The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A.

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

The inhibition of NCS-1 binding to Ric8a rescues fragile X syndrome mice model phenotypes.

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.