American Journal of Medical Genetics, Apr 12, 2023
The sodium leak channel (NALCN) gene encodes a sodium leak channel that plays an important role i... more The sodium leak channel (NALCN) gene encodes a sodium leak channel that plays an important role in the regulation of the resting membrane potential and the control of neuronal excitability. Mutations in the NALCN gene have been reported in patients with infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) and congenital contractures of the limbs and face with hypotonia and developmental delay (CLIFAHDD syndrome). We describe the case of a father with drug‐resistant left temporo‐orbitofrontal epilepsy and his son with mildly‐symptomatic temporal epilepsy (only recurrent déjà vu auras) whose genetic panels identified a likely pathogenic deletion of exon 27 on the NALCN gene. Our study helps broaden the clinical spectrum of diseases associated with mutations in the NALCN gene.
Canadian Journal of Neurological Sciences, Apr 28, 2022
Background: There is limited data on the utility, yield, and cost efficiency of genetic testing i... more Background: There is limited data on the utility, yield, and cost efficiency of genetic testing in adults with epilepsy. We aimed to describe the yield and utility of genetic panels in our adult epilepsy clinic. Methods: We performed a retrospective, cross-sectional study of all patients followed by an epileptologist at a Canadian tertiary care centre's epilepsy clinic between January 2016 and August 2021 for whom a genetic panel was ordered. A panel was generally ordered when the etiology was unknown or in the presence of a malformation of cortical development. We determined the yield of panel positivity and of confirmed genetic diagnoses. We also estimated the proportion of these diagnoses that were clinically actionable. Results: In total, 164 panels were ordered in 164 patients. Most had refractory epilepsy (80%), and few had comorbid intellectual disability (10%) or a positive family history of epilepsy (11%). The yield of panel positivity was 11%. Panel results were uncertain 49% of the time and negative 40% of the time. Genetic diagnoses were confirmed in 7 (4.3%) patients. These genetic conditions involved the following genes: SCARB2, DEPDC5, PCDH19, LGI1, SCN1A, MT-TL1, and CHRNA7. Of the seven genetic diagnoses, 5 (71%) were evaluated to be clinically actionable. Conclusion: We report a lower diagnostic yield for genetic panels in adults with epilepsy than what has so far been reported. Although the field of the genetics of epilepsy is a fast-moving one and more data is required, our findings suggest that guidelines for genetic testing in adults are warranted. RÉSUMÉ : Utilisation de panels génétiques au sein d'une clinique d'épilepsie pour adulte. Contexte : Il existe à l'heure actuelle peu de données portant sur l'utilité, la performance et le rapport coût-efficacité des tests génétiques chez les adultes atteints d'épilepsie. Nous avons ainsi voulu décrire la performance et l'utilité des panels génétiques au sein de notre clinique d'épilepsie pour adultes. Méthodes : Nous avons réalisé une étude rétrospective et transversale de tous les patients suivis par un épileptologue entre janvier 2016 et août 2021 au sein de notre clinique située dans un centre de soins tertiaires, patients pour lesquels un panel génétique avait été demandé. Précisons qu'un tel panel était généralement demandé lorsque l'étiologie de la maladie était inconnue ou lorsqu'on était en présence d'une malformation du développement cortical. Nous avons ensuite déterminé la performance des panels en matière de positivité et de confirmation des diagnostics génétiques. De plus, nous avons estimé la proportion de ces diagnostics qui étaient exploitables sur le plan clinique. Résultats : En tout, 164 panels ont été demandés chez 164 patients. La plupart d'entre eux étaient atteints d'épilepsie réfractaire (80 %) alors qu'ils sont peu nombreux ceux qui présentaient une déficience intellectuelle concomitante (10 %) ou des antécédents familiaux d'épilepsie (11 %). La performance des panels en matière de positivité a atteint 11 %. Les résultats des panels se sont révélés incertains dans 49 % des cas et négatifs dans 40 % des cas. Des diagnostics de nature génétique ont par ailleurs été confirmés chez 7 patients (4,3 %). Ces conditions génétiques impliquaient les gènes suivants : SCARB2, DEPDC5, PCDH19, LGI1, SCN1A, MT-TL1 et CHRNA7. Sur les sept diagnostics génétiques, cinq (71 %) ont été évalués comme pouvant donner lieu à une action clinique. Conclusion : Il nous est donc possible de signaler une performance diagnostique des panels génétiques chez les adultes épileptiques inférieure à ce qui a été rapporté jusqu'à présent. Bien que le domaine de la génétique des épilepsies soit en pleine évolution et que davantage de données soient nécessaires, nos résultats suggèrent que des lignes directrices pour les tests génétiques chez les adultes sont justifiées.
