10 research outputs found
CaractĂ©risation et intĂ©rĂȘt thĂ©rapeutique de potentialisateurs du co-transporteur potassium-chlorure de type 2 (KCC2) dans les Ă©pilepsies du lobe temporal
In neurons, cation-chloride cotransporters (CCCs), such as the potassium-chloride cotransporter type 2 (KCC2) and the sodium-potassium-chloride cotransporter type 1 (NKCC1), control intraneuronal chloride ion homeostasis and, subsequently, the efficiency and polarity of gamma-aminobutyric acid (GABA) synaptic signaling. In many neurological disorders, including epilepsy, reduced KCC2 expression or function may result in depolarizing and paradoxically excitatory GABA signaling that may then contribute to pathological activities and seizures. Compensating for the dysregulation of CCC function in the pathology therefore appears as a promising therapeutic strategy. Bumetanide, an antagonist of the NKCC1 transporter, failed to prevent acute neonatal seizures in clinical trials and instead induced side effects such as ototoxicity. Instead, two compounds - prochlorperazine (PCPZ) and CLP257 â recently identified from library screening as candidate KCC2 enhancers, appear to improve symptoms in several animal models of disorders associated with KCC2 suppression. However, their mode of action in cortical neurons and their therapeutic potential in epilepsy remain unknown or even controversial. During my thesis, I first compared the mode of action of these two compounds in rat hippocampal neurons and showed that both of them enhance KCC2 function. This effect was accompanied for CLP257 by a positive allosteric modulation of extrasynaptic GABAA receptors. Interestingly, both compounds reduce the lateral diffusion of KCC2 in the plasma membrane and increases its clustering, without altering its total or membrane expression or the phosphorylation level of its canonical residues. I demonstrated that both compounds effectively suppress spontaneous interictal-like discharges recorded in postoperative hippocampal tissue resected from intractable, mesial temporal lobe epilepsy (mTLE) patients. Furthermore, chronic administration of PCPZ reduced seizure onset and the frequency of epileptiform activities in a mouse model of mTLE. My results thus decipher the mode of action of two candidate KCC2 enhancers in hippocampal neurons and demonstrate for the first time their therapeutic potential in pharmacoresistant epilepsy.Dans les neurones, les co-transporteurs cation-chlorure (CCCs) tels que le co-transporteur potassium-chlorure de type 2 (KCC2) et le co-transporteur sodium-potassium-chlorure de type 1 (NKCC1), contrĂŽlent l'homĂ©ostasie intraneuronale des ions chlorure et, par consĂ©quent, l'efficacitĂ© et la polaritĂ© de la signalisation synaptique par l'acide gamma-aminobutyrique (GABA). Dans de nombreuses affections neurologiques dont l'Ă©pilepsie, une rĂ©duction de l'expression ou de la fonction de KCC2 peut conduire Ă une signalisation GABAergique dĂ©polarisante et paradoxalement excitatrice, et ainsi contribuer Ă lâĂ©mergence dâactivitĂ©s pathologiques et de crises. Compenser la dĂ©rĂ©gulation de la fonction des CCCs dans la pathologie apparaĂźt donc comme une stratĂ©gie thĂ©rapeutique prometteuse. Le bumĂ©tanide, un antagoniste du transporteur NKCC1, ne parvient pas Ă prĂ©venir les crises nĂ©onatales aiguĂ«s lors dâessais cliniques et au contraire induit des effets secondaires tels quâune ototoxicitĂ©. En revanche, deux composĂ©s â la prochlorperazine (PCPZ) et le CLP257 â rĂ©cemment identifiĂ©s par criblage de banques de petites molĂ©cules et proposĂ©s comme potentialisateurs de KCC2, semblent amĂ©liorer les symptĂŽmes dans diffĂ©rents modĂšles animaux dâaffections associĂ©es Ă une perte dâexpression de KCC2. Cependant, leur mode d'action dans les neurones corticaux et leur potentiel thĂ©rapeutique dans l'Ă©pilepsie restent inconnus, voire controversĂ©s. Au cours de ma thĂšse, jâai tout d'abord comparĂ© le mode d'action de ces composĂ©s dans des neurones hippocampiques de rat et mis en Ă©vidence que tous deux amĂ©liorent la fonction de KCC2. Cet effet s'accompagne pour le CLP257 d'une modulation allostĂ©rique positive des rĂ©cepteurs GABAA extrasynaptiques. De façon intĂ©ressante, ces deux composĂ©s rĂ©duisent la