Elucidating the Molecular Pathway through which L-Lactate potentiates NMDAR Signaling

Abstract

The role of L-Lactate has expanded from an energy metabolite to a signaling molecule in neurons. Studies have shown that L-Lactate plays a role in neuroprotection and in NMDAR-dependent long-term memory formation. The aim of this dissertation is to characterize the role of L-Lactate as a signaling molecule and understand the molecular mechanism through which L-Lactate potentiates NMDAR signal. Using mass spectrometry, I monitored the time-dependent changes in the phosphoproteome of cortical neuronal cultures in response to Lactate. The phosphoproteomic analysis highlighted a number of cytoskeletal proteins involved in synapse remodeling as well as axon guidance that were regulated by L-Lactate. In addition, I found that L-Lactate induced phosphorylation of proteins involved in the MAPK pathway, as reported in an earlier study. I hypothesize the involvement of CaMKII in this mechanism. CaMKII is one of the most abundant kinases in the brain and plays a role in learning and memory via interaction with NMDAR. Using CaMKII inhibitors and mutants of the NMDAR subunit GluN2B, the findings in this dissertation provide evidence for the involvement of CaMKII, specifically, the interaction between CaMKIIa and GluN2B, as a requirement for the L-Lactate mediated potentiation of NMDAR signal. In addition, to gain insight into the evolution of lactate from a metabolite to a signaling molecule, this study explores the evolution of glutamate as a signaling molecule in multicellular organisms so it may serve as a model for evolution of metabolites like lactate into signaling molecules. For this purpose, the model organism Hydra was used, since it belongs to phylum Cnidaria, evolutionarily one of the first phyla to have a nervous system. In order to explore whether glutamate receptors, particularly, NMDAR are functionally expressed in Hydra and are localized in neurons, a line of transgenic Hydra expressing a calcium indicator (GCaMP6s) in neurons was generated. With the transgenic Hydra line, I attempted to measure the in vivo response of neurons in Hydra to glutamate. This study highlights several ground work experiments with an extensive discussion of implications and challenges and an outlook for future investigations.