Bases moléculaires et cellulaires d’un trouble neurodéveloppemental causé par l’haploinsuffisance de SYNGAP1

La déficience intellectuelle est la cause d’handicap la plus fréquente chez l’enfant. De nombreuses évidences convergent vers l’idée selon laquelle des altérations dans les gènes synaptiques puissent expliquer une fraction significative des affections neurodéveloppementales telles que la déficience intellectuelle ou encore l’autisme. Jusqu’à récemment, la majorité des mutations associées à la déficience intellectuelle a été liée au chromosome X ou à la transmission autosomique récessive. D’un autre côté, plusieurs études récentes suggèrent que des mutations de novo dans des gènes à transmission autosomique dominante, requis dans les processus de la plasticité synaptique peuvent être à la source d’une importante fraction des cas de déficience intellectuelle non syndromique. Par des techniques permettant la capture de l’exome et le séquençage de l’ADN génomique, notre laboratoire a précédemment reporté les premières mutations pathogéniques dans le gène à transmission autosomique dominante SYNGAP1. Ces dernières ont été associées à des troubles comportementaux tels que la déficience intellectuelle, l’inattention, des problèmes d’humeur, d’impulsivité et d’agressions physiques. D’autres patients sont diagnostiqués avec des troubles autistiques et/ou des formes particulières d’épilepsie généralisée. Chez la souris, le knock-out constitutif de Syngap1 (souris Syngap1+/-) résulte en des déficits comme l’hyperactivité locomotrice, une réduction du comportement associée à l’anxiété, une augmentation du réflexe de sursaut, une propension à l’isolation, des problèmes dans le conditionnement à la peur, des troubles dans les mémoires de travail, de référence et social. Ainsi, la souris Syngap1+/- représente un modèle approprié pour l’étude des effets délétères causés par l’haploinsuffisance de SYNGAP1 sur le développement de circuits neuronaux. D’autre part, il est de première importance de statuer si les mutations humaines aboutissent à l’haploinsuffisance de la protéine. SYNGAP1 encode pour une protéine à activité GTPase pour Ras. Son haploinsuffisance entraîne l’augmentation des niveaux d’activité de Ras, de phosphorylation de ERK, cause une morphogenèse anormale des épines dendritiques et un excès dans la concentration des récepteurs AMPA à la membrane postsynaptique des neurones excitateurs. Plusieurs études suggèrent que l’augmentation précoce de l’insertion des récepteurs AMPA au sein des synapses glutamatergiques contribue à certains phénotypes observés chez la souris Syngap1+/-. En revanche, les conséquences de l’haploinsuffisance de SYNGAP1 sur les circuits neuronaux GABAergiques restent inconnues. Les enjeux de mon projet de PhD sont: 1) d’identifier l’impact de mutations humaines dans la fonction de SYNGAP1; 2) de déterminer si SYNGAP1 contribue au développement et à la fonction des circuits GABAergiques; 3) de révéler comment l’haploinsuffisance de Syngap1 restreinte aux circuits GABAergiques affecte le comportement et la cognition. Nous avons publié les premières mutations humaines de type faux-sens dans le gène SYNGAP1 (c.1084T>C [p.W362R]; c.1685C>T [p.P562L]) ainsi que deux nouvelles mutations tronquantes (c.2212_2213del [p.S738X]; c.283dupC [p.H95PfsX5]). Ces dernières sont toutes de novo à l’exception de c.283dupC, héritée d’un père mosaïque pour la même mutation. Dans cette étude, nous avons confirmé que les patients pourvus de mutations dans SYNGAP1 présentent, entre autre, des phénotypes associés à des troubles comportementaux relatifs à la déficience intellectuelle. En culture organotypique, la transfection biolistique de l’ADNc de Syngap1 wild-type dans des cellules pyramidales corticales réduit significativement les niveaux de pERK, en fonction de l’activité neuronale. Au contraire les constructions plasmidiques exprimant les mutations W362R, P562L, ou celle précédemment répertoriée R579X, n’engendre aucun effet significatif sur les niveaux de pERK. Ces résultats suggèrent que ces mutations faux-sens et tronquante résultent en