diffusion latĂ©rale de KCC2 Ă la membrane plasmique et augmentent lâagrĂ©gation du transporteur, sans modifier son expression totale ou membranaire, ni le niveau de phosphorylation de ses rĂ©sidus canoniques. Jâai mis en Ă©vidence que ces deux composĂ©s suppriment efficacement les dĂ©charges spontanĂ©es de type interictal, enregistrĂ©es in vitro dans le tissu hippocampique postopĂ©ratoire rĂ©sĂ©quĂ© de patients souffrant dâĂ©pilepsie du lobe temporal (ELT). De plus, l'administration chronique de PCPZ permet de rĂ©duire l'apparition de crises et la frĂ©quence des activitĂ©s Ă©pileptiformes dans un modĂšle souris d'ELT. Mes rĂ©sultats dĂ©cryptent donc le mode d'action de deux candidats potentialisateurs de KCC2 dans des neurones hippocampiques et dĂ©montrent pour la premiĂšre fois leur potentiel thĂ©rapeutique dans une Ă©pilepsie pharmacorĂ©sistante
CaractĂ©risation et intĂ©rĂȘt thĂ©rapeutique de potentialisateurs du co-transporteur potassium-chlorure de type 2 (KCC2) dans les Ă©pilepsies du lobe temporal
In neurons, cation-chloride cotransporters (CCCs), such as the potassium-chloride cotransporter type 2 (KCC2) and the sodium-potassium-chloride cotransporter type 1 (NKCC1), control intraneuronal chloride ion homeostasis and, subsequently, the efficiency and polarity of gamma-aminobutyric acid (GABA) synaptic signaling. In many neurological disorders, including epilepsy, reduced KCC2 expression or function may result in depolarizing and paradoxically excitatory GABA signaling that may then contribute to pathological activities and seizures. Compensating for the dysregulation of CCC function in the pathology therefore appears as a promising therapeutic strategy. Bumetanide, an antagonist of the NKCC1 transporter, failed to prevent acute neonatal seizures in clinical trials and instead induced side effects such as ototoxicity. Instead, two compounds - prochlorperazine (PCPZ) and CLP257 â recently identified from library screening as candidate KCC2 enhancers, appear to improve symptoms in several animal models of disorders associated with KCC2 suppression. However, their mode of action in cortical neurons and their therapeutic potential in epilepsy remain unknown or even controversial. During my thesis, I first compared the mode of action of these two compounds in rat hippocampal neurons and showed that both of them enhance KCC2 function. This effect was accompanied for CLP257 by a positive allosteric modulation of extrasynaptic GABAA receptors. Interestingly, both compounds reduce the lateral diffusion of KCC2 in the plasma membrane and increases its clustering, without altering its total or membrane expression or the phosphorylation level of its canonical residues. I demonstrated that both compounds effectively suppress spontaneous interictal-like discharges recorded in postoperative hippocampal tissue resected from intractable, mesial temporal lobe epilepsy (mTLE) patients. Furthermore, chronic administration of PCPZ reduced seizure onset and the frequency of epileptiform activities in a mouse model of mTLE. My results thus decipher the mode of action of two candidate KCC2 enhancers in hippocampal neurons and demonstrate for the first time their therapeutic potential in pharmacoresistant epilepsy.Dans les neurones, les co-transporteurs cation-chlorure (CCCs) tels que le co-transporteur potassium-chlorure de type 2 (KCC2) et le co-transporteur sodium-potassium-chlorure de type 1 (NKCC1), contrĂŽlent l'homĂ©ostasie intraneuronale des ions chlorure et, par consĂ©quent, l'efficacitĂ© et la polaritĂ© de la signalisation synaptique par l'acide gamma-aminobutyrique (GABA). Dans de nombreuses affections neurologiques dont l'Ă©pilepsie, une rĂ©duction de l'expression ou de la fonction de KCC2 peut conduire Ă une signalisation GABAergique dĂ©polarisante et paradoxalement excitatrice, et ainsi contribuer Ă lâĂ©mergence dâactivitĂ©s pathologiques et de crises. Compenser la dĂ©rĂ©gulation de la fonction des CCCs dans la pathologie apparaĂźt donc comme une stratĂ©gie thĂ©rapeutique prometteuse. Le bumĂ©tanide, un antagoniste du transporteur NKCC1, ne parvient pas Ă prĂ©venir les crises nĂ©onatales aiguĂ«s lors dâessais cliniques et au contraire induit des effets secondaires tels quâune ototoxicitĂ©. En