Supragranular Pyramidal Cells Exhibit Early Metabolic Alterations in the 3xTg-AD Mouse Model of Alzheimer's Disease

Abstract

The impairment of cerebral glucose utilization is an early and predictive biomarker of Alzheimer's disease (AD) that is likely to contribute to memory and cognition disorders during the progression of the pathology. Yet, the cellular and molecular mechanisms underlying these metabolic alterations remain poorly understood. Here we studied the glucose metabolism of supragranular pyramidal cells at an early presymptomatic developmental stage in non-transgenic (non-Tg) and 3xTg-AD mice, a mouse model of AD replicating numerous hallmarks of the disease. We performed both intracellular glucose imaging with a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose biosensor and transcriptomic profiling of key molecular elements of glucose metabolism with single-cell multiplex RT-PCR (scRT-mPCR). We found that juvenile pyramidal cells exhibit active glycolysis and pentose phosphate pathway at rest that are respectively enhanced and impaired in 3xTg-AD mice without alteration of neuronal glucose uptake or transcriptional modification. Given the importance of glucose metabolism for neuronal survival, these early alterations could initiate or at least contribute to the later neuronal dysfunction of pyramidal cells in AD.

Keywords: FRET imaging; glucose uptake; glycolysis; pentose phosphate pathway; single-cell RT-PCR.

FIGURE 1

Expression of the FRET glucose sensor in neocortical slices. Representative single plane confocal image of a slice expressing the FLII12Pglu-700 μδ6 biosensor following sindbis viral transduction. Cells expressing the FRET glucose sensor are identified by their GFP fluorescence (green). Pyramidal cells are immunolabeled for the Satb2 transcription factor (red). Dashed line represents layer I and II border. Note that most layer II and III transduced cells are stab2 positive.

FIGURE 2

Glucose uptake in layer II and III pyramidal cells. (A) The gray scale image shows the YFP fluorescence. Pseudocolor images show the YFP/CFP ratio value [coded by pixel hue, see scale bar in (a)] and the fluorescence intensity (coded by pixel intensity) at different time points before (a) and after (b,c) superfusion of 10 mM glucose. (B) Traces show average YFP/CFP ratio measure at the cytosolic part of the soma of individual pyramidal cells delineated in (A). (a–c) indicate time points corresponding to the pseudocolor images shown in (A). (C) Mean relative YFP/CFP ratio changes in pyramidal cells of non-Tg (black trace) and 3xTg-AD mice (red trace) induced by superfusion of 10 mM glucose. Traces show mean (solid lines) ± standard errors of the mean (color shades). Note the slowly developing increase in mean relative ratio during 10 mM glucose superfusion and the similar temporal profiles between non-Tg and 3xTg-AD mice.

FIGURE 3

Metabolic activity of layer II and III pyramidal cells. (A) Mean relative YFP/CFP ratio changes in pyramidal cells of non-Tg (black trace) and 3xTg-AD mice (red trace) induced by glucose (Glc) restriction (0.2 mM). Traces show mean (solid lines) ± standard errors of the mean (color shades). Note the slowly developing decrease in mean relative ratio during 0.2 mM Glucose superfusion. Note also the difference in the temporal profiles between non-Tg and 3xTg-AD mice. (B) Histograms show the metabolic activity (decrease in relative YFP/CFP ratio) at different time of glucose restriction. After 5 min of glucose restriction pyramidal cells of non-Tg mice exhibit a higher metabolic activity than those of 3xTg-AD mice. In contrast, during longer glucose restriction pyramidal cells of 3xTg-AD mice display a higher metabolic activity. n.s. not statistically significant.

