Amyloid-β42 stimulated hippocampal lactate release is coupled to glutamate uptake

Since brain glucose hypometabolism is a feature of Alzheimer’s disease (AD) progression, lactate utilization as an energy source may become critical to maintaining central bioenergetics. We have previously shown that soluble amyloid-β (Aβ)42 stimulates glutamate release through the α7 nicotinic acetylcholine receptor (α7nAChR) and hippocampal glutamate levels are elevated in the APP/PS1 mouse model of AD. Accordingly, we hypothesized that increased glutamate clearance contributes to elevated extracellular lactate levels through activation of the astrocyte neuron lactate shuttle (ANLS). We utilized an enzyme-based microelectrode array (MEA) selective for measuring basal and phasic extracellular hippocampal lactate in male and female C57BL/6J mice. Although basal lactate was similar, transient lactate release varied across hippocampal subregions with the CA1 > CA3 > dentate for both sexes. Local application of Aβ42 stimulated lactate release throughout the hippocampus of male mice, but was localized to the CA1 of female mice. Coapplication with a nonselective glutamate or lactate transport inhibitor blocked these responses. Expression levels of SLC16A1, lactate dehydrogenase (LDH) A, and B were elevated in female mice which may indicate compensatory mechanisms to upregulate lactate production, transport, and utilization. Enhancement of the ANLS by Aβ42-stimulated glutamate release during AD progression may contribute to bioenergetic dysfunction in AD.


Results
Hippocampal basal and transient lactate analysis. We have previously reported spontaneous or transient release of tonic glutamate in the prefrontal cortex and hippocampus 30,31 . These signals were tetrodotoxin dependent, a sodium channel blocker, supporting release from synaptic signaling 31 . Based on these previous studies and the ANLS hypothesis, we expected to observe spontaneous tonic fluctuations in lactate signaling. Once a stable baseline was reached, MEAs were used to measure extracellular lactate levels for 10 min in each hippocampal subregion prior to pressure ejection recordings. Transient lactate release was observed in all hippocampal subregions and both sexes of C57BL/6J mice. Figure 1A shows representative DG, CA3, and CA1 traces from female mice. The average transient amplitude (Fig. 1B) was similar within a subregion between sexes, but was significantly different across hippocampal subregions. The largest amplitudes occurred within the CA1, followed by the CA3, and then DG of both male and female mice. Likewise, lactate transient peak area (Fig. 1C) varied across subregions with the largest in the CA1 that declined across the CA3 and DG. No differences between sexes were observed for any hippocampal subregion. The elevated transient release of lactate in the CA1 region may be in response to increased energy demands required by glutamatergic pyramidal neurons compared to granule cells types located in the DG. Despite differences in amplitude, the intertransient interval ( Fig. 1D) was similar across all three subregions and sex differences were also not observed. After transient lactate analysis, basal levels were calculated without the detection of transient lactate release. Basal lactate (Fig. 1E) was increased in female C57BL/6 mice particularly within the CA3 hippocampal subregion.
Soluble Aβ 42 stimulates lactate release that is attenuated by blocking glutamate clearance. After transient and basal lactate measurements, pressure ejections studies were performed. All compounds were tested in vitro to confirm they were not electrochemically active at our recording potential prior to local application in vivo. For each compound, consistent volumes between 25 and 75 nl were locally applied into the DG, CA3, and CA1 and no differences were observed between compounds within a brain region ( Table 1). The 0.1 µM Aβ 42 concentration was chosen because this elicited the maximal change in hippocampal glutamate release as shown in our previous research 7 . Representative lactate traces from local application in the CA1 of male C57BL/6J mice for all compounds are shown in Fig. 2A  , and CA1 (red) of female C57BL/6 mice. Note the different scales of the ordinate y-axis in each subregion and color-coordinated triangles identify transient release events. The average lactate transient amplitude (B), peak area (C) and intertransient interval (D) were averaged for each hippocampal region and sex. *p < 0.05; n = 6-7. A measurement without detectable lactate transients was used to calculate basal lactate levels (E). Table 1. Average pressure ejected volumes. Volumes are shown in mean ± SEM for application of each compound for all brain regions and sexes along with the corresponding P value from a one-way ANOVA. Abbreviations -Aβ amyloid-β, TBOA dl-threo-β-Benzyloxyaspartic acid, CHC 2-cyano-3-(4-hydroxyphenyl)-2-propenoic acid.  The legend under the graph indicates the compounds that were locally (co)applied. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 6-8.  2B) and in the CA1 of female mice (Fig. 2C). Next, we coapplied 0.1 µM Aβ 42 with 1 mM 2-cyano-3-(4-hydroxyphenyl)-2-propenoic acid (CHC), a MCT inhibitor that has been shown to block glial lactate efflux at this concentration 33 . Coapplication of Aβ 42 with CHC also blunted extracellular lactate levels in the hippocampus of C57BL/6J male and the CA1 region of female mice (Fig. 2B,C). These data support the increased extracellular lactate levels from local application of Aβ 42 are coupled to clearance of glutamate from the extracellular space through EAATs and efflux from MCT.

