Amp-activated Protein Kinase Mediates Activity-dependent Regulation Of PGC-1α And NRF-1 And Mitochondrial Energy Metabolism Coupling In Rat Visual Cortical Neurons

BackgroundIn development and adult visual cortical neurons, the mitochondrial number, functional capacity, and gene expression are dynamically regulated in accordance with energy demands, namely experience-dependent energy metabolic plasticity. Recent studies show that neuronal activity and functional plasticity are tightly coupled with energy metabolism (energy metabolic coupling). Approximately 90% of adenosine triphosphate (ATP) generated in the brain is synthesized in mitochondria via oxidative phosphorylation (OXPHOS). Thus, brain critically depends upon mitochondrial function and oxygen supply to support its immense energetic demand. However, the signaling mechanisms that couple neuronal activity to energy metabolism are entirely unknown.Nuclear respiratory factors (NRFs), including NRF-1 and NRF-2, are found to act on a number of nuclear genes required for mitochondrial respiratory function. Our previously studies suggest that both NRF-1 and NRF-2 regulate a number of genes required for mitochondrial energy metabolic coupling. The transcription and protein expression of NRF-1 and NRF-2 in visual cortical neurons are positively correlated with neuronal excitability, and consistent with the cytochrome oxidase (COX) activity. Peroxisome proliferator-activated receptorγcoactivator-1-α(PGC-1α), on the other hand, as inducible coregulators of nuclear receptors in the control of cellular energy metabolic pathways, has been adequately represented as a master regulator of mitochondrial biogenesis. PGC-1αhas been shown to coactivate NRFs, which are associated with increased COX activity. Nevertheless, the functional significance of neuronal activity-dependent regulation of PGC-1αand NRFs expression in visual cortical neurons, the signaling pathways that couple neuronal activity to energy metabolism are entirely unknown.AMP-activated protein kinase (AMPK), a metabolic gauge regulating whole-body energy homeostasis, has been reported to regulate mitochondrial biogenesis by sensing the energy state in the cells. Several lines of evidences show that AMPK is a putative upstream activator of NRFs signal transduction pathway. However, the signaling pathway that couples neuronal activity to mitochondrial energy metabolism, particularly whether AMPK mediates activity-dependent regulation of PGC-1αand NRFs for mitochondrial biogenesis and function, has not been examined in visual cortical neurons.AimThe goal of this study was to test our hypothesis that AMPK might be an important mediator of neuronal activity to stimulate mitochondrial biogenesis and function by regulating PGC-1αand NRF-1 gene expression in visual cortical neurons; if so, to detection the functional consequences of these activity-dependent mitochondrial transcriptional regulation. Our study also aims to provide experimental evidences for the treatment of neuronal mitochondrial dysregulation and other related neurodevelopmental dysfunction disease by modulation of AMPK signaling.Results1. Membrane depolarization with 25 mmol/L KCl significantly increases PGC-1α, NRF-1 and mtTFA mRNA and protein level with increased ATP production in primary visual cortical neurons.2. KCl depolarization rapidly increases AMPK phosphorylation in cultured primary visual cortical neurons.3. AMPK inhibition by Compound C, an AMPK inhibitor, completely represses KCl depolarization-induced up-regulation of PGC-1α, NRF-1 and mtTFA mRNA and AMPK ?phosphorylation as well as cellular ATP content.4. AMPK activation by AICAR or resveratrol also markedly increases PGC-1αand NRF-1 mRNA levels as well as cellular ATP content in neuron cultures. All these effects can be completely blocked by an AMPK inhibitor, Compound C.5. Reduction of neuronal activity by one week of MD significantly decreases AMPK phosphorylation and activity, dramatically down-regulates PGC-1αand NRF-1 expression in deprived primary visual cortex.6. Administration of resveratrol in vivo significantly activates AMPK activity and attenuates the effects of MD on mitochondria by significant increase in PGC-1αand NRF-1 levels mitochondria amount, and coupled respiration.Conclusions1. Neuronal activity has a major impact on AMPK signaling, which mediates activity-dependent regulation of mitochondrial energy metabolism by regulating coactivator PGC-1αand its downstream transcription factor NRF-1 and mtTFA in visual cortical neurons. 2. We demonstrated for the first time that pharmacologic interventions, such as resveratrol, that restore AMPK activity in the functional inactivation visual cortices may be a novel means to rescue mitochondrial dysfunction caused by visual deprivation.3. This study provides additional insight into AMPK pathophysiology in the brain, and highlights the importance of AMPK- PGC-1α- NRF-1 networks in activity-dependent regulation of mitochondrial energy metabolism in neurons, which could potentially lead to novel therapeutic targets for the amelioration of neuronal mitochondrial dysfunction and its related disease.