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Epileptic Encephalopathies
Epileptic encephalopathy (EE) carries the notion that “the epileptic activity itself may contribute to severe cognitive and behavioral impairment above and beyond what might be expected from the underlying pathology alone (e.g., cortical malformation), and that these can worsen over time” (Berg et al., 2010). EE are a group of heterogeneous brain disorders that occur in childhood and are characterized by pharmaco-resistance, focal and generalized seizures and severe cognitive and developmental delay often associated to premature death (Cross and Guerrini, 2013). The frequent and intense epileptic activity in young children interferes with normal brain development, induces delays in cognitive maturation and often cognitive regression. It can also have psychiatric and behavioral consequences. The different types of epileptic encephalopathies observed in children are listed in TABLE 1.
Conversely, an important part of the concept of EE treatment is that amelioration of epileptiform activity will improve the developmental consequences of the disorder (Jehi et al., 2015). It has been observed that individuals with EEs who are successfully treated with medications or surgery, display improvements in cognitive function. This demonstrates that seizures and an abnormal EEG play an important role in cognitive outcome (Asarnow et al., 1997; Lee et al., 2014; Matsuzaka et al., 2001).
However, many, if not most, of these disorders are not solely associated with developmental or behavioral deterioration due to epileptiform activity. The term EE is currently being reviewed and debated, because it does not fit to all the EEs. In particular, whether Dravet Syndrome can be classified as an EE has been questioned recently, because development delay may occur in a period were seizures are not very frequent, so this suggests that this delay might have other underlying causes. In fact, the ILAE has recently proposed the new term “development encephalopathy” for particular EE cases (Scheffer et al., 2016).
SCN1A GENE AND TYPE-I VOLTA GE GATED SODIUM CHANNEL
Excitation and electric signaling in the central nervous system involves the flow of ions (sodium, potassium, calcium and chloride) through ion channels. Voltage- gated ion channels underlie the electric properties of the neurons. They are membrane proteins with highly selective pores that can be in open or closed states according to the membrane electrical potential. Changes in membrane electrical potential induce modifications in channel conformation that allow ion tra nsition between the extracellular and intracellular fluid – this property is called ion channel gating. Because these channels allow the ion flux down their electrochemical gradient in response to voltage-gating, they were called voltage- gated ion channels. There are three main types of voltage-gated ion channels with very conserved function similarities, but high selectivity for either sodium, potassium or calcium (Hille, 2001). They are composed by polytopic, transmembrane, pore forming and voltage-sensing α or α1 subunits (Catterall, 1995). The work of Hodgkin and Huxley, 1952 was very important in the description of the action potential’s propagation. Action potentials propagate by voltage- gated activation of Na+ channels that conduct sodium ions inside neurons and allow for activation of potassium- gated channels that reestablish membrane charges by carrying K+ ions out of the cell (FIGURE 6). The authors defined three characteristics of sodium channels: voltage-dependent activation, rapid inactivation and selective ion conductance.
When the voltage depolarizing stimulus arrives to the neuron (orange arrow), the voltage -gated sodium channels open allowing sodium ions to enter the cell (depolarizing state). The potassium (K +) channels open allowing potassium ions to exit the cell wh ile Na+ channels became refractory (repolarizing state).
After a period of hyperpolarization of the cell (hyperpolarizing state), the potential is normalized and the K+ channels close. The role of NaV channels in initiating and propagating action potentials became clear by that time and further research characterized these channels at the ir molecular and functional levels. NaV channels are composed by a la rge α subunit (260kDa) and smaller β subunits (30-40kDa) (FIGURE 7) (Lai and Jan, 2006). The α subunit is encoded by ten genes termed from SCN1A to SCN11A (Sodium Voltage-Gated Channel α Subunit 1-11). These genes code for nine NaV and one sodium channel involved in salt sensing (reviewed in Mantegazza and Catterall, 2012). The genes encoding the nine NaV, called NaV1.1 to Nav1.9, and their primary localization in human and rodent tissues are described in TABLE 2.
A shared common feature: Febrile seizures
It is described that around 5-12% of the children experience febrile seizures (FS) within the range of age 6-60 months with a peak at 2 years (Vestergaard and Christensen, 2009). FS are GTC seizures that happen in childhood associated to an increase in body temperature (~38,5ºC), without CNS infection. They occur in children without history of epilepsy or neurological dysfunction and might be due to a particular sensitivity to fever in the developing brain. It has been clearly demonstrated that there is an increased risk of FS incidence shortly after childhood vaccinations caused by vaccine- induced fever (Brown et al., 2007; Scheffer, 2015). Simple FS last usually less than 15 minutes, are generalized with often loss of consciousness, shakes, and movements in limbs on both sides of the body and do not occur more than once within a 24 hours period (Whelan et al., 2017). FS, first called benign but this term is not accepted anymore by the ILAE, is considered a self- limited condition different from epilepsy. Complex FS are classified in: 1) complex if they last between 15-30 minutes, happen more than once in a period of 24 hours and are focal or localized to a specific part of the brain or 2) febrile status epilepticus (SE) if they last more than 30 minutes (Whelan et al., 2017). FS plus (FS+) were considered as a simple FS that can occur more than once within a 24 hours period or that occur beyond the age of 6 (Grill and Ng, 2013). In terms of duration, the principal ictal* event (seizure) can be confused with the post-ictal events (tonic posture, eye deviation that last for some time post-seizure). So, basically, without EEG recording it is hard to delimitate the end of the seizure. The probability of recurrence after a simple FS is around 2%, while the probability is 2-3 times higher after a complex FS (Camfield and Camfield, 2015). These long-lasting/complex FS are better candidates to cause important modifications in the brain and epileptogenesis (Ellenberg and Nelson, 1978).
