The nocturnal frontal lobe epilepsy (NFLE) comprises a large group of partial epilepsies with heterogeneous origin. Approximately 12% of the families affected by the autosomal dominant form of NFLE (ADNFLE) carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). Attacks arise in the frontal lobe, usually during stage 2 of sleep, and are characterized by clusters of complex and stereotyped hyperkinetic seizures.This is consistent with the widespread expression of nAChRs, and particularly α4β2, in the mammalian brain.Besides directly promoting hyperexcitability in mature networks through cell depolarization and/or altered neurotransmitter release, mutant nAChRs could determine the pathogenetic process during early developmental phases, by affecting synaptic remodeling. Cholinergic signaling has been recently found to affect the development of both GABAergic and glutamatergic systems. . Aberrant cholinergic transmission can lead to an unbalance between excitatory and inhibitory transmission in prefrontal cortex (PFC), therefore facilitating the epileptic fits.We investigated the effect of β2-V287L, a mutant nAChR subunit linked to ADNFLE, in early developmental stages, during which its expression is crucial for the epileptic phenotype to manifest. By using a murine strain which conditionally expresses β2-V287L, we analyzed how the mutant nAChR modifies the balance between excitation and inhibition in the adult brain, leading to the formation of a neuronal network susceptible to seizures. We first considered how β2-V287L (a gain-of-function mutation) affects the development of GABAergic system. By patch-clamp recordings, we observed that mutant nAChRs did not interphere with the GABAergic excitatory/inhibitory transition during early developmental stages (which is known to play a role in synaptic remodelling) nor influenced GABAA receptors’ expression.We then considered the contribute of the mutation to the development of glutamatergic signaling. Our findings revealed that heteromeric nAChRs start to exert their effect on glutamatergic transmission at the end of the first postnatal week in mice. No somatic nicotinic currents have been detected at this as well as at later developmental stages in pyramidal neurons, suggesting that heteromeric nAChRs are mainly located at synaptic level where they stimulate neurotransmitter release. Analysis of transgenic mice highlighted an increase in EPSC frequency both in control condition and following nicotine exposure, compared to control littermates. Cumulative distribution of the EPSC amplitudes showed a larger increase in EPSC amplitude between P7-9 and P10-12 in transgenic mice compared to controls.In our work we also showed how loss of function mutations can lead to a NFLE-like phenotype: in particular, we considered the pathogenic effect of an α2 subunit mutation (Ile297Phe) identified in a cohort including ADNFLE and NFLE patients. A hypofunctional nAChR could hinder the ability of inhibitory interneurons to contain seizure propagation, therefore contributing to seizures.It appears clear that mutations in genes coding for heteromeric nAChRs can contribute to an epileptic phenotype at different levels, promoting excitability in adult neuronal networks (which are still susceptible to remodelling), or affecting the development of a functional cortical circuitry, or both. ADNFLE appears therefore not only to be a channelopaty but a more complex developmental disease.Our aim is to shed new light on the nAChR contribution to brain development and its role in the establishment of an epileptic phenotype, besides its direct effect on excitability in mature prefrontal networks. In this way, we should be able to identify a temporal window for early pharmacological treatment during the pathogenetic process in order to prevent the establishment of ADNFLE

Il termine epilessia notturna del lobo frontale (NFLE) descrive un ampio gruppo di epilessie parziali presentanti origine eterogenea. Gli attacchi originano a livello del lobo frontale, solitamente durante la fase 2 del sonno, e sono caratterizzati da complessi cluster ipercinetici stereotipati. Circa il 12% dei soggetti affetti dalla forma autosomica dominante di questa patologia (ADNFLE) presentano mutazioni nei geni codificanti le subunità dei recettori nicotinici neuronali (nAChRs) eteromerici. Oltre a promuovere direttamente l’eccitabilità neuronale in un network neuronale maturo, nAChRs mutanti possono contribuire alla patogenesi di questo disturbo durante lo sviluppo del SNC, influenzando il rimodellamento sinaptico. Un’alterata trasmissione colinergica potrebbe perciò condurre ad uno sbilancio tra input eccitatori ed inibitori a livello della corteccia prefrontale (PFC) facilitando così l’insorgenza di accessi epilettici. Abbiamo preso in considerazione gli effetti di una mutazione della subunità β2 associata a un fenotipo ADNFLE (β2-V287L) in fasi precoci dello sviluppo cerebrale. L’espressione della mutazione durante le prime due settimane di vita postnatale risulta necessaria per l’instaurarsi del fenotipo epilettico. Sfruttando un modello murino condizionale abbiamo analizzato come nAChRs mutanti contribuiscano a alterare il bilancio tra eccitazione ed inibizione nel cervello. Abbiamo dapprima analizzato come la mutazione β2-V287L influenzi lo sviluppo del sistema GABAergico. Registrazioni di patch-clamp evidenziano che la mutazione non influenza lo switch eccitatorio/inibitorio della trasmissione GABAergica (il quale presenta un ruolo chiave nel remodelling sinaptico), né influenza l’espressione dei recettori GABAA. Abbiamo quindi considerato il contributo della mutazione a carico del sistema glutamatergico. I risultati evidenziano che i nAChRs eteromerici iniziano ad esercitare il loro effetto verso la fine della prima settimana di vita postnatale in topo. Non sono state osservate correnti somatiche nicotiniche in questo stadio né in fasi più avanzate in neuroni piramidali: tale dato suggerisce che i nAChRs eteromerici sono siti principalmente a livello presinaptico, dove la loro attivazione promuove il rilascio di neurotrasmettitore. L’analisi dei mutanti mutazione mette in luce una più alta frequenza delle EPSCs sia in condizioni di controllo che in seguito a somministrazione di nicotina, rispetto al gruppo di controllo. L’analisi della distribuzione delle ampiezze delle EPSC mette in luce un incremento delle stesse tra P7-9 e P10-12 più pronunciato nei topi transgenici. Il nostro lavoro ha inoltre messo in luce come anche mutazioni loss-of-function possono condurre ad un fenotipo NFLE: abbiamo considerato l’effetto patogenico di una mutazione a carico della subunità α2 (Ile297Phe) identificato in una coorte di pazienti affetti da ADNFLE e NFLE. nAChRs ipofunzionali potrebbero limitare la capacità degli interneuroni inibitori di contenere la propagazione degli accessi epilettici, favorendo perciò l’insorgenza degli attacchi. Risulta quindi chiaro che mutazioni in geni codificanti per nAChRs eteromerici possono contribuire ad un fenotipo epilettico a diversi livelli, promuovendo l’eccitabilità in una rete neuronale adulta (che è ancora suscettibile al rimodellamento) o influenzando lo sviluppo di una rete neuronale funzionale, o entrambi. L’ADNFLE risulta perciò non classificabile semplicemente come una canalopatia, ma come un disturbo più complesso dello sviluppo. Il nostro obiettivo è comprendere meglio il ruolo dei nAChRs nello sviluppo del SNC ed il loro contributo nella nella epileptogenesi, oltre che il loro ruolo nella corteccia prefrontale matura. Speriamo così di identificare una finestra temporale precoce per approntare una terapia farmacologica che impedisca l’insorgenza della ADNFLE.

