Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in neurodevelopmental disorders including atypical Rett syndrome (RTT), autism spectrum disorders (ASDs), and early infantile epileptic encephalopathy. The biological function of CDKL5 and its role in the etiology of these disorders, however, remain unclear. Here we report the development of a unique knockout mouse model of CDKL5-related disorders and demonstrate that mice lacking CDKL5 show autistic-like deficits in social interaction, as well as impairments in motor control and fear memory. Neurophysiological recordings reveal alterations in event-related potentials (ERPs) similar to those observed in RTT and ASDs. Moreover, kinome profiling uncovers disruption of multiple signal transduction pathways, including the AKTmammalian target of rapamycin (mTOR) cascade, upon Cdkl5 loss-of-function. These data demonstrate that CDKL5 regulates signal transduction pathways and mediates autistic-like phenotypes and together establish a causal role for Cdkl5 loss-of-function in neurodevelopmental disorders.C yclin-dependent kinase-like 5 (CDKL5) is an X-linked gene associated with early infantile epileptic encephalopathy 2 (EIEE2) (1), atypical Rett syndrome (RTT) (2), and autism spectrum disorders (ASDs) (3, 4). Patients with CDKL5 mutations display a heterogenous array of clinical phenotypes, the most prominent of which include early-onset seizures, intellectual disability, and autistic features (5).CDKL5 is a serine/threonine (S/T) kinase that is highly expressed in the brain (6). In vitro studies have demonstrated that CDKL5 may mediate the phosphorylation of methyl-CpG binding protein 2 (MeCP2) (7), DNA methyltransferase 1 (DNMT1) (8), and netrin-G1 ligand (NGL-1) (9). RNAi-mediated knockdown studies show that CDKL5 can regulate neuronal outgrowth and synapse stability (9, 10). Despite these proposed functions, the exact role of CDKL5 in the phosphorylation of MeCP2 (7, 11) and in dendritic outgrowth (9, 10) remains unclear, and thus requires further investigation. The limited understanding of CDKL5 function and its associated signal transduction pathways has hindered the development of therapeutics for CDKL5-related disorders. Current treatments focus on managing symptoms and reducing seizure frequency, but have limited effectiveness (12).To investigate the function of CDKL5 in a disease model and identify potential avenues of therapeutic intervention, we developed a Cdkl5 knockout mouse. We found that mice lacking CDKL5 show autistic-like behavioral abnormalities, deficits in neural circuit communication, and alterations in multiple signal transduction pathways. We establish a causal link between Cdkl5 loss-of-function and disease-related phenotypes and identify the AKT-mammalian target of rapamycin (mTOR) pathway as a unique candidate for targeted therapeutic intervention of CDKL5-related disorders. ResultsGeneration of Cdkl5 Knockout Mice. To investigate the pathophysiology underlying CDKL5-related disorders, we generated ...

Mutations in the MECP2 gene cause the autism spectrum disorder Rett Syndrome (RTT). One of the most common mutations associated with RTT occurs at MeCP2 Threonine 158 converting it to Methionine (T158M) or Alanine (T158A). To understand the role of T158 mutation in the pathogenesis of RTT, we generated knockin mice recapitulating MeCP2 T158A mutation. Here we show a causal role for T158A mutation in the development of RTT-like phenotypes including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those in Mecp2-null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. Importantly, the age-dependent development of event-related neuronal responses are disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials may serve as a biomarker for RTT and treatment evaluation.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory.

CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autistic features, and CDKL5-deficient mice exhibit a constellation of behavioral phenotypes reminiscent of the human disorder. We previously found that CDKL5 dysfunction in forebrain glutamatergic neurons results in deficits in learning and memory. However, the pathogenic origin of the autistic features of CDD remains unknown. Here, we find that selective loss of CDKL5 in GABAergic neurons leads to autistic-like phenotypes in mice accompanied by excessive glutamatergic transmission, hyperexcitability, and increased levels of postsynaptic NMDA receptors. Acute, low-dose inhibition of NMDAR signaling ameliorates autistic-like behaviors in GABAergic knockout mice, as well as a novel mouse model bearing a CDD-associated nonsense mutation, CDKL5 R59X, implicating the translational potential of this mechanism. Together, our findings suggest that enhanced NMDAR signaling and circuit hyperexcitability underlie autistic-like features in mouse models of CDD and provide a new therapeutic avenue to treat CDD-related symptoms.