Fret-based real time ATP measurements in sensory neurons of normal or diabetic rats

P Fernyhough, M-R Aghanoori, V Margulets, L Kirshenbaum, D Gitler

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

he distal dying-back of nerve fibers is a hallmark of diabeticneuropathy. The provision of energy in the form of ATP ischallenging for neurons with long axons. We hypothesized thatenergy supplementation via glycolysis and/or mitochondrial oxida-tive phosphorylation is compromised in nerve endings thuscontributing to axonal degeneration in diabetic conditions. DRGneuron cultures from age-matched control or streptozotocin (STZ)-induced type 1 diabetic rats were used for in vitro studies. Threeplasmids containing ATP sensors of varying affinities (detectable byFRET technology and live cell confocal imaging) were transfectedinto neurons to study endogenous ATP levels in real time. FRETefficiency (YFP/CFP ratio) of the ATP sensors AT1.03 (lowaffinity) and AT1.03 YEMK (medium affinity) were significantlyhigher than the mutant (AT1.03 R122/6K) in DRG neurons in bothcell bodies and neurites (p < 0.0001). Using the AT1.03 YEMKconstruct, treatment with oligomycin (an ATP synthase inhibitor inmitochondria) decreased the ATP levels in neurites and cell bodiesof DRG neurons (p < 0.05). Blockade of glycolysis using 2-deoxy-D-glucose (2-DG: a glucose analog) also lowered ATP levels(p < 0.001). Both neurites and cell bodies of DRGs from diabeticrats showed a diminishment of ATP levels when compared toneurons from control rats (p < 0.01). In conclusion, low ATP levelsin cell bodies and distal axons may contribute to the energy deficit innerve in diabetes and could trigger distal dying-back nervedegeneration. Funded by St Boniface Research
Original languageEnglish
Title of host publicationJOURNAL OF NEUROCHEMISTRY
Pages196-196
Number of pages1
Volume150
DOIs
StatePublished - 2019

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