revanche, deux composĂ©s â la prochlorperazine (PCPZ) et le CLP257 â rĂ©cemment identifiĂ©s par criblage de banques de petites molĂ©cules et proposĂ©s comme potentialisateurs de KCC2, semblent amĂ©liorer les symptĂŽmes dans diffĂ©rents modĂšles animaux dâaffections associĂ©es Ă une perte dâexpression de KCC2. Cependant, leur mode d'action dans les neurones corticaux et leur potentiel thĂ©rapeutique dans l'Ă©pilepsie restent inconnus, voire controversĂ©s. Au cours de ma thĂšse, jâai tout d'abord comparĂ© le mode d'action de ces composĂ©s dans des neurones hippocampiques de rat et mis en Ă©vidence que tous deux amĂ©liorent la fonction de KCC2. Cet effet s'accompagne pour le CLP257 d'une modulation allostĂ©rique positive des rĂ©cepteurs GABAA extrasynaptiques. De façon intĂ©ressante, ces deux composĂ©s rĂ©duisent la diffusion latĂ©rale de KCC2 Ă la membrane plasmique et augmentent lâagrĂ©gation du transporteur, sans modifier son expression totale ou membranaire, ni le niveau de phosphorylation de ses rĂ©sidus canoniques. Jâai mis en Ă©vidence que ces deux composĂ©s suppriment efficacement les dĂ©charges spontanĂ©es de type interictal, enregistrĂ©es in vitro dans le tissu hippocampique postopĂ©ratoire rĂ©sĂ©quĂ© de patients souffrant dâĂ©pilepsie du lobe temporal (ELT). De plus, l'administration chronique de PCPZ permet de rĂ©duire l'apparition de crises et la frĂ©quence des activitĂ©s Ă©pileptiformes dans un modĂšle souris d'ELT. Mes rĂ©sultats dĂ©cryptent donc le mode d'action de deux candidats potentialisateurs de KCC2 dans des neurones hippocampiques et dĂ©montrent pour la premiĂšre fois leur potentiel thĂ©rapeutique dans une Ă©pilepsie pharmacorĂ©sistante
Characterization and therapeutic potential of potassium-chloride co-transporter type 2 (KCC2) enhancers in temporal lobe epilepsy
Dans les neurones, les co-transporteurs cation-chlorure (CCCs) tels que le co-transporteur potassium-chlorure de type 2 (KCC2) et le co-transporteur sodium-potassium-chlorure de type 1 (NKCC1), contrĂŽlent l'homĂ©ostasie intraneuronale des ions chlorure et, par consĂ©quent, l'efficacitĂ© et la polaritĂ© de la signalisation synaptique par l'acide gamma-aminobutyrique (GABA). Dans de nombreuses affections neurologiques dont l'Ă©pilepsie, une rĂ©duction de l'expression ou de la fonction de KCC2 peut conduire Ă une signalisation GABAergique dĂ©polarisante et paradoxalement excitatrice, et ainsi contribuer Ă lâĂ©mergence dâactivitĂ©s pathologiques et de crises. Compenser la dĂ©rĂ©gulation de la fonction des CCCs dans la pathologie apparaĂźt donc comme une stratĂ©gie thĂ©rapeutique prometteuse. Le bumĂ©tanide, un antagoniste du transporteur NKCC1, ne parvient pas Ă prĂ©venir les crises nĂ©onatales aiguĂ«s lors dâessais cliniques et au contraire induit des effets secondaires tels quâune ototoxicitĂ©. En revanche, deux composĂ©s â la prochlorperazine (PCPZ) et le CLP257 â rĂ©cemment identifiĂ©s par criblage de banques de petites molĂ©cules et proposĂ©s comme potentialisateurs de KCC2, semblent amĂ©liorer les symptĂŽmes dans diffĂ©rents modĂšles animaux dâaffections associĂ©es Ă une perte dâexpression de KCC2. Cependant, leur mode d'action dans les neurones corticaux et leur potentiel thĂ©rapeutique dans l'Ă©pilepsie restent inconnus, voire controversĂ©s. Au cours de ma thĂšse, jâai tout d'abord comparĂ© le mode d'action de ces composĂ©s dans des neurones hippocampiques de rat et mis en Ă©vidence que tous deux amĂ©liorent la fonction de KCC2. Cet effet s'accompagne pour le CLP257 d'une modulation allostĂ©rique positive des rĂ©cepteurs GABAA extrasynaptiques. De façon intĂ©ressante, ces deux composĂ©s rĂ©duisent la diffusion latĂ©rale de KCC2 Ă la membrane plasmique et augmentent lâagrĂ©gation du transporteur, sans modifier son expression totale ou membranaire, ni le niveau de phosphorylation de ses rĂ©sidus canoniques. Jâai mis en Ă©vidence que ces deux composĂ©s suppriment efficacement les dĂ©charges spontanĂ©es de type interictal, enregistrĂ©es in vitro dans le tissu hippocampique postopĂ©ratoire rĂ©sĂ©quĂ© de patients souffrant dâĂ©pilepsie du lobe temporal (ELT). De plus, l'administration chronique de PCPZ permet de rĂ©duire l'apparition de crises et la frĂ©quence des activitĂ©s Ă©pileptiformes dans un modĂšle