<p><b>A,</b> Touch-evoked escape response of hitch mutants versus their sibling... more <p><b>A,</b> Touch-evoked escape response of hitch mutants versus their siblings at 7 dpf show a dysfunction of motor coordination of hic-/- larvae. Indeed, hic-/- larvae are not able to bend their tail in a coordinated manner in order to initiate an escape swimming response (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0216159#pone.0216159.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0216159#pone.0216159.s002" target="_blank">S2</a> Movies for full recordings). <b>B,</b> Hic-/- larvae at 7 dpf show a reduction of the trunk size as well as defects of the notochord and a change in birefringence of trunk muscles. <b>C,</b> Measurement of the trunk size at different age shows that the reduction in trunk length starts at 3 dpf and aggravates at 7 dpf presumably due to the motor dysfunction.</p
Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques
ABSTRACT:Background:There is limited data on the utility, yield, and cost efficiency of genetic t... more ABSTRACT:Background:There is limited data on the utility, yield, and cost efficiency of genetic testing in adults with epilepsy. We aimed to describe the yield and utility of genetic panels in our adult epilepsy clinic.Methods:We performed a retrospective, cross-sectional study of all patients followed by an epileptologist at a Canadian tertiary care centre’s epilepsy clinic between January 2016 and August 2021 for whom a genetic panel was ordered. A panel was generally ordered when the etiology was unknown or in the presence of a malformation of cortical development. We determined the yield of panel positivity and of confirmed genetic diagnoses. We also estimated the proportion of these diagnoses that were clinically actionable.Results:In total, 164 panels were ordered in 164 patients. Most had refractory epilepsy (80%), and few had comorbid intellectual disability (10%) or a positive family history of epilepsy (11%). The yield of panel positivity was 11%. Panel results were uncert...
Seizures are the outward manifestation of abnormally excessive or synchronous brain activity. Whi... more Seizures are the outward manifestation of abnormally excessive or synchronous brain activity. While seizures can be somewhat symptomatically managed with anti-epileptic drugs (AEDs), many patients are still refractory to the currently available AEDs. As a result, there is a need to identify new molecules with anti-seizure properties. Curcumin is the principle curcuminoid of Curcuma longa, or colloquially turmeric, and has been experimentally proven to have anti-convulsive properties, but its poor bioavailability has dampened further therapeutic interest. Hence, this study aimed to ask if structural analogues of curcumin with an adequate bioavailability could have an anti-seizure effect in vivo. To do so, we tested these analogues following a multipronged approach combining the use of several zebrafish seizure models (chemically-induced and genetic) and complementary assays (behavioural and brain activity). Overall, from the 68 analogues tested, we found 15 different derivatives that were able to significantly decrease the behavioural hyperactivity induced by pentylenetetrazol. Of those, only a few showed an effect on the hyperactivity phenotype of two genetic models of brain seizures that are the gabra1 and gabrg2 knockouts. Two analogues, CA 80(1) and CA 74(1), were able to significantly alleviate brain seizures of gabrg2-mutant larvae. As a result, these analogues are good candidates as novel anti-seizure agents.
The genetic diagnosis of patients with seizure disorders has been improved significantly by the d... more The genetic diagnosis of patients with seizure disorders has been improved significantly by the development of affordable next-generation sequencing technologies. Indeed, in the last 20 years, dozens of causative genes and thousands of associated variants have been described and, for many patients, are now considered responsible for their disease. However, the functional consequences of these mutations are often not studied in vivo, despite such studies being central to understanding pathogenic mechanisms and identifying novel therapeutic avenues. One main roadblock to functionally characterizing pathogenic mutations is generating and characterizing in vivo mammalian models carrying clinically relevant variants in specific genes identified in patients. Although the emergence of new mutagenesis techniques facilitates the production of rodent mutants, the fact that early development occurs internally hampers the investigation of gene function during neurodevelopment. In this context, functional genomics studies using simple animal models such as flies or fish are advantageous since they open a dynamic window of investigation throughout embryonic development. In this review, we will summarize how the use of simple animal models can fill the gap between genetic diagnosis and functional and phenotypic correlates of gene function in vivo. In particular, we will discuss how these simple animals offer the possibility to study gene function at multiple scales, from molecular function (i.e., ion channel activity), to cellular circuit and brain network dynamics. As a result, simple model systems offer alternative avenues of investigation to model aspects of the disease phenotype not currently possible in rodents, which can help to unravel the pathogenic substratum in vivo.
