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.
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.
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.
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.
Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, ...molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates 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.
Intellectual disability (ID) is the unavoidable hallmark of Down syndrome (DS), with a heavy impact on public health. Accumulating evidence shows that DS is characterized by numerous ...neurodevelopmental alterations among which the reduction of neurogenesis, dendritic hypotrophy and connectivity alterations appear to play a particularly prominent role. Although the mechanisms whereby gene triplication impairs brain development in DS have not been fully clarified, it is theoretically possible to correct trisomy-dependent defects with targeted pharmacotherapies. This review summarizes what we know about the effects of pharmacotherapies during different life stages in mouse models of DS. Since brain alterations in DS start to be present prenatally, the prenatal period represents an optimum window of opportunity for therapeutic interventions. Importantly, recent studies clearly show that treatment during the prenatal period can rescue overall brain development and behavior and that this effect outlasts treatment cessation. Although late therapies are unlikely to exert drastic changes in the brain, they may have an impact on the hippocampus, a brain region where neurogenesis continues throughout life. Indeed, treatment at adult life stages improves or even rescues hippocampal neurogenesis and connectivity and hippocampal-dependent learning and memory, although the duration of these effects still remains, in the majority of cases, a matter of investigation. The exciting discovery that trisomy-linked brain abnormalities can be prevented with early interventions gives us reason to believe that treatments during pregnancy may rescue brain development in fetuses with DS. For this reason we deem it extremely important to expedite the discovery of additional therapies practicable in humans in order to identify the best treatment/s in terms of efficacy and paucity of side effects. Prompt achievement of this goal is the big challenge for the scientific community of researchers interested in DS.
Abstract Mutations in the X-linked cyclin-dependent kinase-like 5 ( CDKL5 ) gene have been identified in a neurodevelopmental disorder characterized by early-onset intractable seizures, severe ...developmental delay, intellectual disability, and Rett's syndrome-like features. Since the physiological functions of CDKL5 still need to be elucidated, in the current study we took advantage of a new Cdkl5 knockout (KO) mouse model in order to shed light on the role of this gene in brain development. We mainly focused on the hippocampal dentate gyrus, a region that largely develops postnatally and plays a key role in learning and memory. Looking at the process of neurogenesis, we found a higher proliferation rate of neural precursors in Cdkl5 KO mice in comparison with wild type mice. However, there was an increase in apoptotic cell death of postmitotic granule neuron precursors, with a reduction in total number of granule cells. Looking at dendritic development, we found that in Cdkl5 KO mice the newly-generated granule cells exhibited a severe dendritic hypotrophy. In parallel, these neurodevelopmental defects were associated with impairment of hippocampus-dependent memory. Looking at the mechanisms whereby CDKL5 exerts its functions, we identified a central role of the AKT/GSK-3β signaling pathway. Overall our findings highlight a critical role of CDKL5 in the fundamental processes of brain development, namely neuronal precursor proliferation, survival and maturation. This evidence lays the basis for a better understanding of the neurological phenotype in patients carrying mutations in the CDKL5 gene.
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.
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.
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.
•Dendritic pathology appears early during postnatal development in Cdkl5 KO mice.•Microglia overactivation appears early during postnatal development in Cdkl5 KO mice.•Cdkl5 KO mice are characterized by an impairment in their early behavioral development.•Results indicate precocious onset of brain defects in Cdkl5 KO mice.
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.
Abstract 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 3β (GSK3β) a crucial inhibitory regulator of many neurodevelopmental processes. The goal of the current study was to establish whether inhibition of GSK3β corrects hippocampal developmental defects due to Cdkl5 loss. We found that treatment with the GSK3β 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 GSK3β inhibitors as potential therapeutic tools for the improvement of abnormal brain development in CDKL5 disorder.
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