The Development of Genetic and Molecular Tools To Identify Activators of egl-30/GNAQ That Enhance Memory with Age

Abstract

Cognitive decline is a prominent feature of aging. We previously showed that activating the Gαq pathway enhances memory in young animals, and the expression of neuronal gain-of-function EGL-30/GNAQ improves memory in aged C. elegans and mice. Therefore, GNAQ represents a promising target for therapeutics. Given that EGL-30/GNAQ and Gαq pathways are conserved between C. elegans and mammals, C. elegans is a good model organism to investigate the efficacy of various therapies to treat age-related memory decline. Although EGL-30 and GNAQ share 82% amino acid identity, we wanted to be sure that our chemical screens are specific for mammalian GNAQ. To address this, we aimed to generate a GNAQ-humanized C. elegans for drug screening purposes. We used Gibson assembly to create plasmids with human GNAQ gain-of-function mutations under the control of a general neuronal promoter. After the plasmids were injected into wild-type C. elegans, no changes in memory were observed. Subsequently, we generated plasmids expressing GNAQ and GFP under egl-30 promoters. These plasmids are to be integrated into the C. elegans genome using a Recombinase-Mediated Cassette Exchange procedure. Separately, we performed memory assays on wild-type C. elegans treated with the GNAQ-activating chemical aluminum fluoride; however, no improvement in memory was observed. Finally, Gαq signaling in the AWC neuron results in neuropeptide release that then activates CREB in AIM neurons which is responsible for activating genes associated with long-term memory. We created a list of neuropeptides released from the AWC neuron and receptors in the AIM. However, whether these neuropeptide-receptor pairs are responsible for extending memory has yet to be determined. We used RNA interference (RNAi) to knock down select neuropeptides and receptors in worms with egl-30 gain-of-function mutations. However, the extrachromosomal array containing the egl-30 gain-of-function did not transmit well, therefore both the RNAi knockdown worms and the positive control did not have memory. We then generated an integrated line by treating the C. elegans containing the extrachromosomal array with UV radiation. While none of the lines were fully integrated, the transmittance of the extrachromosomal array was significantly improved. We performed a memory assay on the lines exhibiting a high transmission rate to verify that exposure to UV radiation did not result in any memory deficits; both lines maintained memory at 18 hours, suggesting that the lines are competent for memory assays. In the future, an outcross will be performed in order to remove any background mutations caused by UV integration and subsequently the worms will be used to test the previously identified neuropeptide-receptor pairs. My thesis focused on generating humanized C. elegans to screen for drug candidates that have the ability to target the Gαq signaling pathway and improve memory. I also sought to investigate the genes downstream of the Gαq signaling pathway, specifically, the neuropeptides and receptors that are activated as a result of signaling.