Epilepsy is the most common primary neurological disorder characterized by the chronic tendency o... more Epilepsy is the most common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and whilst about two thirds of epilepsies were classically considered idiopathic, a majority of those is now believed to be of genetic origin. Mutations in genes involved in GABA-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene (GABRG2), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss-of-function depict clinical features of the disease, however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we gener...
25 Glycine encephalopathy (GE) or Non-Ketotic Hyperglycinemia (NKH), is a rare recessive genetic ... more 25 Glycine encephalopathy (GE) or Non-Ketotic Hyperglycinemia (NKH), is a rare recessive genetic 26 disease caused by defective glycine cleavage and characterized by an increased accumulation of 27 glycine in all tissues. Here, based on new case-reports of GLDC loss-of-function mutations in GE 28 patients, we aimed at generating a zebrafish model of severe GE in order to unravel the 29 molecular substratum of the disease. Using CRISPR/CAS9, we knocked-out gldc gene and 30 showed that -/fish recapitulate GE on a molecular level and depict a motor phenotype 31 reminiscent of severe GE symptoms. The molecular characterization of gldc -/mutants showed 32 a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, 33 with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell 34 proliferation in the brain of gldc -/-, the main brain networks are not affected thus suggesting 35 that GE pathogenicity is mainly due to me...
L’acide rétinoïque (AR) est le dérivé actif majeur de la vitamine A et a de multiples rôles au ni... more L’acide rétinoïque (AR) est le dérivé actif majeur de la vitamine A et a de multiples rôles au niveau cellulaire ainsi que pendant le développement. L’AR agit via deux familles de récepteurs nucléaires : les Récepteurs de l’Acide Rétinoïque (RAR) et les Récepteurs X des Rétinoïdes (RXR). Ces récepteurs sont des facteurs de transcription dépendants du ligand et leur activité est régulée par des phosphorylations via des kinases activées par l’AR. Durant ma thèse, je me suis intéressé à l’étude fonctionnelle et évolutive de la voie de l’AR et de la phosphorylation des RAR chez le poisson-zèbre Danio rerio. En étudiant l’activité des différents sous-types de RAR chez le poisson-zèbre, nous avons mis en avant qu’il existe une activité transcriptionnelle propre à chaque sous-type dans un embryon précoce de poisson-zèbre. De plus, mes travaux ont montré qu’au cours de l’évolution, l’acquisition d’un site de phosphorylation chez RARα permet une régulation fine de son activité chez les mammi...
Superimposition of tooth germs, marked by GFP and tooth being calcified, stained with alizarin re... more Superimposition of tooth germs, marked by GFP and tooth being calcified, stained with alizarin red in control (A) (120 hpf) and in RA exposed embryos (24-36 hpf) (B). Only is B can we detect ectopic anterior teeth.
(A) Strong expression of pitx2a in 4V1 at 80 hpf. (B) At 96 hpf pitx2a expression in faintly dete... more (A) Strong expression of pitx2a in 4V1 at 80 hpf. (B) At 96 hpf pitx2a expression in faintly detected in 4V1 mesenchyme. (C) At 120 hpf pitx2a is strongly expressed in 4V2, while its expression is very faint in this tooth at 132 hpf (D). Note that at no point during zebrafish embryogenesis did we detect pitx2a expression in 3 or 5 V1. (E) Table recapitulative of the selected genes expressed during normal tooth development in 4V1, 3/5V1 and 4V2 and which of these genes are found to be expressed in the newly formed 4V1-like tooth. (A,C) insert: high magnification image of the tooth germ
(A,B) Strong expression of hoxb5a in the ventral posterior and mid-pharyngeal region at 52 hpf (A... more (A,B) Strong expression of hoxb5a in the ventral posterior and mid-pharyngeal region at 52 hpf (Arrow). Exposure to RA from 24-36 hpf does not modify the expression of hoxb5a at 52 hpf (Arrow). A,C lateral views; B,D ventral views.