(2017). Mutant heteromeric nicotinic receptors in brain development and sleep-related epilepsy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

Mutant heteromeric nicotinic receptors in brain development and sleep-related epilepsy

BRUSCO, SIMONE
2017

Abstract

The nocturnal frontal lobe epilepsy (NFLE) comprises a large group of partial epilepsies with heterogeneous origin. Approximately 12% of the families affected by the autosomal dominant form of NFLE (ADNFLE) carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). Attacks arise in the frontal lobe, usually during stage 2 of sleep, and are characterized by clusters of complex and stereotyped hyperkinetic seizures.This is consistent with the widespread expression of nAChRs, and particularly α4β2, in the mammalian brain.Besides directly promoting hyperexcitability in mature networks through cell depolarization and/or altered neurotransmitter release, mutant nAChRs could determine the pathogenetic process during early developmental phases, by affecting synaptic remodeling. Cholinergic signaling has been recently found to affect the development of both GABAergic and glutamatergic systems. . Aberrant cholinergic transmission can lead to an unbalance between excitatory and inhibitory transmission in prefrontal cortex (PFC), therefore facilitating the epileptic fits.We investigated the effect of β2-V287L, a mutant nAChR subunit linked to ADNFLE, in early developmental stages, during which its expression is crucial for the epileptic phenotype to manifest. By using a murine strain which conditionally expresses β2-V287L, we analyzed how the mutant nAChR modifies the balance between excitation and inhibition in the adult brain, leading to the formation of a neuronal network susceptible to seizures. We first considered how β2-V287L (a gain-of-function mutation) affects the development of GABAergic system. By patch-clamp recordings, we observed that mutant nAChRs did not interphere with the GABAergic excitatory/inhibitory transition during early developmental stages (which is known to play a role in synaptic remodelling) nor influenced GABAA receptors’ expression.We then considered the contribute of the mutation to the development of glutamatergic signaling. Our findings revealed that heteromeric nAChRs start to exert their effect on glutamatergic transmission at the end of the first postnatal week in mice. No somatic nicotinic currents have been detected at this as well as at later developmental stages in pyramidal neurons, suggesting that heteromeric nAChRs are mainly located at synaptic level where they stimulate neurotransmitter release. Analysis of transgenic mice highlighted an increase in EPSC frequency both in control condition and following nicotine exposure, compared to control littermates. Cumulative distribution of the EPSC amplitudes showed a larger increase in EPSC amplitude between P7-9 and P10-12 in transgenic mice compared to controls.In our work we also showed how loss of function mutations can lead to a NFLE-like phenotype: in particular, we considered the pathogenic effect of an α2 subunit mutation (Ile297Phe) identified in a cohort including ADNFLE and NFLE patients. A hypofunctional nAChR could hinder the ability of inhibitory interneurons to contain seizure propagation, therefore contributing to seizures.It appears clear that mutations in genes coding for heteromeric nAChRs can contribute to an epileptic phenotype at different levels, promoting excitability in adult neuronal networks (which are still susceptible to remodelling), or affecting the development of a functional cortical circuitry, or both. ADNFLE appears therefore not only to be a channelopaty but a more complex developmental disease.Our aim is to shed new light on the nAChR contribution to brain development and its role in the establishment of an epileptic phenotype, besides its direct effect on excitability in mature prefrontal networks. In this way, we should be able to identify a temporal window for early pharmacological treatment during the pathogenetic process in order to prevent the establishment of ADNFLE
BECCHETTI, ANDREA
nAChRs; ADNFLE; Epilepsy; NFLE; GABA
nAChRs; ADNFLE; Epilepsy; NFLE; GABA
MED/04 - PATOLOGIA GENERALE
Italian
20-mar-2017
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
29
2015/2016
open
(2017). Mutant heteromeric nicotinic receptors in brain development and sleep-related epilepsy. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/153196
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