Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: Both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission

Abstract

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as GluN1 (Grin1) and GluN2B (Grin2b) subunits of NMDA receptors. We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level.

Transcriptional Regulation of Brain-derived Neurotrophic Factor Coding Exon IX

“…In agreement with our previous findings (26), the promoter of cytochrome c oxidase subunit 6b (COX6b) formed a specific DNA/NRF-2 shift and a DNA/NRF-2/NRF-2 antibody supershift complex when incubated with visual cortical nuclear extract (Fig. 3, lanes 1 and 3, respectively).…”

“…Our laboratory has shown that NRF-2 regulates all 10 nuclear-encoded and all three mitochondrial-encoded subunit genes of cytochrome c oxidase, an important energy generating enzyme in the mitochondria (25). Moreover, we found that NRF-2 links energy metabolism and neuronal activity at the transcriptional level by also regulating specific subunits of glutamatergic receptors (26,27). The present study adds a third component to this coupling, i.e.…”

“…On the other hand, it enhances glutamate release and increases the abundance of NMDA and AMPA receptors and their delivery to the plasma membrane, thereby up-regulating receptor activity in hippocampal and cortical neurons (59 -61). Previously, we have found that NRF-2 functionally regulates key NMDA and AMPA receptor subunit genes (26,27). With our current discovery of direct regulation of BDNF by NRF-2 in visual cortical neurons, it is highly possible that the actions of BDNF on energy metabolism and glutamatergic synapses result from upstream activation by NRF-2.…”

“…We have shown previously that KCl depolarization up-regulated COX and specific excitatory glutamatergic neurotransmitter receptor subunits, and that this involved NRF-2 (25,26,29). Indeed, 20 mM KCl for 5 h induced an up-regulation of COX7c (to 152%) as well as Bdnf exon IX (to 142%) in cultured visual cortical neurons (p Ͻ 0.05 and p Ͻ 0.01, respectively) ( Fig.…”

“…Previous studies in our laboratory showed that TTX-induced impulse blockade decreased the transcript levels of COX subunits as well as those of NMDA, AMPA, and GABA A receptor subunit genes (25)(26)(27)30). To determine whether NRF-2 overexpression could rescue Bdnf transcripts from being down-regulated by TTX, primary cultured neurons that were transfected with empty vector controls or with NRF-2␣/␤ overexpression vectors were subjected to 0.4 M TTX treatments for 3 days.…”