la perte de la fonction de SYNGAP1 ayant fort probablement pour conséquences d’affecter la régulation du développement cérébral. Plusieurs études publiées suggèrent que les déficits cognitifs associés à l’haploinsuffisance de SYNGAP1 peuvent émerger d’altérations dans le développement des neurones excitateurs glutamatergiques. Toutefois, si, et auquel cas, de quelle manière ces mutations affectent le développement des interneurones GABAergiques résultant en un déséquilibre entre l’excitation et l’inhibition et aux déficits cognitifs restent sujet de controverses. Par conséquent, nous avons examiné la contribution de Syngap1 dans le développement des circuits GABAergiques. A cette fin, nous avons généré une souris mutante knockout conditionnelle dans laquelle un allèle de Syngap1 est spécifiquement excisé dans les interneurones GABAergiques issus de l’éminence ganglionnaire médiale (souris Tg(Nkx2.1-Cre);Syngap1flox/+). En culture organotypique, nous avons démontré que la réduction de Syngap1 restreinte aux interneurones inhibiteurs résulte en des altérations au niveau de leur arborisation axonale et dans leur densité synaptique. De plus, réalisés sur des coupes de cerveau de souris Tg(Nkx2.1-Cre);Syngap1flox/+, les enregistrements des courants inhibiteurs postsynaptiques miniatures (mIPSC) ou encore de ceux évoqués au moyen de l’optogénétique (oIPSC) dévoilent une réduction significative de la neurotransmission inhibitrice corticale. Enfin, nous avons comparé les performances de souris jeunes adultes Syngap1+/-, Tg(Nkx2.1-Cre);Syngap1flox/+ à celles de leurs congénères contrôles dans une batterie de tests comportementaux. À l’inverse des souris Syngap1+/-, les souris Tg(Nkx2.1-Cre);Syngap1flox/+ ne présentent pas d’hyperactivité locomotrice, ni de comportement associé à l’anxiété. Cependant, elles démontrent des déficits similaires dans la mémoire de travail et de reconnaissance sociale, suggérant que l’haploinsuffisance de Syngap1 restreinte aux interneurones GABAergiques dérivés de l’éminence ganglionnaire médiale récapitule en partie certains des phénotypes cognitifs observés chez la souris Syngap1+/-. Mes travaux de PhD établissent pour la première fois que les mutations humaines dans le gène SYNGAP1 associés à la déficience intellectuelle causent la perte de fonction de la protéine. Mes études dévoilent, également pour la première fois, l’influence significative de ce gène dans la régulation du développement et de la fonction des interneurones. D’admettre l’atteinte des cellules GABAergiques illustre plus réalistement la complexité de la déficience intellectuelle non syndromique causée par l’haploinsuffisance de SYNGAP1. Ainsi, seule une compréhension raffinée de cette condition neurodéveloppementale pourra mener à une approche thérapeutique adéquate.Intellectual deficiency is the most frequent cause of severe handicap in children. A growing body of work indicates that disruption of synaptic genes explains a significant fraction of neurodevelopmental disorders, such as intellectual deficiency and autism. Until recently, the majority of known mutations associated with non-syndromic intellectual deficiency were either X-linked or autosomal recessive. On the other hand, more recent studies suggest that de novo mutations in autosomal genes required for synaptic plasticity may explain an important fraction of cases of non-syndromic intellectual deficiency. Using exome capture and genomic DNA sequencing technics, our laboratory reported for the first time pathogenic mutations in the autosomal dominant gene of SYNGAP1. Pathogenic mutations in SYNGAP1 have been associated with behavioral abnormalities such as intellectual deficiency, inattention, mood problem, impulsivity, and physical aggression. In addition, a subset of these patients show acquired microcephaly, autism and/or specific forms of generalized epilepsy. Syngap1 germline knockout (Syngap1+/-) mice show several behavioral abnormalities, such as locomotor hyperactivity, decreased anxiety-like behavior, enhanced startle reactivity, lack of social memory, a propensity toward isolation, deficits