Early-onset brain alterations during postnatal development in a mouse model of CDKL5 deficiency disorder.

Mutations in the CDKL5 gene are the cause of CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental condition characterized by early-onset epilepsy, motor impairment, intellectual disability, and autistic features. A mouse model of CDD, the Cdkl5 KO mouse, that recapitulates several aspects of CDD symptomology, has helped to highlight brain alterations leading to CDD neurological defects. Studies of brain morphogenesis in adult Cdkl5 KO mice showed defects in dendritic arborization of pyramidal neurons and in synaptic connectivity, a hypocellularity of the hippocampal dentate gyrus, and a generalized microglia over-activation. Nevertheless, no studies are available regarding the presence of these brain alterations in Cdkl5 KO pups, and their severity in early stages of life compared to adulthood. A deeper understanding of the CDKL5 deficient brain during an early phase of postnatal development would represent an important milestone for further validation of the CDD mouse model, and for the identification of the optimum time window for treatments that target defects in brain development. In sight of this, we comparatively evaluated the dendritic arborization and spines of cortical pyramidal neurons, cortical excitatory and inhibitory connectivity, microglia activation, and proliferation and survival of granule cells of the hippocampal dentate gyrus in hemizygous Cdkl5 KO male (-/Y) mice aged 7, 14, 21, and 60 days. We found that most of the structural alterations in Cdkl5 -/Y brains are already present in pups aged 7 days and do not worsen with age. In contrast, the difference in the density of excitatory and inhibitory terminals between Cdkl5 -/Y and wild-type mice changes with age, suggesting an age-dependent cortical excitatory/inhibitory synaptic imbalance. Confirming the precocious presence of brain defects, Cdkl5 -/Y pups are characterized by an impairment in neonatal sensory-motor reflexes.

Cardiac Functional and Structural Abnormalities in a Mouse Model of CDKL5 Deficiency Disorder.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental disease that mostly affects girls, who are heterozygous for mutations in the X-linked CDKL5 gene. Mutations in the CDKL5 gene lead to a lack of CDKL5 protein expression or function and cause numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, gastrointestinal problems, and severe neurodevelopmental impairment. Mouse models of CDD recapitulate several aspects of CDD symptomology, including cognitive impairments, motor deficits, and autistic-like features, and have been useful to dissect the role of CDKL5 in brain development and function. However, our current knowledge of the function of CDKL5 in other organs/tissues besides the brain is still quite limited, reducing the possibility of broad-spectrum interventions. Here, for the first time, we report the presence of cardiac function/structure alterations in heterozygous Cdkl5 +/- female mice. We found a prolonged QT interval (corrected for the heart rate, QTc) and increased heart rate in Cdkl5 +/- mice. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Interestingly, Cdkl5 +/- hearts showed increased fibrosis, altered gap junction organization and connexin-43 expression, mitochondrial dysfunction, and increased ROS production. Together, these findings not only contribute to our understanding of the role of CDKL5 in heart structure/function but also document a novel preclinical phenotype for future therapeutic investigation.

Luteolin Treatment Ameliorates Brain Development and Behavioral Performance in a Mouse Model of CDKL5 Deficiency Disorder.

CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene, is characterized by early-onset epilepsy, intellectual disability, and autistic features. Although pharmacotherapy has shown promise in the CDD mouse model, safe and effective clinical treatments are still far off. Recently, we found increased microglial activation in the brain of a mouse model of CDD, the Cdkl5 KO mouse, suggesting that a neuroinflammatory state, known to be involved in brain maturation and neuronal dysfunctions, may contribute to the pathophysiology of CDD. The present study aims to evaluate the possible beneficial effect of treatment with luteolin, a natural flavonoid known to have anti-inflammatory and neuroprotective activities, on brain development and behavior in a heterozygous Cdkl5 (+/-) female mouse, the mouse model of CDD that best resembles the genetic clinical condition. We found that inhibition of neuroinflammation by chronic luteolin treatment ameliorates motor stereotypies, hyperactive profile and memory ability in Cdkl5 +/- mice. Luteolin treatment also increases hippocampal neurogenesis and improves dendritic spine maturation and dendritic arborization of hippocampal and cortical neurons. These findings show that microglia overactivation exerts a harmful action in the Cdkl5 +/- brain, suggesting that treatments aimed at counteracting the neuroinflammatory process should be considered as a promising adjuvant therapy for CDD.

A GABAB receptor antagonist rescues functional and structural impairments in the perirhinal cortex of a mouse model of CDKL5 deficiency disorder.

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.

Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder.

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.

