Mechanisms of coupling energy metabolism and neuronal activity at the molecular level

The brain is a highly energy-intensive organ that represents only 2% of total body weight, but consumes 20% of total body oxygen and 25% of total body glucose. Neuronal activity and energy metabolism are coupled at the cellular level, as most of the energy is used to perform the neurons' main function -- communicate via neurotransmitters. Glutamate is the main excitatory neurotransmitter, and the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the two main glutamatergic receptors.;The AMPA and NMDA receptors mediate the majority of excitatory synaptic transmission in the brain and are both heterotetrameric proteins. The AMPA receptors are composed of the GluA1, GluA2, GluA3, and GluA4 subunits, and most of them contain the Ca2+-impermeable GluA2 subunit with either the GluA1 or GluA3 subunit. The NMDA receptors are composed of the ubiquitous GluN1 subunit in various combinations with the GluN2A-D and GluN3A-B subunits. Most NMDA receptors are composed of two GluN1 subunits and two GluN2A or GluN2B subunits. The properties of the AMPA and NMDA receptors are dictated by their subunit composition.;Recently, our laboratory has found that the coupling between energy metabolism and neuronal activity exists at the molecular level. Nuclear respiratory factor 1 (NRF-1) is a transcription factor that co-regulates all 13 subunits of cytochrome c oxidase (COX), the terminal enzyme of the oxidative metabolic pathway that generates energy, as well as critical subunits of NMDA (GluN1 and GluN2B) and AMPA (GluA2) receptors. The goal of my research is to determine if other transcription factors are also capable of coregulating neuronal energy metabolism and neurotransmitter receptors mediating glutamatergic neurotransmission.;Nuclear respiratory factor 2 (NRF-2) is a transcription factor in the E26 transformation-specific (ETS) family of transcription factors. The functional NRF-2 protein is composed of alpha and beta subunits. The alpha subunit contains the DNA-binding domain that recognizes the 'GGAA' motif, whereas the beta subunit contains the transactivating domain. Our laboratory has recently found that NRF-2 also regulates all 13 subunit genes of COX. We hypothesize that NRF-2 also regulates subunits of the NMDA and AMPA receptors, and that it does so via one of three mechanisms with respect to NRF-1: complementary, concurrent and parallel, and a combination of the complementary and concurrent/parallel mechanisms. The first aim of this dissertation is to determine the mechanism of NRF-2 co-regulation in relationship to NRF-1.;Recently, our laboratory has also found that the specificity protein (Sp) family of zinc-finger transcription factors also regulates all subunits of COX. The Sp family binds to GC-rich regulatory regions and three of its members, Sp1, Sp3, and Sp4, compete for the same cis- motif. Sp1 and Sp3 are ubiquitously expressed, but Sp4 is expressed in neurons and testes only. We hypothesize that Sp factors, especially Sp4, also co-regulate energy metabolism and mediators of neurotransmission. The second aim of this dissertation is to determine the mechanism of Sp-factor co-regulation in relationship to NRF-1.;Using multiple approaches, including in silico analyses, EMSA and supershift assays, ChIP in primary visual cortical tissue, promoter mutational analysis, overexpression and shRNA knock-down, as well as KCl-depolarization and TTX-impulse blockage experiments, this dissertation shows that NRF-2 and Sp4 regulate the expression of the GluA2 subunit of the AMPA receptor in a concurrent and parallel manner with NRF-1. NRF-2 also functionally regulates the expression of NMDA receptor subunits GluN1 and GluN2B in a concurrent and parallel manner with NRF-1, whereas Sp4 functionally regulates the GluN1, GluN2A, and GluN2B receptor subunits in a combination of the complementary and concurrent/parallel mechanism with NRF-1.;Defective energy metabolism underlies many neurodegenerative diseases. By elucidating the molecular mechanisms coupling neuronal activity and energy metabolism, this dissertation paves the way for future therapeutic measures to prevent and/or ameliorate neurodegenerative diseases.