in cued fear conditioning, and impaired reference and working memories. Thus, Syngap1+/- mice might represent a reliable experimental model to study the pathological effects of Syngap1 haploinsufficiency on neuronal circuit development. On the other hand, it is important to first establish whether mutations in Syngap1 found in human lead in fact to protein haploinsufficiency. SYNGAP1 codes for a Ras GTPase-activating protein. Its haploinsufficiency results in an increased level of Ras activity and ERK phosphorylation, abnormal dendritic spine morphogenesis, and an excess of postsynaptic AMPA receptors in excitatory neurons. Specifically, it has been suggested that prematurely increased insertion of AMPA receptors into excitatory synapses could explain some of the phenotypes observed in Syngap1+/- mice. Conversely, the role of Syngap1 haploinsufficiency in GABAergic circuits is unknown. The aims of my thesis project are: 1), to identify the impact of human mutations on SYNGAP1 function; 2) to determine whether SYNGAP1 plays a role in GABAergic circuit development and function, and 3) to reveal how Syngap1 haploinsufficiency specifically in GABAergic circuits affects cognitive behavior. We reported the first missense mutations (c.1084T>C [p.W362R]; c.1685C>T [p.P562L]), as well as two novel truncating mutations (c.2212_2213del [p.S738X]; c.283dupC [p.H95PfsX5]) in SYNGAP1 in humans. These mutations are de novo, except c.283dupC, which was inherited from a mosaic parent. In this study, we confirmed that patients with these mutations in SYNGAP1 showed, among other phenotypes, behavioral disorders associated with intellectual deficiency. Biolistic transfection of wild-type Syngap1 cDNA in pyramidal cells from cortical organotypic cultures significantly reduced neural activity-dependent pERK levels. In contrast, plasmid constructs expressing W362R, P562L or the previously described R579X mutations had no significant effect on pERK levels. These experiments suggest that these mutations in SYNGAP1 result in a loss of function, most probably disrupting brain development via a mechanism of haploinsufficiency. Several studies suggest that the cognitive deficits associated with Syngap1 haploinsufficiency may arise from alterations in the developmental trajectory of glutamatergic excitatory neurons. Whether and to what extent Syngap1 haploinsufficiency affects the synaptic network development of GABAergic cells, thus contributing to excitation/inhibition imbalance and cognitive abnormalities, remains elusive. Therefore, we examined the involvement of Syngap1 in the development of GABAergic circuits. To this goal, we generated conditional knockout mice where one Syngap1 allele was removed specifically in GABAergic interneurons derived from the medial ganglionic eminence (MGE) (Tg(Nkx2.1-Cre);Syngap1flox/+ mice). In organotypic cultures, we found that reduction of Syngap1 specifically in GABAergic interneurons resulted in alterations of their axonal arborisation and synapse density. Moreover, miniature inhibitory postsynaptic currents (mIPSC) and optogenetically-evoked IPSC (oIPSC) recordings in acute brain slices showed a significant reduction in cortical inhibitory neurotransmission in Tg(Nkx2.1-Cre);Syngap1flox/+ mice. Furthermore, we compared the performance of young adult Syngap1+/-, Tg(Nkx2.1-Cre);Syngap1flox/+ mice and their respective control littermates in several behavioral tests. Contrary to Syngap1+/- mice, Tg(Nkx2.1-Cre);Syngapflox/+ mice were not hyperactive, and did not demonstrate any anxiety-like behavior. They, however, showed the same impairment in working and social recognition memory, indicating that Syngap1 disruption in MGE-derived GABAergic neurons recapitulate at least some of the cognitive deficits observed in Syngap1+/- mice. All together, my PhD work first, demonstrated that human mutations in the SYNGAP1 gene associated with intellectual deficiency likely cause Syngap1 haploinsufficiency, and second, revealed for the first time a significant role of SYNGAP1 in the regulation of the development and function of GABAergic interneurons