souris d'ELT. Mes rĂ©sultats dĂ©cryptent donc le mode d'action de deux candidats potentialisateurs de KCC2 dans des neurones hippocampiques et dĂ©montrent pour la premiĂšre fois leur potentiel thĂ©rapeutique dans une Ă©pilepsie pharmacorĂ©sistante.In neurons, cation-chloride cotransporters (CCCs), such as the potassium-chloride cotransporter type 2 (KCC2) and the sodium-potassium-chloride cotransporter type 1 (NKCC1), control intraneuronal chloride ion homeostasis and, subsequently, the efficiency and polarity of gamma-aminobutyric acid (GABA) synaptic signaling. In many neurological disorders, including epilepsy, reduced KCC2 expression or function may result in depolarizing and paradoxically excitatory GABA signaling that may then contribute to pathological activities and seizures. Compensating for the dysregulation of CCC function in the pathology therefore appears as a promising therapeutic strategy. Bumetanide, an antagonist of the NKCC1 transporter, failed to prevent acute neonatal seizures in clinical trials and instead induced side effects such as ototoxicity. Instead, two compounds - prochlorperazine (PCPZ) and CLP257 â recently identified from library screening as candidate KCC2 enhancers, appear to improve symptoms in several animal models of disorders associated with KCC2 suppression. However, their mode of action in cortical neurons and their therapeutic potential in epilepsy remain unknown or even controversial. During my thesis, I first compared the mode of action of these two compounds in rat hippocampal neurons and showed that both of them enhance KCC2 function. This effect was accompanied for CLP257 by a positive allosteric modulation of extrasynaptic GABAA receptors. Interestingly, both compounds reduce the lateral diffusion of KCC2 in the plasma membrane and increases its clustering, without altering its total or membrane expression or the phosphorylation level of its canonical residues. I demonstrated that both compounds effectively suppress spontaneous interictal-like discharges recorded in postoperative hippocampal tissue resected from intractable, mesial temporal lobe epilepsy (mTLE) patients. Furthermore, chronic administration of PCPZ reduced seizure onset and the frequency of epileptiform activities in a mouse model of mTLE. My results thus decipher the mode of action of two candidate KCC2 enhancers in hippocampal neurons and demonstrate for the first time their therapeutic potential in pharmacoresistant epilepsy
Cotransporteurs cation-chlorure et polarité de la signalisation GABAergique dans les interneurones à parvalbumine de l'hippiocampe chez la souris
International audienceKEY POINTS:Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAAR)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABAAR activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAAR-mediated currents often above action potential threshold. However, during repetitive GABAAR activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride buildup is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons.ABSTRACT:Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAAR activation in adult mouse hippocampal parvalbumin interneurons (PV INs) and pyramidal cells (PCs). Loose cell attached, tight-seal and gramicidin-perforated patch recordings all show GABAAR-mediated transmission is slightly depolarizing and yet inhibitory in both PV INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAAR-mediated currents in PV INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAARs. Finally, we show that repetitive 10 Hz activation of GABAARs in both PV INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride buildup may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types
Cation-chloride cotransporters and the polarity of GABA signaling 2 in mouse hippocampal parvalbumin interneurons Running title: GABA signaling in hippocampal parvalbumin interneurons
