Gloire, Mélanie

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Reciprocal influence between APP expression and glucose metabolism in the hippocampusMélanie GLOIRE, Agnès VILLERS, Jean-Marie COLET and Laurence RISDepartment of Neuroscience, University of Mons, Belgium29Working hypothesis and Mouse Model⇧ [APP]Glucose hypometabolism[APP]Glucose metabolismGlycolysis (GAPDH) ATP productionGlutamate transport…⇧ APP mRNA[APP]Alzheimer’s diseaseAgeingBrain insulin resistance⇧ [APP]Glucose hypometabolismDown SyndromeRat primary cortical astroglialcells deprived of 95% of their glucose for 24hType 2 Diabetes⇧ [APP]Hyperglycemia, hypoglycemia and insulin resistanceGenotype Level of expressionWT +/+ Normal expressionHT +/- Half expressionKO -/- No expressionAdvantages• Allowing to study the physiological roles of APP and theimportance of its expression level• Excluding the role of Aβ oligomerizationInconvenient• No possible APP overexpression (without glucose metabolism orgenetic modifications)2. APP overexpression is correlated with glucose metabolism disruption in pathologies or ageing3. Upregulation of APP mRNA when glucose is reduced is also found in the literature…4. …but our experiments did not show any overexpression of the APP protein when glucose supply is reduced in WT mice1. APP is involved in glucose metabolism 5. So, until now, we mostly focused on the 3 APP expression levels available thanks to APP knockout miceororElectrophysiological recordings : synaptic activity in restricted glucose supply hippocampal slices[D-glucose] Condition10mM Control5mM Mild glucose restriction2.5mM Severe glucose restriction1. Hippocampal slices preparation (APP +/+, +/- and -/- mice)2. Resting period in an interface recording chamber (1h30). aCSF(32°C) contains 10mM, 5mM or 2.5mM of D-glucose3. Electrodes positioning in CA1 area. Stimulation of the Schaffercollaterals and recording in the stratum radiatum of CA14. Evaluation of synaptic activity by fEPSP slope recording5. Input-Output curve resulting from an increasing electrical potentialdifference from 2V to 10V in increments of 1V1 2 3 4 52. The level of APP expression modulates the sensitivityto restriction in glucose supply in the hippocampus1. Glucose restriction reduces synaptic transmissionin a concentration dependant way in CA1 area ofhippocampal slices3. Ageing reduces the basal synaptic activity of theneuronal network and the tolerance to restriction inglucose supply in the hippocampusI-O curves distributions presented in 1 and 3. are similar in the other two genotypes.HT mice always present an intermediate phenotype. 1HNMR: metabolic activity in the hippocampusPhosphate buffer 100% D2O TSP (0ppm)H2O + MethanolChloroformeCell debrisAqueous phaseOrganic phase1. Hippocampi extraction, dissociation andmetabolites extraction in methanol, H2O andchloroform (4°C)2. Centrifugation and phases separation3. Evaporation of the aqueous phase (speedvac)4. Metabolites resuspension in phosphate buffer100% D2O and a reference compound : TSP5. Sample magnetization in a 500 mHz 1HNMRspectrometer and spectra acquisition (FourierTransform)6. Spectra normalization (Mestre Renova) andppm separation (loading and score plots) byPrincipal Component Analysis (PCA) in Simca.Corresponding metabolites are finallyidentified thanks to Chenomx and tables12 3 456Hippocampusppm4. The level of APP expression modifies the metabolic function in the hippocampusScore plot Loading plotPrincipal Component Analysis (PC1 and PC2)1HNMR: metabolic activity in the hippocampus7. Metabolic modifications are more important between WT and KO mice while WT and HT mice present a similar hippocampus metabolism HT mice present an intermediate phenotype5. Identification of aqueous metabolites detectable on a 1HNMR spectrum obtained from the extraction of the hippocampi of an APP KO mouse 6. APP plays a role in neurotransmitters homeostasis in the hippocampusThe absence of APP increases the abundance of glutamate but decreases the abundance of GABAQuestions left and conclusion APP protein quantification (Western-Blotting)  from hippocampi submitted to an in vivo hypoglycemia  APP mRNA quantification  (RT qPCR) from our culture model and hippocampi submitted to an in vivo hypoglycemia2. Will we be able to observe that APP is upregulated and/or overexpressed when glucose supply is restricted in one of our models ?1. What kind of modifications could we observe in the hippocampus energy metabolism submitted to an in vivo hypoglycemia ? Continuous and controlled insulin administration to induce an in vivohypoglycemia (50mg/dL) Hippocampi extraction Comparison of metabolite profiles modifications (1HNMR) between sham condition and hypoglycemia for each genotypesWe can conclude that even if APP overexpression has not been observed in this project yet, APP expression and glucose metabolism are linked in the hippocampus and that further investigations need to be conducted to better understand this relationship

Reciprocal influence between APP expression and glucose metabolism in the hippocampus (short talk)

https://orbi.umons.ac.be/bitstream/20.500.12907/28828/1/eposterVF.pdf

Reciprocal influence between APP expression and glucose metabolism in the hippocampus (short talk)

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