(A, B) In situ hybridization for dlx3b at 96 hpf reveals four spots in controls corresponding to ... more (A, B) In situ hybridization for dlx3b at 96 hpf reveals four spots in controls corresponding to the induction of 3/5V1 teeth (arrows) whereas only one pair of spots is present after blockage of FGF signalling by SU5402 from 40 to 80 hpf and after washing it out afterwards. This unique pair of spots corresponds to a delayed pair of initiator teeth (4V1-like) that is forming on the 5th pharyngeal arch (D) whereas three pairs of teeth (4/3/5V1) have already developed in the control condition at 132 hpf (C).
The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locatio... more The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locations in the oral and pharyngeal regions. The first-formed tooth has been hypothesized to serve as an initiator controlling the formation of the subsequent teeth. In zebrafish, the existence of the first tooth (named 4 V<sup>1</sup>) is puzzling as its replacement is induced before the opening of the mouth. Functionally, it has been shown that 4 V<sup>1</sup> formation requires fibroblast growth factor (FGF) and retinoic acid (RA) signalling. Here, we show that the ablation of 4 V<sup>1</sup> prevents the development of the dental row demonstrating its dependency over it. If endogenous levels of FGF and RA are restored after 4 V<sup>1</sup> ablation, embryonic dentition starts again by <i>de novo</i> formation of a first tooth, followed by the dental row. Similarly, induction of anterior ectopic teeth induces subsequent tooth formation, demonstrating that the initiator tooth is necessary and sufficient for dental row formation, likely via FGF ligands released by 4 V<sup>1</sup> to induce the formation of subsequent teeth. Our results show that by modifying the formation of the initiator tooth it is possible to control the formation of a dental row. This could help to explain the diversity of tooth patterns observed in actinopterygians and more broadly, how diverse traits emerged evolved through molecular fine-tuning.
Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which... more Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA ...
Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmissi... more Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/-(hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.
American Journal of Medical Genetics, Apr 12, 2023
The sodium leak channel (NALCN) gene encodes a sodium leak channel that plays an important role i... more The sodium leak channel (NALCN) gene encodes a sodium leak channel that plays an important role in the regulation of the resting membrane potential and the control of neuronal excitability. Mutations in the NALCN gene have been reported in patients with infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) and congenital contractures of the limbs and face with hypotonia and developmental delay (CLIFAHDD syndrome). We describe the case of a father with drug‐resistant left temporo‐orbitofrontal epilepsy and his son with mildly‐symptomatic temporal epilepsy (only recurrent déjà vu auras) whose genetic panels identified a likely pathogenic deletion of exon 27 on the NALCN gene. Our study helps broaden the clinical spectrum of diseases associated with mutations in the NALCN gene.
Canadian Journal of Neurological Sciences, Apr 28, 2022
Background: There is limited data on the utility, yield, and cost efficiency of genetic testing i... more Background: There is limited data on the utility, yield, and cost efficiency of genetic testing in adults with epilepsy. We aimed to describe the yield and utility of genetic panels in our adult epilepsy clinic. Methods: We performed a retrospective, cross-sectional study of all patients followed by an epileptologist at a Canadian tertiary care centre's epilepsy clinic between January 2016 and August 2021 for whom a genetic panel was ordered. A panel was generally ordered when the etiology was unknown or in the presence of a malformation of cortical development. We determined the yield of panel positivity and of confirmed genetic diagnoses. We also estimated the proportion of these diagnoses that were clinically actionable. Results: In total, 164 panels were ordered in 164 patients. Most had refractory epilepsy (80%), and few had comorbid intellectual disability (10%) or a positive family history of epilepsy (11%). The yield of panel positivity was 11%. Panel results were uncertain 49% of the time and negative 40% of the time. Genetic diagnoses were confirmed in 7 (4.3%) patients. These genetic conditions involved the following genes: SCARB2, DEPDC5, PCDH19, LGI1, SCN1A, MT-TL1, and CHRNA7. Of the seven genetic