Identification of RasGEFs counteracting neuronal SynGAP1 function

Abstract

SynGAP syndrome refers to an under-diagnosed group of diseases caused by the mutation of the SynGAP1 gene encoding the predominant synaptic protein SynGAP1. SynGAP1 is a Ras GTPase activating protein that is a major regulator of synaptic ERK signaling regulating neurotransmitter receptor traffic and synaptic plasticity. Mutation in SynGAP1 can lead to hyperactivation of the Ras signaling pathway. This may contribute to the development of symptoms related to neurodevelopmental disorders such as intellectual disability, autism spectrum disorder (ASD), and a variety of other conditions such as epilepsy, depending on the specific mutation. SynGAP1 (GAPs) constrains Ras signaling in neurons by stimulating the hydrolysis of Ras bound GTP to GDP. On the contrary, guanine nucleotide exchange factors (GEFs) activate Ras signaling by stimulating the exchange of Ras bound GDP (inactive) for GTP (active). This study was aimed to identify possible druggable targets for SynGAP1 deficiencies in human disease using rodent neurons as a model utilizing a high throughput neuronal ERK reporter imaging platform and identify the RasGEFs counterbalancing SynGAP1 function. The SynGAP1 and RasGEF deficiency were achieved with artificial miRNA-mediated knockdown of the corresponding RNAs. The response was measured by translocation of the ERK reporter from the nucleus to cytoplasm at baseline and after induction of synaptic activity with Picrotoxin. Our results have recapitulated the effects of SynGAP1 deficiency in neuronal cultures and have suggested possible candidates of RasGEF that cause a reduction in the ERK signaling pathway. Taken together, this approach may have the potential to identify RasGEFs whose elimination counteract SynGAP1 deficiency and this may provide a basis for drug discovery in the future.