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

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.

Keyphrases: SYNGAP1, neurotransmission GABAergique, Non-syndromic intellectual deficiency, SYNGAP1, de novo truncating and missense mutations, inhibitory perisomatic innervation, Tg(Nkx2.1-Cre);Syngap1flox/+ mouse, GABAergic neurotransmission, behavior and cognition, medial ganglionic eminence derived GABAergic interneuron