Decrease of SYNGAP1 in GABAergic cells impairs inhibitory synapse connectivity, synaptic inhibition and cognitive function

Haploinsufficiency of the SYNGAP1 gene, which codes for a Ras GTPase-activating protein, impairs cognition both in humans and in mice. Decrease of Syngap1 in mice has been previously shown to cause cognitive deficits at least in part by inducing alterations in glutamatergic neurotransmission and premature maturation of excitatory connections. Whether Syngap1 plays a role in the development of cortical GABAergic connectivity and function remains unclear. Here, we show that Syngap1 haploinsufficiency significantly reduces the formation of perisomatic innervations by parvalbumin-positive basket cells, a major population of GABAergic neurons, in a cell-autonomous manner. We further show that Syngap1 haploinsufficiency in GABAergic cells derived from the medial ganglionic eminence impairs their connectivity, reduces inhibitory synaptic activity and cortical gamma oscillation power, and causes cognitive deficits. Our results indicate that Syngap1 plays a critical role in GABAergic circuit function and further suggest that Syngap1 haploinsufficiency in GABAergic circuits may contribute to cognitive deficits

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Summary: Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced as-trocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual do-nors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.Peer reviewe

Antenatal suppression of il-1 protects against inflammation-induced fetal injury and improves neonatal and developmental outcomes in mice

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1β induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1β, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1β, IL-6, and IL-8, increased IL-1β, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.Mathieu Nadeau-Vallée, Peck-Yin Chin, Lydia Belarbi, Marie-Ève Brien, Sheetal Pundir, Martin H. Berryer, Alexandra Beaudry-Richard, Ankush Madaan, David J. Sharkey, Alexis Lupien-Meilleur, Xin Hou, Christiane Quiniou, Alexandre Beaulac, Ines Boufaied, Amarilys Boudreault, Adriana Carbonaro, Ngoc-Duc Doan, Jean-Sebastien Joyal, William D. Lubell, David M. Olson, Sarah A. Robertson, Sylvie Girard and Sylvain Chemto

Correction to: Novel and de novo mutations in pediatric refractory epilepsy

Following publication of the original article [1], the authors reported that one of the authors’ names is spelled incorrectly

Novel and de novo mutations in pediatric refractory epilepsy

Abstract Pediatric refractory epilepsy is a broad phenotypic spectrum with great genetic heterogeneity. Next-generation sequencing (NGS) combined with Sanger sequencing could help to understand the genetic diversity and underlying disease mechanisms in pediatric epilepsy. Here, we report sequencing results from a cohort of 172 refractory epilepsy patients aged 0–14 years. The pathogenicity of identified variants was evaluated in accordance with the American College of Medical Genetics and Genomics (ACMG) criteria. We identified 43 pathogenic or likely pathogenic variants in 40 patients (23.3%). Among these variants, 74.4% mutations (32/43) were de novo and 60.5% mutations (26/43) were novel. Patients with onset age of seizures ≤12 months had higher yields of deleterious variants compared to those with onset age of seizures > 12 months (P = 0.006). Variants in ion channel genes accounted for the greatest functional gene category (55.8%), with SCN1A coming first (16/43). 81.25% (13/16) of SCN1A mutations were de novo and 68.8% (11/16) were novel in Dravet syndrome. Pathogenic or likely pathogenic variants were found in the KCNQ2, STXBP1, SCN2A genes in Ohtahara syndrome. Novel deleterious variants were also found in West syndrome, Doose syndrome and glucose transporter type 1 deficiency syndrome patients. One de novo MECP2 mutation were found in a Rett syndrome patient. TSC1/TSC2 variants were found in 60% patients with tuberous sclerosis complex patients. Other novel mutations detected in unclassified epilepsy patients involve the SCN8A, CACNA1A, GABRB3, GABRA1, IQSEC2, TSC1, VRK2, ATP1A2, PCDH19, SLC9A6 and CHD2 genes. Our study provides novel insights into the genetic origins of pediatric epilepsy and represents a starting-point for further investigations into the molecular pathophysiology of pediatric epilepsy that could eventually lead to better treatments