The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons

Giulia M Serratto,Silvia Bassani,Maria Passafaro,Sara Mazzoleni,Erika Pizzi,Silvia Pelucchi,Luca Murru,Michele Mazzanti,Elena Marcello

doi:10.1007/s12035-020-02099-7

Giulia M Serratto, Silvia Bassani + Show 7 more

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https://doi.org/10.1007/s12035-020-02099-7

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Abstract

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

Highlights

IntroductionMutations in the human gene PCDH19 result in an epileptic syndrome known as EIEE9 (epileptic encephalopathy, early infantile, 9; OMIM # 300088), characterized by early-onset seizures, cognitive impairment, and autistic features, in addition to a variety of behavioral problems and sleep dysregulation [1,2,3]
Mutations in the human gene PCDH19 result in an epileptic syndrome known as EIEE9, characterized by early-onset seizures, cognitive impairment, and autistic features, in addition to a variety of behavioral problems and sleep dysregulation [1,2,3]
As the first step of our study, we assessed whether PCDH19 downregulation might affect the GABAA receptors (GABAARs)-mediated tonic current in primary hippocampal neurons

Summary

Introduction

Mutations in the human gene PCDH19 result in an epileptic syndrome known as EIEE9 (epileptic encephalopathy, early infantile, 9; OMIM # 300088), characterized by early-onset seizures, cognitive impairment, and autistic features, in addition to a variety of behavioral problems and sleep dysregulation [1,2,3]. PCDH19 is supposed to mediate cell-cell recognition within neuronal circuits and their correct assembly, in accordance with PCDH19 adhesive properties [9] and recent findings that supported PCDH19 involvement in neuronal migration and sorting [10,11,12]. Despite these advances, most aspects of PCDH19 biological functions remain elusive, and the link between PCDH19 neuronal role and EIEE9 pathological phenotype. There are evidences supporting the contribution to tonic transmission of both delta and gamma subunits, and of all of the five different alpha subunits expressed in the hippocampus (alpha 1 to 5) [20,21,22]

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