Research Articles: Neurobiology of Disease ROCK/PKA inhibition rescues hippocampal hyperexcitability and GABAergic neuron alterations in Oligophrenin-1 Knock-out mouse model of X-linked intellectual disability https://doi.org/10.1523/JNEUROSCI.0462-19.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0462-19.2020 Received: 27 February 2019 Revised: 28 January 2020 Accepted: 3 February 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 ROCK/PKA inhibition rescues hippocampal hyperexcitability 2 and GABAergic neuron alterations 3 in Oligophrenin-1 Knock-out mouse model of X-linked intellectual disability 4 5 Irene Busti1,2,*, Manuela Allegra1,*,§, Cristina Spalletti1, Chiara Panzi1, Laura Restani1, 6 Pierre Billuart3, Matteo Caleo4,1 7 8 1 Neuroscience Institute, National Research Council (CNR), via G.Moruzzi 1, 56124 Pisa, 9 Italy 10 2 NEUROFARBA, University of Florence, via G. Pieraccini 6, 50134 Florence, Italy 11 3Institute of Psychiatry and Neuroscience of Paris, INSERM UMR1266, Paris Descartes 12 University, 102-108 rue de la Santé, 75014 Paris, France 13 4 Department of Biomedical Sciences, University of Padua, via G. Colombo 3, 35121 Padua, 14 Italy 15 *I.B. and M.A. share equal contribution as first authors 16 §M.A. present address: Institut Pasteur, 25 Rue du Dr Roux, 75015 Paris, France 17 18 Abbreviated title: Rescue of hyperexcitability and GABAergic defects in Ophn1 KO mice 19 Corresponding author: 20 Matteo Caleo 21 Department of Biomedical Sciences, University of Padua, via G. Colombo 3, 35121 Padua, 22 Italy - CNR Neuroscience Institute, via G. Moruzzi 1, 56124 Pisa, Italy 23 e-mail: [email protected] 24 Number of text pages: 30; Number of Figures: 9; Number of words (Abstract): 180; Number 25 of words (Introduction): 620; Number of words (Discussion): 1.514 26 Conflict of interest: The authors declare no competing financial interests. 27 Acknowledgements: We thank Francesca Biondi (CNR Pisa) for animal care and Elena 28 Novelli (CNR Pisa) for microscopy technical support. This work was funded by Telethon 29 Foundation (project #GGP11116 to M.C.) and by Fondazione Cassa di Risparmio di Padova 30 e Rovigo (Foundation Cariparo, project #52000 to M.C.). 1 31 Abstract 32 Oligophrenin-1 (Ophn1) encodes a Rho GTPase activating protein whose mutations cause 33 X-linked intellectual disability (XLID) in humans. Loss of function of Ophn1 leads to 34 impairments in the maturation and function of excitatory and inhibitory synapses, causing 35 deficits in synaptic structure, function and plasticity. Epilepsy is a frequent co-morbidity in 36 patients with Ophn1-dependent XLID, but the cellular bases of hyperexcitability are poorly 37 understood. Here we report that male mice knock-out (KO) for Ophn1 display hippocampal 38 epileptiform alterations, which are associated with changes in parvalbumin-, somatostatin- 39 and neuropeptide Y-positive interneurons. Since loss of function of Ophn1 is related to 40 enhanced activity of Rho-associated protein kinase (ROCK) and protein kinase A (PKA), we 41 attempted to rescue Ophn1-dependent pathological phenotypes by treatment with the 42 ROCK/PKA inhibitor Fasudil. While acute administration of Fasudil had no impact on seizure 43 activity, seven weeks of treatment in adulthood were able to correct electrographic, 44 neuroanatomical and synaptic alterations of Ophn1 deficient mice. These data demonstrate 45 that hyperexcitability and the associated changes in GABAergic markers can be rescued at 46 the adult stage in Ophn1-dependent XLID through ROCK/PKA inhibition. 47 48 Key words: hippocampus, interneurons, epilepsy, Fasudil, synapses 49 50 Significance Statement: In this study we demonstrate enhanced seizure propensity and 51 impairments in hippocampal GABAergic circuitry in Ophn1 mouse model of XLID. 52 Importantly, the enhanced susceptibility to seizures, accompanied by an alteration of 53 GABAergic markers were rescued by ROCK/PKA inhibitor Fasudil, a drug already tested on 54 humans. Since seizures can significantly impact the quality of life of XLID patients, the 55 present data suggest a potential therapeutic pathway to correct alterations in GABAergic 56 networks and dampen pathological hyperexcitability in adults with XLID. 57 58 59 Introduction 60 Oligophrenin-1 (Ophn1) is a gene whose mutations cause X-linked intellectual disability 61 (XLID) in humans. Ophn1 encodes for a Rho GTPase activating protein (RhoGAP) which 62 negatively regulates Rac, RhoA and Cdc42 (Billuart et al., 1998; Fauchereau et al., 2003; 63 Khelfaoui et al., 2007). Ophn1 is expressed in several brain regions, including the cerebral 64 cortex and the hippocampus, where it contributes to synapse maturation and plasticity 65 (Govek et al., 2004; Khelfaoui et al., 2007; Powell et al., 2012; Powell et al., 2014). Ophn1 66 knock-out (KO) mice represent an excellent model of Ophn1 mutations in humans (Khelfaoui 67 et al., 2007). These mice exhibit impairments in spatial memory and social behavior, 2 68 alterations in adult neurogenesis, and defects in dendritic spines associated with altered 69 synaptic plasticity (Khelfaoui et al., 2013; Meziane et al., 2016; Redolfi et al., 2016; Allegra et 70 al., 2017; Zhang et al., 2017). 