The polarity of GABA signaling in the brain relies on transmembrane chloride gradients which are themselves regulated by cation/chloride cotransporters (CCCs). Intraneuronal chloride buildup promotes pathological activities in the human epileptic cortex and animal models of epilepsy. It is therefore crucial to develop novel approaches to prevent intraneuronal chloride accumulation in the pathological brain. We have recently identified an unsuspected role of membrane gangliosides in the control of CCC function in neurons. Interestingly, gangliosides, including monosialotetrahexosylganglioisde (GM1), are largely represented in neuronal lipid rafts and regulate several cellular processes including the membrane trafficking of neuronal proteins. Reduced GM1 levels are observed in mouse epilepsy models as well as in peritumoral tissue from human brain resections and participate in the development of seizures. Moreover, ganglioside metabolism deregulation is associated with epilepsy in animals and humans. Conversely, increasing ganglioside levels reduces brain trauma and SE-related damages. We propose to dissect the molecular mechanisms underlying the modulation of CCCs function by GM1 and to evaluate its therapeutic potential. Our preliminary results show a specific interaction of GM1 with both KCC2 and NKCC1 and regulations of their membrane mobility. In addition, we have identified a point mutation in KCC2 that abolishes GM1-KCC2 interaction. Importantly, this point-mutation in the KCC2 GM1-binding domain is associated with human epilepsy. The GABGANG consortium gathers leaders in their respective fields to address three main aims via a multidisciplinary, 4-year program. Our first aim is to elucidate the role of gangliosides in the regulation of KCC2 and NKCC1 membrane diffusion, nanoscale organization, membrane stability and function, and its consequences on chloride homeostasis and GABA signaling. Our second aim is to evaluate the contribution of GM1-CCC interaction in an animal model of temporal lobe epilepsy (TLE). In particular, we will evaluate the impact of status epilepticus on gangliosides and CCC expression. Then, we will study the impact of altered interaction with GM1 on CCC membrane diffusion and hyperexcitability in the epileptic hippocampus. If such effects are demonstrated, we will then test whether increasing GM1 levels can rescue CCC function and prevent interictal/ictal activity in the pilocarpine rodent TLE model. Our last aim is to address the impact of GM1 manipulations on GABA signaling and network activity in human postoperative cortical tissue. To achieve this, we will combine several state-of-the-art approaches including advanced single particle tracking, live 2-photon chloride imaging, mass-spectroscopy imaging, in vivo intrahippocampal multichannel electrophysiological recordings, GRIN-lens based endoscopic calcium imaging combined with behavior and multielectrode-array recordings from human organotypic cultures. This work is bound to disclose entirely novel mechanisms regulating neuronal chloride homeostasis and GABA signaling and identify novel therapeutically relevant targets that will be useful for intractable epilepsy as well as other neurological and psychiatric conditions associated with altered neuronal chloride transport.