CDKL5 deficiency disorder (CDD), a developmental encephalopathy caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene, is characterized by a complex and severe clinical picture, including early-onset epilepsy and cognitive, motor, visual, and gastrointestinal disturbances. This disease still lacks a medical treatment to mitigate, or reverse, its course and improve the patient’s quality of life. Although CDD is primarily a genetic brain disorder, some evidence indicates systemic abnormalities, such as the presence of a redox imbalance in the plasma and skin fibroblasts from CDD patients and in the cardiac myocytes of a mouse model of CDD. In order to shed light on the role of oxidative stress in the CDD pathophysiology, in this study, we aimed to investigate the therapeutic potential of Coenzyme Q10 (CoQ10), which is known to be a powerful antioxidant, using in vitro and in vivo models of CDD. We found that CoQ10 supplementation not only reduces levels of reactive oxygen species (ROS) and normalizes glutathione balance but also restores the levels of markers of DNA damage (γ-H2AX) and senescence (lamin B1), restoring cellular proliferation and improving cellular survival in a human neuronal model of CDD. Importantly, oral supplementation with CoQ10 exerts a protective role toward lipid peroxidation and DNA damage in the heart of a murine model of CDD, the Cdkl5 (+/−) female mouse. Our results highlight the therapeutic potential of the antioxidant supplement CoQ10 in counteracting the detrimental oxidative stress induced by CDKL5 deficiency.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset, intractable epilepsy, motor and cognitive impairment, and autistic-like features. Although constitutive Cdkl5 knockout (KO) models have established the importance of CDKL5 during early brain development, CDKL5’s role in the mature brain remains poorly defined. Here, we employed an inducible, conditional KO model in which Cdkl5 is selectively deleted from forebrain glutamatergic neurons in adult mice to investigate the postdevelopmental functions of CDKL5. Using a total of 48 adult male mice, including Cdkl5^flox/Y(Cre⁺) (n = 30) and Cdkl5^flox/Y(Cre⁻) littermate controls (n = 18), we found that tamoxifen-induced Cdkl5 deletion led to prominent behavioral impairments, including deficits in motor coordination, reduced sociability, and impaired hippocampus-dependent spatial memory, while behavioral features such as hyperactivity and stereotypic jumping, typically present in germline KOs, were absent. Sensory functions, including olfaction and pain perception, were also preserved. At the cellular level, the loss of Cdkl5 resulted in a marked reduction in excitatory synapse density in the cortex and hippocampus, accompanied by increased numbers of immature dendritic spines and decreased mature spines. Neuronal loss in the hippocampal CA1 region and selective microglial activation in the cortex were also observed. These alterations closely resemble those seen in constitutive KO models, underscoring the ongoing requirement for CDKL5 expression in excitatory neurons for maintaining synaptic integrity and neuronal homeostasis in the adult brain. This study underscores the importance of temporally controlled models for investigating the mechanisms underlying CDD pathophysiology in the adult brain.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset, intractable epilepsy, motor and cognitive impairment, and autistic-like features. Although constitutive Cdkl5 knock-out (KO) models have established the importance of CDKL5 during early brain develop-ment, its role in the mature brain remains poorly defined. Here, we used an inducible, conditional KO model in which Cdkl5 is selectively deleted from forebrain glutamatergic neurons in adult mice to investigate the postdevelopmental functions of CDKL5. Tamoxi-fen-induced Cdkl5 deletion in adult Cdkl5flox/Y(Cre+) males led to prominent behavioral impairments, including deficits in motor coordination, reduced sociability, and impaired hippocampus-dependent spatial memory, while behavioral features such as hyperactivity and stereotypic jumping, typically present in germline KOs, were absent. Sensory func-tions, including olfaction and pain perception, were also preserved. At the cellular level, the loss of Cdkl5 resulted in a marked reduction of excitatory synapse density in the cortex and hippocampus, accompanied by increased numbers of immature dendritic spines and decreased mature spines. Neuronal loss in the hippocampal CA1 region and selective mi-croglial activation in the cortex were also observed. These alterations closely resemble those seen in constitutive KO models, underscoring the ongoing requirement for CDKL5 expression in excitatory neurons for maintaining synaptic integrity and neuronal homeo-stasis in the adult brain. This study underscores the importance of temporally controlled models for investigating the mechanisms underlying CDD pathophysiology in the adult brain.

Efficient delivery of biological material to the central nervous system (CNS) remains a key limitation of conventional gene therapies. Recently, we developed a novel strategy based on a secretable and cell-penetrating TATk-CDKL5 fused protein which enhances the brain biodistribution and the therapeutic efficiency of the gene therapy approach in a mouse model of CDKL5 Deficiency Disorder (CDD). Here, to compare the efficacy of the TATk-CDKL5 gene therapy with a conventional approach in correcting the CDD pathological phenotype, we employed cortical organoids generated from CDD patient-derived iPSCs as a human model of CDD. We found greater therapeutic efficacy of the recombinant TATk-CDKL5 protein compared to the CDKL5 protein alone in improving or ameliorating defects caused by the absence of CDKL5, such as abnormal hyperexcitability evaluated with microelectrode arrays (MEA). Interestingly, CDD cortical organoids exhibited reduced cell proliferation and increased neuronal cell death compared to control cortical organoids; defects that were only restored by the expression of the recombinant TATk-CDKL5 protein. These findings suggest the superior efficiency of a gene therapy based on a cross-correction approach for treating CDD.

CDKL5 Deficiency Disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early disruptions of synaptic maturation and network stability, leading to persistent motor, cognitive, and behavioral impairments. Given the role of the endocannabinoid system in synaptic development, neuroinflammation, and neuronal resilience, we investigated whether the sustained enhancement of endogenous 2-arachidonoylglycerol (2-AG) signaling via monoacylglycerol lipase (MAGL) inhibition could mitigate key pathological features in adult Cdkl5 knockout (KO) mice. Using an intermittent 6-week treatment, the MAGL inhibitor JZL184 robustly increased plasma 2-AG levels, reduced MAGL protein levels, and activated CB1-AKT signaling without evidence of receptor desensitization. Despite this clear pharmacodynamic efficacy, behavioral effects were domain-specific: neither dose ameliorated core behavioral deficits, although the higher dose selectively reduced stereotypic jumping and modestly improved cue-dependent associative memory. At the cellular level, JZL184 induced biologically meaningful effects, partially restoring dendritic spine maturation in the primary somatosensory cortex and increasing neuronal survival in the vulnerable CA1 hippocampal region. In contrast, microglial responses were dose-dependent and divergent, with the lower dose exerting anti-inflammatory effects, while the higher dose increased cortical microglial density and Allograft Inflammatory Factor-1 (AIF-1) expression, suggesting engagement of compensatory or off-target mechanisms. Overall, these findings show that MAGL inhibition activates neuroprotective pathways and ameliorates select structural deficits in adult Cdkl5 KO mice, but is insufficient to produce broad behavioral recovery, highlighting the domain-specific effects of selective 2-AG enhancement via MAGL inhibition and the need for developmentally informed or multimodal therapeutic strategies in CDD.

CDKL5 Deficiency Disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early disruptions of synaptic maturation and network stability, leading to persistent motor, cognitive, and behavioral impairments. Given the role of the endocannabinoid system in synaptic development, neuroinflammation, and neuronal resilience, we investigated whether chronic enhancement of endogenous 2-arachidonoylglycerol (2-AG) signaling via monoacylglycerol lipase (MAGL) inhibition could mitigate key pathological features in Cdkl5 knockout (KO) mice. Using an intermittent 6-week treatment, the MAGL inhibitor JZL184 robustly increased plasma 2-AG levels, inhibited MAGL activity, and activated CB1-AKT signaling without evidence of receptor desensitization. Despite this clear pharmacodynamic efficacy, behavioral improvement was limited: neither dose rescued core behavioral deficits, although the higher dose selectively reduced stereotypic jumping and modestly improved cue-dependent associative memory. At the cellular level, JZL184 induced biologically meaningful effects, partially restoring dendritic spine maturation and increasing neuronal survival in the vulnerable CA1 hippocampal region. In contrast, microglial responses were dose-dependent and divergent, with the lower dose exerting anti-inflammatory effects, while the higher dose increased cortical microglial density and AIF-1 expression, suggesting engagement of compensatory or off-target mechanisms. Overall, these findings show that MAGL inhibition activates neuroprotective pathways and ameliorates select structural deficits in Cdkl5 KO mice, but is insufficient to reverse established behavioral abnormalities, highlighting the limits of endocannabinoid enhancement and the need for developmentally timed or multimodal therapeutic strategies in CDD.