71 At the electrophysiological level, the loss of function of Ophn1 leads to alterations of both 72 excitatory and inhibitory synaptic transmission. Patch-clamp recordings from the 73 hippocampus of Ophn1 KO mice have shown reductions in evoked and spontaneous 74 excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs; Powell et al., 2012, 75 2014). By contrast, the Ophn1 deficiency leads to an increased spontaneous activity in the 76 medial prefrontal cortical (mPFC) neurons, where Zhang and coworkers (2017) found a 77 higher frequency of excitatory postsynaptic potentials (EPSPs). Synaptic deficits were 78 rapidly rescued by inhibition of ROCK/PKA, which are over-activated after loss of Ophn1 79 (Meziane et al., 2016; Compagnucci et al., 2016; Zhang et al., 2017). Gamma oscillations 80 were also found to be reduced in Ophn1 deficient hippocampal slices, pointing to deficits in 81 synaptic inhibition (Powell et al., 2014). However, a detailed analysis of inhibitory circuits in 82 Ophn1-dependent XLID is still lacking. A growing body of evidence has highlighted the 83 crucial role of GABAergic interneurons in the pathophysiology of XLID (Papale et al., 2017; 84 Zapata et al., 2017). In this context, distinct subsets of GABAergic interneurons such as 85 parvalbumin (PV)-positive basket cells, somatostatin (SOM)-positive cells and neuropeptide 86 Y-positive interneurons play distinct roles in fine-tuning and synchronization of the 87 hippocampal network (Pelkey et al., 2017). 88 89 Epilepsy is a frequent co-morbidity of Ophn1-dependent XLID and may significantly impact 90 quality of life in the patients (Bergmann et al., 2003). The electrographic analysis has 91 demonstrated seizure episodes as well as interictal epileptic activity in Ophn1-mutated 92 subjects (Bergmann et al., 2003; des Portes et al., 2004; Santos-Reboucas et al., 2014). 93 However, the mechanisms by which mutations in Ophn1 affect the balance between 94 excitation and inhibition, with consequent cognitive impairment and network hyperexcitability 95 remain still incompletely understood. 96 In subjects with refractory epilepsy and intellectual disability, the treatment of epileptic 97 symptoms is normally carried out by conventional anti-epileptic drugs (AEDs). However, 98 these treatments can lead to adverse events and, importantly, a proportion of the patients 99 remains pharmacoresistant (Jackson et al., 2015). This prompts the need for alternative 100 treatments to reduce seizure burden and epileptic activity in subjects with XLID. 101 Lack of Ophn1 leads to high level of ROCK/PKA activity, and several pathological deficits of 102 Ophn1 KO mice are at least partially rescued by treatment with ROCK/PKA inhibitor Fasudil 103 (Khelfaoui et al, 2013; Meziane et al., 2016; Redolfi et al., 2016; Allegra et al., 2017), an 3 104 isoquinoline derivative drug approved for use in humans in China and Japan, and currently 105 tested in multiple clinical trials in the United States and Europe. 106 107 In this manuscript, we have used recordings of local field potentials (LFPs) to describe the 108 electrographic alterations of Ophn1 KO mice and their association with alterations in 109 GABAergic hippocampal networks. We have also explored the possibility of rescuing the 110 pathological hyperexcitability and GABAergic network defects induced by loss of function of 111 Ophn1 via treatment with Fasudil in adulthood. 112 113 114 Materials and Methods 115 116 Animals and treatment 117 All experiments were performed in compliance with ARRIVE guidelines and the EU Council 118 Directive 2010/63/EU on the protection of animals used for scientific purposes and were 119 approved by the Italian Ministry of Health. All experiments and analyses were performed 120 blind to the genotype and treatment. The animals used for all experiments were of the 121 C57BL/6-J strain. Mice were housed in cages in a controlled environment (21°C and 60% of 122 humidity) with 12 hour/12 hour light/dark cycle, with food and water available ad libitum. All 123 experiments were performed using Ophn1෥\ knock-out (KO) mice and Ophn1+/y wild-type 124 (WT) littermates of two months of age, generated by breeding heterozygote females 125 (Ophn1෥) with WT males (Ophn1+/y). Since the Ophn1 gene is located on X chromosome, 126 only male mice were used for our experiments because they develop X-linked ID, while 127 females are not-affected carriers. The genotype was revealed through polymerase chain 128 reaction (PCR) analysis on small samples of tail tissue taken from pups at post-natale stage 129 P10, as described by Khelfaoui et al.