Inhibition of microglia overactivation restores neuronal survival in a mouse model of CDKL5 deficiency disorder.

BACKGROUND: CDKL5 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. 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Treatment with a GSK-3ß/HDAC Dual Inhibitor Restores Neuronal Survival and Maturation in an In Vitro and In Vivo Model of CDKL5 Deficiency Disorder.

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-3ß 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-3ß/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3ß 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-3ß/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.

CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of 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.

HDAC4: a key factor underlying brain developmental alterations in CDKL5 disorder.

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.

Treatment with the GSK3-beta inhibitor Tideglusib improves hippocampal development and memory performance in juvenile, but not adult, Cdkl5 knockout mice.

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 GSK3ß, an important inhibitory regulator of many neuronal functions. Pharmacological inhibition of GSK3ß activity was able to recover hippocampal defects and cognitive performance in juvenile Cdkl5 KO mice, suggesting that GSK3ß inhibitors might be a potential therapeutic option for CDKL5 disorder. As GSK3ß inhibitors have been shown to have differential medication responses in young people and adults, this study was designed to examine whether GSK3ß is a possible therapeutic target both in juvenile and in adult CDKL5 patients. We found that treatment with the GSK3ß 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 GSK3ß 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.

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder.

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.

Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome.

Neurogenesis impairment is considered a major determinant of the intellectual disability that characterizes Down syndrome (DS), a genetic condition caused by triplication of chromosome 21. Previous evidence obtained in the Ts65Dn mouse model of DS showed that the triplicated gene APP (amyloid precursor protein) is critically involved in neurogenesis alterations. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain) resulting from APP cleavage by gamma-secretase increase the transcription of Ptch1, a Sonic Hedgehog (Shh) receptor that keeps the mitogenic Shh pathway repressed. Previous evidence showed that neonatal treatment with ELND006, an inhibitor of gamma-secretase, reinstates the Shh pathway and fully restores neurogenesis in Ts65Dn pups. In the framework of potential therapies for DS, it is extremely important to establish whether the positive effects of early intervention are retained after treatment cessation. Therefore, the goal of the current study was to establish whether early treatment with ELND006 leaves an enduring trace in the brain of Ts65Dn mice. Ts65Dn and euploid pups were treated with ELND006 in the postnatal period P3-P15 and the outcome of treatment was examined at ~one month after treatment cessation. We found that in treated Ts65Dn mice the pool of proliferating cells in the hippocampal dentate gyrus (DG) and total number of granule neurons were still restored as was the number of pre- and postsynaptic terminals in the stratum lucidum of CA3, the site of termination of the mossy fibers from the DG. Accordingly, patch-clamp recording from field CA3 showed functional normalization of the input to CA3. Unlike in field CA3, the number of pre- and postsynaptic terminals in the DG of treated Ts65Dn mice was no longer fully restored. The finding that many of the positive effects of neonatal treatment were retained after treatment cessation provides proof of principle demonstration of the efficacy of early inhibition of gamma-secretase for the improvement of brain development in DS.

CDKL5 deficiency entails sleep apneas in mice.

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.

Inhibition of GSK3ß rescues hippocampal development and learning in a mouse model of CDKL5 disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in a rare neurodevelopmental disorder characterized by early-onset seizures, severe developmental delay, intellectual disability and Rett syndrome-like features. CDKL5 is highly expressed in the brain during early postnatal stages, suggesting its importance for brain maturation. Using a newly-generated Cdkl5 knockout (Cdkl5 -/Y) mouse, we recently found that loss of Cdkl5 impairs postnatal hippocampal development with a reduction in neuronal precursor survival and maturation. These defects were accompanied by increased activity of the glycogen synthase kinase 3ß (GSK3ß) a crucial inhibitory regulator of many neurodevelopmental processes. The goal of the current study was to establish whether inhibition of GSK3ß corrects hippocampal developmental defects due to Cdkl5 loss. We found that treatment with the GSK3ß inhibitor SB216763 restored neuronal precursor survival, dendritic maturation, connectivity and hippocampus-dependent learning and memory in the Cdkl5 -/Y mouse. Importantly, these effects were retained one month after treatment cessation. At present, there are no therapeutic strategies to improve the neurological defects of subjects with CDKL5 disorder. Current results point at GSK3ß inhibitors as potential therapeutic tools for the improvement of abnormal brain development in CDKL5 disorder.

Inhibition of APP gamma-secretase restores Sonic Hedgehog signaling and neurogenesis in the Ts65Dn mouse model of Down syndrome.

Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3-P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower ßamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.