diagnoses, 5 (71%) were evaluated to be clinically actionable. Conclusion: We report a lower diagnostic yield for genetic panels in adults with epilepsy than what has so far been reported. Although the field of the genetics of epilepsy is a fast-moving one and more data is required, our findings suggest that guidelines for genetic testing in adults are warranted. RÉSUMÉ : Utilisation de panels génétiques au sein d'une clinique d'épilepsie pour adulte. Contexte : Il existe à l'heure actuelle peu de données portant sur l'utilité, la performance et le rapport coût-efficacité des tests génétiques chez les adultes atteints d'épilepsie. Nous avons ainsi voulu décrire la performance et l'utilité des panels génétiques au sein de notre clinique d'épilepsie pour adultes. Méthodes : Nous avons réalisé une étude rétrospective et transversale de tous les patients suivis par un épileptologue entre janvier 2016 et août 2021 au sein de notre clinique située dans un centre de soins tertiaires, patients pour lesquels un panel génétique avait été demandé. Précisons qu'un tel panel était généralement demandé lorsque l'étiologie de la maladie était inconnue ou lorsqu'on était en présence d'une malformation du développement cortical. Nous avons ensuite déterminé la performance des panels en matière de positivité et de confirmation des diagnostics génétiques. De plus, nous avons estimé la proportion de ces diagnostics qui étaient exploitables sur le plan clinique. Résultats : En tout, 164 panels ont été demandés chez 164 patients. La plupart d'entre eux étaient atteints d'épilepsie réfractaire (80 %) alors qu'ils sont peu nombreux ceux qui présentaient une déficience intellectuelle concomitante (10 %) ou des antécédents familiaux d'épilepsie (11 %). La performance des panels en matière de positivité a atteint 11 %. Les résultats des panels se sont révélés incertains dans 49 % des cas et négatifs dans 40 % des cas. Des diagnostics de nature génétique ont par ailleurs été confirmés chez 7 patients (4,3 %). Ces conditions génétiques impliquaient les gènes suivants : SCARB2, DEPDC5, PCDH19, LGI1, SCN1A, MT-TL1 et CHRNA7. Sur les sept diagnostics génétiques, cinq (71 %) ont été évalués comme pouvant donner lieu à une action clinique. Conclusion : Il nous est donc possible de signaler une performance diagnostique des panels génétiques chez les adultes épileptiques inférieure à ce qui a été rapporté jusqu'à présent. Bien que le domaine de la génétique des épilepsies soit en pleine évolution et que davantage de données soient nécessaires, nos résultats suggèrent que des lignes directrices pour les tests génétiques chez les adultes sont justifiées.
<p><b>A,</b> Touch-evoked escape response of hitch mutants versus their sibling... more <p><b>A,</b> Touch-evoked escape response of hitch mutants versus their siblings at 7 dpf show a dysfunction of motor coordination of hic-/- larvae. Indeed, hic-/- larvae are not able to bend their tail in a coordinated manner in order to initiate an escape swimming response (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0216159#pone.0216159.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0216159#pone.0216159.s002" target="_blank">S2</a> Movies for full recordings). <b>B,</b> Hic-/- larvae at 7 dpf show a reduction of the trunk size as well as defects of the notochord and a change in birefringence of trunk muscles. <b>C,</b> Measurement of the trunk size at different age shows that the reduction in trunk length starts at 3 dpf and aggravates at 7 dpf presumably due to the motor dysfunction.</p
Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques
ABSTRACT:Background:There is limited data on the utility, yield, and cost efficiency of genetic t... more ABSTRACT:Background:There is limited data on the utility, yield, and cost efficiency of genetic testing in adults with epilepsy. We aimed to describe the yield and utility of genetic panels in our adult epilepsy clinic.Methods:We performed a retrospective, cross-sectional study of all patients followed by an epileptologist at a Canadian tertiary care centre’s epilepsy clinic between January 2016 and August 2021 for whom a genetic panel was ordered. A panel was generally ordered when the etiology was unknown or in the presence of a malformation of cortical development. We determined the yield of panel positivity and of confirmed genetic diagnoses. We also estimated the proportion of these diagnoses that were clinically actionable.Results:In total, 164 panels were ordered in 164 patients. Most had refractory epilepsy (80%), and few had comorbid intellectual disability (10%) or a positive family history of epilepsy (11%). The yield of panel positivity was 11%. Panel results were uncert...