Transmembrane chloride gradients govern the efficacy and polarity of GABA signaling in neurons and are usually maintained by the activity of cation chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAAR activation in adult mouse hippocampal parvalbumin interneurons (PV INs) and pyramidal cells (PCs). Loose cell attached, tight-seal and gramicidin-perforated patch recordings all show GABAAR-mediated transmission is slightly depolarizing and yet inhibitory in both PV INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAAR-mediated currents in PV INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAARs. Finally, we show that repetitive 10 Hz activation of GABAARs in both PV INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride buildup may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signaling in both cell types
KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels
International audienceKCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention
La géphyrine interagit avec le co-transporteur K-Cl KCC2 pour réguler son expression de surface et sa fonction dans les neurones corticaux.
International audienceThe K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling
Epilepsie liée à une lipofuscinose neuronale focale : localisation extra-frontale, signatures EEG et implication du GABA
International audienceFocal neuronal lipofuscinosis (FNL) is an uncommon epileptic disorder related to an excess of lipofuscin accumulation within dysmorphic-appearing neurons (DANs), whose epileptogenic mechanisms are still poorly understood. It shares some clinical and neuroimaging similarities with focal cortical dysplasia of type IIb (FCDIIb), but it represents a different pathological entity. Here, we identified two patients with FNL among a 10-year cohort of 323 patients who underwent neurosurgery for a focal pharmacoresistant epilepsy. We describe the electroclinical, metabolic and neuropathological features of both patients with FNL who benefited from a comprehensive presurgical investigation. While the previous reports showed frontal lobe localization of the lesion, FNL was identified in the temporal lobe, in one of our patients. EEG investigations in both patients showed striking focal and rich interictal activity resembling that described in FCDIIb. Besides focal intraneuronal lipofuscin accumulation, the neuropathological analysis demonstrated that somata of DANs were surrounded by a large amount of GABAergic presynaptic buttons, suggesting the involvement of interneurons in the epileptogenicity of FNL. To further explore the role of GABAergic transmission in the generation of epileptiform activity in FNL, we performed in vitro multi-electrode array recordings on the post-surgery tissue from one patient. Spontaneous interictal-like discharges (IILDs) were identified only in the restricted area displaying the highest density of lipofuscin-containing DANs, suggesting a close correlation between the density of lipofuscin-containing neurons and epileptogenicity. Moreover, IILDs were blocked by the GABAA receptor antagonist gabazine. All together, these findings showed how GABA signaling may contribute to the generation of interictal-like activity in FNL tissue
Hippocampal and neocortical BRAF mutant nonâexpansive lesions in focal epilepsies
International audienceAbstract Objective Mesial Temporal Lobe Epilepsyâassociated Hippocampal Sclerosis (MTLEâHS) is a syndrome associated with various aetiologies. We previously identified CD34âpositive extravascular stellate cells (CD34+ cells) possibly related to BRAF V600E oncogenic variant in a subset of MTLEâHS. We aimed to identify the BRAF V600E oncogenic variants and characterise the CD34+ cells. Methods We analysed BRAF V600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLEâHS samples (25 with CD34+ cells) and nine nonâexpansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multiâelectrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAF wildtype MTLEâHS and BRAF V600E mutant nonâexpansive lesion of hippocampus and/or neocortex. Results We identified a BRAF V600E oncogenic variant in five MTLEâHS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAF V600E mutant samples. The coâexpression of the oncogeneâinduced senescence marker p16 INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. Interpretation BRAF V600E underlies a subset of MTLEâHS and epileptogenic nonâexpansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies
Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19
BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old