FIGURE 4

Glucose metabolic pathways in layer II and III pyramidal cells. Left panels show the mean relative YFP/CFP ratio changes in pyramidal cells of non-Tg (black traces) and 3xTg-AD mice (red traces) induced by glycolysis inhibition with IAA (A1), pentose phosphate pathway inhibition with 6-AN (B1), and combined inhibition with both IAA and 6-AN (C1). Traces show mean (solid lines) ± standard errors of the mean (color shades). Histograms show the metabolic activity (decrease in relative YFP/CFP ratio) at different time of glycolysis inhibition (A2), pentose phosphate pathway inhibition (B2), and combined inhibition (C2). n.s. not statistically significant. (A1) Note the delayed increase in mean relative ratio in non-Tg pyramidal cells during IAA application. Note also the early decrease followed by a late increase in pyramidal cells of 3xTg-AD mice. (A2) After 10 min of IAA application pyramidal cells of 3xTg-AD mice develop a transient increase in metabolic activity. Longer application reveals a larger impact of glycolysis inhibition in pyramidal cells of 3xTg-AD mice than in pyramidal cells of non-Tg mice. (B1) Note the very low increase in mean relative ratio during 6-AN application in pyramidal cells of 3xTg-AD mice. (B2) The mean increase in relative ratio is higher in pyramidal cells of non-Tg mice at 10, 20, and 30 min of 6-AN superfusion. (C1) Note the transient decrease in mean relative ratio during combined IAA and 6-AN application more pronounced in pyramidal cells of 3xTg-AD mice than in non-Tg neurons. (C2) After 20 min of combined inhibition pyramidal cells of 3xTg-AD mice exhibit a marked transient increase in metabolic activity. Longer application reveals an accumulation of glucose smaller in pyramidal cells from 3xTg-AD than from non-Tg mice.

FIGURE 5

Sensitivity of the RT-mPCR. Total whole brain RNAs (1 ng) were subjected to a RT-PCR protocol. The PCR products were resolved in separate lanes by agarose gel electrophoresis in parallel with Φ×174 digested by HaeIII as molecular marker and stained with ethidium bromide. The amplified fragments correspond to neuronal markers (left), glucose transporters and key enzymes of glucose metabolic pathways (right).

FIGURE 6

Characterization of key molecular elements of glucose metabolism in layer II and III pyramidal cells. (A) Voltage responses (upper traces) induced by injection of current pulses (bottom traces) in a non-Tg layer II and III pyramidal cell. In response to just-above-threshold current pulse, this neuron fired an action potential with a long lasting biphasic AHP (middle trace). Near saturation it showed the typical firing of a regular spiking neuron with marked frequency adaptation and spike amplitude accommodation (shaded trace). (B) The pyramidal cell shown in (A) expressed vGluT1, GluT1, GluT3, HK1, pfkfb3, PFK1m, PFK1l, PFK1p, and G6PDx. (C) Histograms showing the expression profiles of key molecular elements of glucose metabolism in layer II and III pyramidal cells of non-Tg (black) and 3xTg-AD mice (red). No statistically significant difference in the occurrence of genes was observed. Note the very low occurrence of pfkfb3 and Gys1 (glycogen synthase) in pyramidal neurons.

FIGURE 7

Resting glucose metabolism of juvenile pyramidal cells and its alterations in 3xTg-AD mice. Glucose uptake is facilitated via glucose transporters (GluT). Once in the cytoplasm glucose phosphorylation by Hexokinase1 (HK1) yields to glucose 6-phosphate (G6P), the common substrate of different metabolic pathways. Glucose-6-phosphate dehydrogenase (G6PDH) is the rate limiting enzyme of the pentose phosphate pathway leading to ribose-5-phosphate (R5P). Pentose phosphate pathway is blocked by the G6PDH inhibitor, 6-aminonicotinamide (6-AN). G6P can also be isomerized into fructose-6-phosphate (F6P), a common intermediate of the glycolysis and the hexosamine biosynthetic pathway. Glycolysis is blocked by Iodoacetic acid (IAA) inhibiting glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme of the hexosamine biosynthetic pathway, converts fructose-6-phosphate and glutamine into glucosamine-6-phosphate (GlcN6P) and glutamate (glutamine and glutamate are omitted for the sake of simplicity). G6P is also the precursor of glycogen whose synthesis is normally absent in neurons (gray arrow). Dashed arrows indicate multisteps reactions. Functional changes in 3xTg-AD neurons are depicted by red arrows. Thicker and thinner arrows denote increased and decreased pathways, respectively.