Seizures are the outward manifestation of abnormally excessive or synchronous brain activity. Whi... more Seizures are the outward manifestation of abnormally excessive or synchronous brain activity. While seizures can be somewhat symptomatically managed with anti-epileptic drugs (AEDs), many patients are still refractory to the currently available AEDs. As a result, there is a need to identify new molecules with anti-seizure properties. Curcumin is the principle curcuminoid of Curcuma longa, or colloquially turmeric, and has been experimentally proven to have anti-convulsive properties, but its poor bioavailability has dampened further therapeutic interest. Hence, this study aimed to ask if structural analogues of curcumin with an adequate bioavailability could have an anti-seizure effect in vivo. To do so, we tested these analogues following a multipronged approach combining the use of several zebrafish seizure models (chemically-induced and genetic) and complementary assays (behavioural and brain activity). Overall, from the 68 analogues tested, we found 15 different derivatives that were able to significantly decrease the behavioural hyperactivity induced by pentylenetetrazol. Of those, only a few showed an effect on the hyperactivity phenotype of two genetic models of brain seizures that are the gabra1 and gabrg2 knockouts. Two analogues, CA 80(1) and CA 74(1), were able to significantly alleviate brain seizures of gabrg2-mutant larvae. As a result, these analogues are good candidates as novel anti-seizure agents.
The genetic diagnosis of patients with seizure disorders has been improved significantly by the d... more The genetic diagnosis of patients with seizure disorders has been improved significantly by the development of affordable next-generation sequencing technologies. Indeed, in the last 20 years, dozens of causative genes and thousands of associated variants have been described and, for many patients, are now considered responsible for their disease. However, the functional consequences of these mutations are often not studied in vivo, despite such studies being central to understanding pathogenic mechanisms and identifying novel therapeutic avenues. One main roadblock to functionally characterizing pathogenic mutations is generating and characterizing in vivo mammalian models carrying clinically relevant variants in specific genes identified in patients. Although the emergence of new mutagenesis techniques facilitates the production of rodent mutants, the fact that early development occurs internally hampers the investigation of gene function during neurodevelopment. In this context, functional genomics studies using simple animal models such as flies or fish are advantageous since they open a dynamic window of investigation throughout embryonic development. In this review, we will summarize how the use of simple animal models can fill the gap between genetic diagnosis and functional and phenotypic correlates of gene function in vivo. In particular, we will discuss how these simple animals offer the possibility to study gene function at multiple scales, from molecular function (i.e., ion channel activity), to cellular circuit and brain network dynamics. As a result, simple model systems offer alternative avenues of investigation to model aspects of the disease phenotype not currently possible in rodents, which can help to unravel the pathogenic substratum in vivo.
Epilepsy is the most common primary neurological disorder characterized by the chronic tendency o... more Epilepsy is the most common primary neurological disorder characterized by the chronic tendency of a patient to experience epileptic seizures, which are abnormal body movements or cognitive states that result from excessive, hypersynchronous brain activity. Epilepsy has been found to have numerous etiologies and whilst about two thirds of epilepsies were classically considered idiopathic, a majority of those is now believed to be of genetic origin. Mutations in genes involved in GABA-mediated inhibitory neurotransmission have been associated with a broad range of epilepsy syndromes. Mutations in the GABA-A receptor gamma 2 subunit gene (GABRG2), for example, have been associated with absence epilepsy and febrile seizures in humans. Several rodent models of GABRG2 loss-of-function depict clinical features of the disease, however, alternative genetic models more amenable for the study of ictogenesis and for high-throughput screening purposes are still needed. In this context, we gener...
25 Glycine encephalopathy (GE) or Non-Ketotic Hyperglycinemia (NKH), is a rare recessive genetic ... more 25 Glycine encephalopathy (GE) or Non-Ketotic Hyperglycinemia (NKH), is a rare recessive genetic 26 disease caused by defective glycine cleavage and characterized by an increased accumulation of 27 glycine in all tissues. Here, based on new case-reports of GLDC loss-of-function mutations in GE 28 patients, we aimed at generating a zebrafish model of severe GE in order to unravel the 29 molecular substratum of the disease. Using CRISPR/CAS9, we knocked-out gldc gene and 30 showed that -/fish recapitulate GE on a molecular level and depict a motor phenotype 31 reminiscent of severe GE symptoms. The molecular characterization of gldc -/mutants showed 32 a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, 33 with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell 34 proliferation in the brain of gldc -/-, the main brain networks are not affected thus suggesting 35 that GE pathogenicity is mainly due to me...
