Tracing the metabolic rewiring of glycolytically compromised neurons. (20th December 2022) - Sei, Yoshitaka J
Abstract: Background: While a reduction in cerebral glucose uptake is a diagnostic trait of Alzheimer's Disease, the neuronal requirement for the uptake and metabolism of glucose is not well understood. To dissect how neurons metabolize glucose, we tracked [U‐ 13 C]glucose metabolism in human iPSC‐derived neurons. Method: Human WTC‐11 iPSCs expressing the CRISPR inhibition machinery dCas9‐KRAB were differentiated into neurons using a doxycycline‐inducible NGN2. After a 2 week maturation period, neurons were incubated with [U‐ 13 C]glucose at either a physiologically relevant 1.5 mM or a glucose‐scarce 0.15 mM concentration for 24 hours prior to metabolite extraction. To determine the importance of glucose uptake to neurons, a guide for the neuronal glucose transporter GLUT3 was added to the CRISPR machinery to limit the neuronal uptake of glucose (GLUT3 KD). These GLUT3 KD neurons were compared to neurons with uninhibited glucose uptake (non‐target). Student t‐tests or two‐way ANOVAs with Bonferonni correction were used to compare metabolite levels between neuronal groups and the concentration of extracellular [U‐ 13 C]glucose. Result: Under physiologically relevant glucose conditions, GLUT3 KD neurons had decreased levels of labeled glucose. Despite the difference in internalized glucose, GLUT3 KD did not affect the amount of [U‐ 13 C]glucose‐derived citrate that feeds the TCA cycle. The labeled level of a critical pentose phosphate pathway (PPP) metabolite, R5P, was decreased in GLUT3 KD neurons. When measuring the total levels, both labeled and unlabeled, of R5P and citrate, we found there to be no significant effect of GLUT3 KD. This trend was also observed in a 0.15 mM [U‐ 13 C]glucose condition. With the change in R5P levels exclusively in the amount derived from [U‐ 13 C]glucose, we found that neurons prioritize the usage of glucose to feed the TCA cycle over the PPP. Conclusion: Neurons rewire their metabolism in response to glucose shortages, demonstrating their sensitivity to and reliance on the availability of glucose. During limited access to glucose, glucose utilization was pulled away from the PPP and shunted to the TCA cycle. In the context of AD, the connection between glucose shortages in neurons and their bioenergetic dysfunction is a critical point of discussion centered on understanding the mechanistic initiation of neurodegeneration.