The mechanism by which FS are generated is unknown. Yet four theories have been proposed for FS generation: 1) the neuronal hyperexcitability induced by the increased temperature (Fisher and Wu, 2002), possibly by acting directly on Na V gating at the AIS (Thomas et al., 2009), 2) the neuronal hyperexcitability caused by the release of inflammatory mediators during fever (eg.: Il-1β) (Alheim and Bartfai, 1998; Dubé et al., 2005), 3) the neuronal hyperexcitability caused by brain alkalosis during high ventilation in hyperthermic conditions (Aram and Lodge, 1987; Balestrino and Somjen, 1988; Schuchmann et al., 2006, 2011) or 4) a result from inefficient thermoregulation (Feng et al., 2014; Richmond, 2003). Among the many risk factors for FS, the genetic predisposition strongly confers the epidemiological link in many families (Baulac et al., 2004). Mantegazza et al., 2005 identified a mild loss-of- function mutation in NaV1.1 responsible for the familial FS condition. Very few children exhibit more than three FS during their childhood (reviewed in Camfield and Camfield, 2015), and there is little evidence for a correlation of FS in these children with long- lasting consequences like brain damage or cognitive problems (Sillanpää et al., 2011). Also, the incidence of FS does not indicate that epilepsy will develop (15% of the children have a febrile seizure event and do not develop epilepsy). FS are common to all diseases characterized by NaV1.1 channel haploinsufficiency* thus it is considered that FS is a mild phenotypic consequence of the mutations in SCN1A gene.
Epileptic syndromes associated to Na V1.1 channel mutation: genetic and phenotypic variants.
SCN1A gene mutations associated to Na V1.1 loss of function cause a vast range of epilepsy syndromes in humans. The associated phenotypes range from simple FS as described in the previous part (Mantegazza et al., 2005), en passant par generalized epilepsy with febrile seizures plus (GEFS+), a mild epileptic syndrome with high phenotypic variability (Escayg et al., 2000a), to an extremely severe condition – Dravet Syndrome (DS) (Claes et al., 2001) (FIGURE 8C). The diseases associated to the NaV1.1 spectrum are categorized according to patient’s phenotype (mainly to seizures history). The first identified mutation in the NaV1.1 α-subunit was the SCN1A-R1648H missense mutation* in one large family presenting GEFS+ (Escayg et al., 2000a). Since then, more than 30 other missense mutations have been identified that cause GEFS+, a disease usually associated to the partial loss of the channel’s function. Most displayed GEFS+ cases present autosomal-dominant transmission with high variability of phenotypes. Following the first SCN1A mutations that accounted for 10% of GEFS+ patients, a new report of SCN1A mutation was described in children presenting the sporadic severe myoclonic epilepsy in infants (SMEI) later called Dravet Syndrome (DS) (Claes et al., 2001). In SMEI children the mutations were de novo mutations since none of the parents were affected. More than 80% of DS patients present SCN1A gene mutations and more than 900 different types had been described by 2013 (Parihar and Ganesh, 2013)(see also www.gzneurosci.com/scn1adatabase/). The types of mutation in DS are: 1) severe truncating mutations* (and less often splicing* or deletion* mutations) causing loss of NaV1.1 function or 2) severe missense mutations that prevent the channel’s expression or severely impair its function (Brunklaus et al., 2014). The duplication and deletion of sequences in the gene have also been pointed to impair the channel’s function (Marini et al., 2009).
Type of mutatio ns and the ir localization in the SC N 1A gene.
FIGURE 8A&B represents some of the mutations found in GEFS+ and DS patients, respectively (but by now they are many more). In FIGURE 8B one can observe that the DS-causing missense mutations (green circles) are concentrated within the transmembrane segment where they probably prevent the channel’s folding and function. In fact, a recent study on large cohort of Japanese DS patients showed that the missense mutations found in DS were in the majority concentrated at the S4 voltage sensor and pore loops (S5 and S6) (Ishii et al., 2016). It was described that disease phenotype is worse when there is a substantial change in the physicochemical properties of the amino acids as measured by the Grantham score (A formula for the difference between amino acids that correlate better with protein residues substitution frequencies: composition, polarity, and molecular volume) (Grantham, 1974).
