, we generated a human neuronal model of CDKL5 deficiency, using CRISPR/Cas9-mediated genome editing. We demonstrated that CDKL5 deletion in human neuroblastoma SH-SY5Y cells not only impairs neuronal maturation but also reduces cell proliferation and survival, with alterations in the AKT and ERK signaling pathways and an increase in the proapoptotic BAX protein and in DNA damage-associated biomarkers (i.e., γH2AX, RAD50, and PARP1). Furthermore, CDKL5-deficient cells were hypersensitive to DNA damage-associated stress, accumulated more DNA damage foci (γH2AX positive) and were more prone to cell death than the controls. Importantly, increased kainic acid-induced cell death of hippocampal neurons of Cdkl5 KO mice correlated with an increased γH2AX immunostaining. The results suggest a previously unknown role for CDKL5 in DNA damage response that could underlie the pro-survival function of CDKL5.

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 femaleCdkl5knockout (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 heterozygousCdkl5+/− mice. We found thatCdkl5+/− 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 femaleCdkl5+/− mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

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.

The mitochondrial genome (mtDNA) is assembled into nucleo-protein structures termed nucleoids and maintained differently compared to nuclear DNA, the involved molecular basis remaining poorly understood. In yeast (Saccharomyces cerevisiae), mtDNA is a ∼80 kbp linear molecule and Abf2p, a double HMG-box protein, packages and maintains it. The protein binds DNA in a non-sequence-specific manner, but displays a distinct ‘phased-binding’ at specific DNA sequences containing poly-adenine tracts (A-tracts). We present here two crystal structures of Abf2p in complex with mtDNA-derived fragments bearing A-tracts. Each HMG-box of Abf2p induces a 90° bend in the contacted DNA, causing an overall U-turn. Together with previous data, this suggests that U-turn formation is the universal mechanism underlying mtDNA compaction induced by HMG-box proteins. Combining this structural information with mutational, biophysical and computational analyses, we reveal a unique DNA binding mechanism for Abf2p where a characteristic N-terminal flag and helix are crucial for mtDNA maintenance. Additionally, we provide the molecular basis for A-tract mediated exclusion of Abf2p binding. Due to high prevalence of A-tracts in yeast mtDNA, this has critical relevance for nucleoid architecture. Therefore, an unprecedented A-tract mediated protein positioning mechanism regulates DNA packaging proteins in the mitochondria, and in combination with DNA-bending and U-turn formation, governs mtDNA compaction., Ministry of Economy and Competitiveness (MINECO) [BFU2012-33516, BFU2015-70645-R to M.S; BIO2012- 32868, BFU2014-61670-EXP to M.O.; BFU2011-25697, BFU2014-60550-P to J.V.; FEDER CSIC13-4E-2065 to the Molecular imaging platform IBMB-PCB; Biomolecular and Bioinformatics Resources Platform (ISCIII PT 13/0001/0030) cofunded by the Fondo Europeo de Desarrollo Regional (FEDER) to M.O.]; Generalitat de Catalunya [SGR2009-1366, 2014-SGR-997 to M.S., SGR2009-1348, 2014 SGR-134 to M.O.]; Instituto Nacional de Bioinformatica; European Union [FP7- ´ HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2 to M.S.; H2020-EINFRA2015-1-675728, H2020-EINFRA-2015-676556 to M.O.]; European Research Council [ERC-2011-ADG 20110209- 291433 to M.O.]; ITN Fellowship [FP7-PEOPLE-2011- 290246 to A.C.]; Structural Biology Unit at Molecular Biology Institute of Barcelona (IBMB) is a ‘Maria de Maeztu’ Unit of Excellence awarded by MINECO [MDM2014-0435]; Severo Ochoa Award of Excellence from MINECO (to IRB Barcelona); ICREA Academia Research (to M.O.). Funding for open access charge: Ministry of Economy and Competitiviness [BFU2015-70645-R].

The introduction of Information Technologies in modern production systems is a consequence of the large amount of information that must be managed to gain an adequate control of an automated plant.