Inhibition of GSK3β rescues hippocampal development and learning in a mouse model of CDKL5 disorder Claudia Fuchs a , Roberto Rimondini b,1 , Rocchina Viggiano a,1 , Stefania Trazzi a , Marianna De Franceschi a , Renata Bartesaghi a , Elisabetta Ciani a, ⁎ a Department of Biomedical and Neuromotor Sciences, University of Bologna, Piazza di Porta San Donato 2, 40126 Bologna, Italy b Department of Medical and Clinical Sciences, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy abstract article info Article history: Received 10 March 2015 Revised 10 June 2015 Accepted 24 June 2015 Available online 2 July 2015 Keywords: CDKL5 disorder Encephalopathy Pharmacotherapy GSK3β inhibitor Rescue of hippocampal developmental 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 pre- cursor 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. Impor- tantly, these effects were retained one month after treatment cessation. At present, there are no therapeutic strat- egies 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. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been found in individuals with a rare neurodevelopmental disorder characterized by early-onset epileptic encephalopathy (Fehr et al., 2013). CDKL5 mutation-linked primary clinical features are severe intellectual disability and gross motor impairment. Moreover, patients manifest features overlapping with Rett syndrome, including micro- cephaly, hyperventilation, hand apraxia, and stereotypies. CDKL5 disor- der affects mainly females due to the X-chromosome location of the CDKL5 gene (Fehr et al., 2013). Females are heterozygous for CDKL5 de- ficiency and are mosaic for mutated CDKL5 gene due to random X- chromosome inactivation. Males may also be affected, although with a lower incidence, and have a more severe phenotype (Fehr et al., 2013; Guerrini and Parrini, 2012). CDKL5, also known as STK9, is a serine/threonine protein kinase that is highly expressed in the brain, mainly in neurons with both a nuclear and dendrite localization (Chen et al., 2010; Ricciardi et al., 2012; Rusconi et al., 2008; Zhu et al., 2013). In the early postnatal period, CDKL5 brain expression exhibits a peak (Ricciardi et al., 2012; Zhu et al., 2013), suggesting its potential importance in brain maturation and function. Recently, Cdkl5 knockout mouse models have been creat- ed (Amendola et al., 2014; Wang et al., 2012) in order to understand the etiology of the CDKL5 disorder phenotype. Deficiency of Cdkl5 in mice leads to autistic-like behavior, dendritic hypotrophy and impairment of neural circuit communication, indicating that CDKL5 loss of function severely impairs brain development (Amendola et al., 2014; Wang et al., 2012). Using a Cdkl5 knockout mouse model, we recently showed that CDKL5 plays a fundamental role in postnatal hippocampal neurogenesis, affecting neural precursor proliferation as well as the survival and matu- ration of newborn granule cells (Fuchs et al., 2014). Disruption of neurogenesis and dendritic development was associated with alterations in hippocampal-dependent cognitive performance (Fuchs et al., 2014). Looking at the molecular mechanisms whereby loss of Cdkl5 may alter brain development, we found disruption of the AKT/GSK3β signaling pathway in neural precursor cells of Cdkl5 KO mice, leading to reduced phosphorylation at Ser9 of GSK3β (Fuchs et al., 2014), a status that in- creases GSK3β activity (Cross et al., 1995). Normalization of GSK3β phos- phorylation in cultures of neural precursors by treatment with lithium restored neuron survival and maturation (Fuchs et al., 2014). Neurobiology of Disease 82 (2015) 298–310 ⁎ Corresponding author at: Department of Biomedical and Neuromotor Sciences, Piazza di Porta San Donato 2, 40126 Bologna, Italy. E-mail address: eelisabetta.ciani@unibo.it (E. Ciani). 1 Authors labeled with an asterisk contributed equally to the work. Available online on ScienceDirect (www.sciencedirect.com). http://dx.doi.org/10.1016/j.nbd.2015.06.018 0969-9961/© 2015 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi