Distinct Regulation of Bioenergetics and Translation by Group I mGluR and NMDAR

Abstract

21Neuronal activity is responsible for disproportionally large consumption of energy by 22 the brain. However, little is known about the processes within neurons responsible for 23 such abundant ATP outlay. Here we provide evidence that major fraction of ATP 24 consumed on glutamate stimulation is due to activation of global de-novo protein 25 synthesis mediated by group I mGluRs in cultured cortical neurons. We found mGluR 26 and NMDAR stimulation impacts translation with distinct kinetics; while mGluR lead to 27 rapid and sustained translation activation as measured by eEF2 phosphorylation and 28 metabolic labeling, NMDAR stimulation induced a robust translation inhibition. Our 29 observations established an inverse correlation between the kinetics of translation and 30 change in ATP level for both receptors. We observed an immediate mGluR mediated 31 reduction in neuronal ATP level while NMDA lead to a delayed (≥15 mins) protein 32 synthesis dependent ATP expenditure demonstrating a possibility for NMDAR dependent 33 delayed translation activation. Interestingly, we identified that AMP activated protein 34 kinase (AMPK) has a central role in regulating downstream effects of both mGluR and 35 NMDAR. AMPK activity was reduced on mGluR stimulation even under low ATP levels 36 while there was a rapid increase in AMPK activity on NMDAR stimulation. Perturbing 37 AMPK function prior to either stimulation led to a complete loss of stimulation specific 38 effects on eEF2 phosphorylation and global translation. Our observation therefore 39 established a pivotal role for AMPK-eEF2 signaling axis on receptor specific regulation 40 of global translation.41 42 109 dependent reduction was absent in presence of protein synthesis inhibitors anisomycin 110 (25μM) or cycloheximide (100μg/ml) (Figure 1B) while drugs themselves had no 111 significnat impact on the ATP/(ATP+ADP) ratio. These observations suggest a large 112 increase in protein synthesis on stimulation, responsible for consuming signifcant 113 fraction of neuronal ATP. The identical effects of both anisomycin and cycloheximide 114 negates the possibility of these observations due to non-specific effects of these drugs. 115 Further, to understand the contributions from individual glutamate receptors, we 116 repeated glutamate stimulation inhibiting either ionotropic NMDA and AMPA receptors 117 with a cocktail of D-AP5 (25μM) + CNQX (40μM) (referred to as inotropic glutamate 118 receptor (iGluR) blocker cocktail hereafter) or inhibiting mGluRs with MPEP (10μM). Co-119 stimulation with iGluR blocker cocktail led to significant reduction in ATP/(ATP+ADP) 120 ratio similar to glutamate alone while stimulation along with MPEP abolished the 121 glutamate mediated reduction significantly (Figure 1C). These results therefore, 122 indicate that the observed reduction in ATP on glutamate stimulation is majorly due to 123 mGluR activity with minimal contributions from the iGluRs. Furthermore, Stimulation 124 in presence iGluR blocker cocktail along with protein synthesis inhib...