Down-regulation of RalBP1 expression reduces seizure threshold and synaptic inhibition in mice

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Abstract

Idiopathic epilepsy is characterized by seizures without a clear etiology and is believed to have a strong genetic component but exhibits a complex inheritance pattern. Genetic factors seem to confer a low seizure threshold to susceptible individuals and thereby enhance epileptogenesis. However, the identity of susceptibility genes and the mechanisms regulating seizure threshold are still poorly understood. Here, we describe that reduced expression of RalBP1, a downstream effector of the small GTPases RalA and RalB, lowers the seizure threshold in mice. The intraperitoneal injection of the chemoconvulsant pentylenetetrazol induced more severe seizures in RalBP1 hypomorphic mice than in their wild-type littermates. The reduction of RalBP1 in the brain has no effect on neuronal excitability, but does decrease the inhibitory synaptic transmission onto CA1 pyramidal neurons. This impaired synaptic inhibition was associated with the loss of GABAergic interneurons in the CA1 subfield of the hippocampus. The present study identifies RalBP1 as a gene regulating the seizure threshold in mice and provides direct evidence for the role of RalBP1 in synaptic inhibition in vivo.

Highlights

► RalBP1 hypomorphic mice display reduced seizure threshold to convulsant stimulation. ► Reduced RalBP1 expression decreases synaptic inhibition onto CA1 pyramidal neurons. ► Impaired synaptic inhibition was associated with the loss of GABAergic interneurons.

Introduction

Epilepsy is a common, highly heritable neurological disorder that is characterized by recurrent and unprovoked seizures [1]. Genetic studies have identified some of the genes responsible for monogenic epilepsy; these genes predominantly encode ion channels or their accessory subunits [2]. These genes have provided opportunities for understanding the mechanisms that cause neuronal hyperexcitability or an imbalance in excitatory and inhibitory synaptic transmission, which are major causes of monogenic epilepsy. However, the genetic etiology of the majority of epilepsy is still unknown because most common epilepsies do not follow a Mendelian pattern of inheritance. Monogenic inheritance accounts for only a few unusual types of epilepsy that exhibit a prevalence of ∼1%. In contrast, common epilepsies – idiopathic epilepsies – occur without a clear cause and exhibit complex inheritance patterns [3], [4]. In addition, it is well known that environmental factors also influence the onset of common epilepsies [5]. According to this multifactorial threshold model of inheritance, the epileptic phenotype is expressed beyond a critical threshold. The combined action of risk genes and environmental factors determines whether an individual surpasses the critical threshold and develops epileptic disease [6]. In line with this concept, the seizure threshold is partly determined by genetic background. Defects in susceptible genes seem to lower the seizure threshold [7], but the identity of these susceptibility genes and the underlying mechanisms that link genetic background and altered seizure threshold are still poorly understood. These susceptibility genes are of particular interest because they may provide new insights into the mechanism underlying idiopathic epilepsies.

Here, we report that reduced expression of RalBP1 (RLIP76) lowers the seizure threshold in mice. RalBP1 is a downstream effector of the small GTPases RalA and RalB that mediates signaling from Ral GTPases to Rho pathways [8]. RalBP1 contains a Ral binding domain and a RhoGAP domain. The former domain mediates binding to Ral GTPases and the latter domain confers GAP activity to Rac1 and Cdc42. In addition, the N-terminal and C-terminal regions of RalBP1 bind to the AP2 adapter complex and POB1, respectively [9], [10]. RalBP1 has been implicated in various cellular processes, including receptor-mediated endocytosis, membrane transport, and mitochondrial fission [11], [12], [13]. In neurons, RalBP1 is known to mediate long-term synaptic depression through interactions with RalA and PSD-95 [14]. However, link between RalBP1 and seizure threshold has not been studied.

In the present study, we identified RalBP1 as a regulator of seizure threshold and investigated the neural mechanisms associated with altered seizure threshold in mice hypomorphic for RalBP1.

Section snippets

Animals

Heterozygous RalBP1 (RalBP1+/−) mice were generated as previously described [14] and backcrossed with C57BL/6J mice for at least 10 generations. All of the experiments were performed with littermates of both genotypes that were derived from heterozygote crosses. All of the animal maintenance and related experiments were performed according to Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of animals in research and were approved by the IACUC of Seoul

RalBP1−/− mice show a reduced seizure threshold to convulsant stimulation

To test the association between RalBP1 and epilepsy, we examined spontaneous seizure activity inRalBP1 hypomorphic (RalBP1−/−) mice. The level of RalBP1 protein in the RalBP1−/− brains was reduced to ∼18% of the level in wild-type (WT) brains by the insertion of a gene trapping cassette in the RalBP1 gene [14]. This reduced expression of RalBP1 causes modest growth retardation and a reduced body size. As shown in Fig. 1A, the male RalBP1−/− mice weighed ∼20% less than WT littermates at 8 weeks

Discussion

RalBP1 is expressed in the mammalian brain, but the behavioral and neurophysiological roles of RalBP1 in vivo are largely unknown. In the present study, we demonstrated that mice hypomorphic for RalBP1 (RalBP1−/− mice) are vulnerable to chemoconvulsant stimulation. The intraperitoneal injection of PTZ induced more severe seizures in RalBP1−/− mice compared to WT mice. We also found that reduced RalBP1 expression decreases the inhibitory synaptic transmission onto CA1 pyramidal neurons and that

Acknowledgments

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare (A120476), by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0014661), and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MESF) [2011-0030737] (to M-H.K).

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