Analisis of the cellular and molecular organization of the lactate shuttle in the inner retina
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2022-01
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Universidad de Valparaíso
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Facultad de Ciencias
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Programa de Doctorado en Ciencias con mencion en Neurociencias
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Doctor en Ciencias con Mención en Neurociencia
Resumen
Metabolism is a fundamental issue for cell function. This becomes an important concern for the nervous system and, therefore, for neurons, since excitatory synapses consume almost 50% of ATP to restore the ion gradients challenged by depolarization. The retina shares several features with the brain, including its high energy demands. Nonetheless, it is known that the retina metabolizes glucose mainly by the energetic-inefficient aerobic glycolysis pathway, which is 18- fold less productive than oxidative phosphorylation and has as its final step the production of lactate. Many investigations have suggested the existence of a retinal lactate shuttle, but to date the possible cells that consume this extracellular lactate which is produced by aerobic glycolysis remain unclear. The aim of this study was to better understand the energy metabolism in the retina and especially propose a consumption of extracellular lactate as an alternative energy source for inner retinal cells.
Immunofluorescence experiments showed that the inner retina massively expressed a specific monocarboxylate transporter isoform (MCT2). Moreover, this transporter colocalizes with the rod bipolar cell (RBC) marker, PKCα, and the amacrine cell marker, calretinin. To test the functionality of this expression we cultured retinal explants, and we manipulated pharmacologically the lactate metabolism for 4 days. These different treatments altered the death cell rate and number of RBCs. Furthermore, calcium and sodium imaging experiments reveal that the inhibition of lactate transport disrupts the ion gradients, resulting in changes in intracellular levels of basal calcium and sodium, that alter the amplitude, and kinetics parameters of calcium and sodium responses, suggesting a disruption in ion pumps. Interestingly, single-cell experiments reveal that the exposure of retinal slices to an extracellular solution with lactate, maintains the outward current, calcium current, and membrane potential. But the addition of an MCT2 blocker decreases the outward current, and calcium current and depolarizes RBCs. Finally, the study of intracellular lactate dynamics expressing the genetically-encoded FRET nanosensor Laconic, suggests that inner retinal neurons are capable of consuming extracellular lactate through MCT2 under culture basal conditions, which is exacerbated when the retina is subjected to general depolarization. Conversely, Müller cells display a producer profile under culture basal condition, which is reversed under a general depolarization.
In conclusion, our data support the notion that inner retinal neurons can import a portion of the extracellular lactate through MCT2 to meet their physiological demands, specifically cellular survival, ion homeostasis, and currents. In parallel, other observations suggest that Müller cells produce lactate under basal culture conditions, so they could be one of the cell types performing aerobic glycolysis in the retina. Likely, this extracellular lactate is used by retinal bipolar cells to produce ATP by classic oxidative phosphorylation.
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RETINA, GLUCOSA, L-LACTATO DESHIDROGENASA