Table of contents :
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
LIST OF DEFINITIONS
REVISION OF CONCEPTS
I- Introduction
Chapter 1- Epilepsy and genetic epilepsies
1. Historical, definition and epidemiology
2. Classification of Seizures, Epilepsies and comorbidities
2.1 Seizures Definition and Classification
2.2 Co-morbidities in epilepsy
2.2.1 Epileptic Encephalopathies
2.3 Etiology of epilepsy
Chapter 2- Genetic epilepsies associated to SCN1A gene mutation
1. SCN1A gene and type-I voltage gated sodium channel
2. NaV 1.1 channel phenotypic/genotypic spectrum
2.1 A shared common feature: Febrile seizures
2.2 Epileptic syndromes associated to NaV1.1 channel mutation: genetic and phenotypic variants.
2.2.1 Type of mutations and their localization in the SCN1A gene.
2.2.2 Inherited transmission of SCN1A-DS mutations. The concept of mosaicism.
2.2.3 Phenotypic variability in GEFS+ and DS: genetic and environmental factors.
3. Genetic epilepsy with febrile seizures plus (GEFS+)
3.1 GEFS+ historical, definition and epidemiology
3.2 The genetics of GEFS+
3.3 Clinical characterization of GEFS+:epileptic history
3.4 Therapeutic strategy and long-term outcome
4. Dravet Syndrome (DS)
4.1 DS historical, Definition and Epidemiology
4.2 The genetics of DS
4.3 Clinical characterization of DS: Epileptic history
4.4 Neuroimaging and neuropathology
4.5 Therapeutic strategy and long term outcome
Chapter 3- Behavioral and cognitive: Long-term outcome in Dravet Syndrome :
1. Neurological Abnormalities in Dravet syndrome
2. Cognitive delay and regression in Dravet syndrome
3. Psychiatric/behavioral abnormalities in Dravet Syndrome
Chapter 4- Animal models of epilepsy and seizures consequences on cognition
1. Models of recurrent seizures at early-age on the developing brain and their consequences on behavioral and cognitive phenotypes
1.1 Hyperthermia-induced seizures: the model of febrile seizures, its epileptic and behavioral consequences
1.2 Flurothyl-induced seizures: its epileptic and behavioral consequences .
2. Animal models harboring the Scn1a mutation
2.1 Using animal models of Scn1a mutation
2.2 Impaired neuronal inhibition in Scn1a mouse models: Role of interneurons
2.3 Strain and age dependent – phenotypic severity: mortality, seizure frequency and electrophysiological properties.
2.4 Behavioral/cognitive phenotypes in mouse models of Scn1a gene mutation
2.4.1 DS mouse model
2.4.2 GEFS+ mouse model
Chapter 5- Seizures and Cognitive phenotype in DS: Epileptic encephalopathy or channelopathy?
1. Can seizure severity be correlated with cognitive/behavioral outcome in DS patients?
2. Do NaV1.1 dysfunctional mice support the channelopathy theory?
II- PROBLEMATIC AND OBJECTIVES
III- MATERIAL AND METHODS
1. Animal models , breeding and housing conditions
1.1 Scn1a+/- (NaV1.1 knock-out): Dravet Syndrome’s mouse model:
1.2 Scn1aRH/+ (NaV 1.1 knock-in): GEFS+ mouse model
1.3 Housing conditions
2. Experimental timeline
2.1 Genotyping
2.1.1 DNA extraction
2.1.2 DNA amplification
2.1.3 DNA revelation and sequencing
2.2 Repeated Seizures induction
2.2.1 Seizures induction by hyperthermia
2.2.2 Seizures induction by flurothyl
2.2.3 Behavioral characterization of seizures
2.3 Electrocorticogram recordings
2.3.1 Electrods implantation
2.3.2 Video Electrocorticogram recordings
2.3.3 Signal analysis
2.4 Electrophysiological recordings in the hippocampus
2.4.1 Field potential recordings
2.4.2 Patch-clamp recordings (performed by Doctor Pousinha)
2.5 Immunohistochemical analysis in brain slices
2.5.1 Intracardiac paraformaldeyde perfusion
2.5.2 Antibody staining
2.6 Behavioral analysis
2.6.1 Openfield test
2.6.2 Dark↔light exploration test
2.6.3 Three-chamber social interaction
2.6.4 Morris water maze
2.6.5 Contextual-Fear conditioning
2.6.6 Eight-arm radial maze
2.6.7 Actimeter
3. Therapeutic effect of drug X in decreasing the spontaneous seizures frequency in Scn1a+/- mouse model
3.1 Drug X administration
3.2 Protocol of seizure induced by hyperthermia and monitoring
4. Statistical Analysis
IV- RESULTS.