L’acide rétinoïque (AR) est le dérivé actif majeur de la vitamine A et a de multiples rôles au ni... more L’acide rétinoïque (AR) est le dérivé actif majeur de la vitamine A et a de multiples rôles au niveau cellulaire ainsi que pendant le développement. L’AR agit via deux familles de récepteurs nucléaires : les Récepteurs de l’Acide Rétinoïque (RAR) et les Récepteurs X des Rétinoïdes (RXR). Ces récepteurs sont des facteurs de transcription dépendants du ligand et leur activité est régulée par des phosphorylations via des kinases activées par l’AR. Durant ma thèse, je me suis intéressé à l’étude fonctionnelle et évolutive de la voie de l’AR et de la phosphorylation des RAR chez le poisson-zèbre Danio rerio. En étudiant l’activité des différents sous-types de RAR chez le poisson-zèbre, nous avons mis en avant qu’il existe une activité transcriptionnelle propre à chaque sous-type dans un embryon précoce de poisson-zèbre. De plus, mes travaux ont montré qu’au cours de l’évolution, l’acquisition d’un site de phosphorylation chez RARα permet une régulation fine de son activité chez les mammi...
Superimposition of tooth germs, marked by GFP and tooth being calcified, stained with alizarin re... more Superimposition of tooth germs, marked by GFP and tooth being calcified, stained with alizarin red in control (A) (120 hpf) and in RA exposed embryos (24-36 hpf) (B). Only is B can we detect ectopic anterior teeth.
(A) Strong expression of pitx2a in 4V1 at 80 hpf. (B) At 96 hpf pitx2a expression in faintly dete... more (A) Strong expression of pitx2a in 4V1 at 80 hpf. (B) At 96 hpf pitx2a expression in faintly detected in 4V1 mesenchyme. (C) At 120 hpf pitx2a is strongly expressed in 4V2, while its expression is very faint in this tooth at 132 hpf (D). Note that at no point during zebrafish embryogenesis did we detect pitx2a expression in 3 or 5 V1. (E) Table recapitulative of the selected genes expressed during normal tooth development in 4V1, 3/5V1 and 4V2 and which of these genes are found to be expressed in the newly formed 4V1-like tooth. (A,C) insert: high magnification image of the tooth germ
(A,B) Strong expression of hoxb5a in the ventral posterior and mid-pharyngeal region at 52 hpf (A... more (A,B) Strong expression of hoxb5a in the ventral posterior and mid-pharyngeal region at 52 hpf (Arrow). Exposure to RA from 24-36 hpf does not modify the expression of hoxb5a at 52 hpf (Arrow). A,C lateral views; B,D ventral views.
(A, B) In situ hybridization for dlx3b at 96 hpf reveals four spots in controls corresponding to ... more (A, B) In situ hybridization for dlx3b at 96 hpf reveals four spots in controls corresponding to the induction of 3/5V1 teeth (arrows) whereas only one pair of spots is present after blockage of FGF signalling by SU5402 from 40 to 80 hpf and after washing it out afterwards. This unique pair of spots corresponds to a delayed pair of initiator teeth (4V1-like) that is forming on the 5th pharyngeal arch (D) whereas three pairs of teeth (4/3/5V1) have already developed in the control condition at 132 hpf (C).
The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locatio... more The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locations in the oral and pharyngeal regions. The first-formed tooth has been hypothesized to serve as an initiator controlling the formation of the subsequent teeth. In zebrafish, the existence of the first tooth (named 4 V<sup>1</sup>) is puzzling as its replacement is induced before the opening of the mouth. Functionally, it has been shown that 4 V<sup>1</sup> formation requires fibroblast growth factor (FGF) and retinoic acid (RA) signalling. Here, we show that the ablation of 4 V<sup>1</sup> prevents the development of the dental row demonstrating its dependency over it. If endogenous levels of FGF and RA are restored after 4 V<sup>1</sup> ablation, embryonic dentition starts again by <i>de novo</i> formation of a first tooth, followed by the dental row. Similarly, induction of anterior ectopic teeth induces subsequent tooth formation, demonstrating that the initiator tooth is necessary and sufficient for dental row formation, likely via FGF ligands released by 4 V<sup>1</sup> to induce the formation of subsequent teeth. Our results show that by modifying the formation of the initiator tooth it is possible to control the formation of a dental row. This could help to explain the diversity of tooth patterns observed in actinopterygians and more broadly, how diverse traits emerged evolved through molecular fine-tuning.
Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which... more Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA ...
Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmissi... more Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/-(hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.
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Papers by Eric SAMARUT