mRNA expression of glutamate and lactate transporters.
To determine if sex differences in lactate release were due to varying expression levels of genes involved with the ANLS, RT-PCR was conducted on tissue from a separate, naïve, cohort of male and female mice (Fig. 3). CHRNA7 expression levels were increased in female C57BL/6J mice while glial EAATs (SLC1A2 and SLC1A3) were similar between sexes. This suggests enhanced Aβ 42 -stimulated glutamate release without a change in the glutamate clearance in female mice. Next we examined expression levels involved with lactate production or utilization. Lactate dehydrogenase (LDH) is a tetramer consisting of either LDHA or LDHB subunits to form LDH-5 and LDH-1, respectively. In glial cells, homomeric LDHA catalyzes the reduction of pyruvate to lactate, while in neurons the homomeric LDHB isoform drives oxidation of lactate to pyruvate for oxidative phosphorylation 34 . Expression of both isoforms was increased in female mice indicating enhanced glial lactate production and neuronal utilization, respectively. While no sex differences were observed in neuronal SLC16A7 expression, we observed glial SLC16A1 expression was increased in female C57BL/6J mice. BSG is known to chaperone glial MCT1 and regulate basal and glutamate evoked lactate release through this transporter 35 . No BSG expression differences were observed between sexes.

Discussion
Glucose enters the brain through transporters located on astrocytic endfeet that cover the surface of blood vessels. Glucose metabolism is the predominant fuel source for the brain serving as a substrate not only for ATP production in aerobic glycolysis but also providing the carbon backbone for maintaining the neurotransmitter pools of glutamate and γ-aminobutyric acid. This ATP production is necessary to reestablish the resting membrane potential for neuronal signaling which accounts for ~ 70% of the brain's energy expenditure with excitatory neurons accounting for 80-85% of this usage 36 . Lactate is a byproduct of aerobic glycolysis and during periods of increased neuronal activity, can serve as an additional energy substrate through mitochondrial oxidative phosphorylation 11 . In this process, neuronally released glutamate is cleared from the extracellular space through EAAT located on glia. Uptake of glutamate is coupled to glucose transport into astroglia and a subsequent increase in glycolysis. The lactate formed is shuttled from astrocytes to neurons through MCT thereby coupling glutamatergic neurotransmission with bioenergetics to support synaptic signaling.
Once thought of as a metabolic waste product, the role of lactate in brain health and disease has garnered much attention over the last two decades. During acute neurotrauma, such as with traumatic brain injury or cerebral ischemia, astrocytic release of lactate provides neuroprotection 37 . Lactate release is also important for memory consolidation 12,17 and promotes hippocampal neurogenesis 38 . However, during normal aging, lactate levels increase 39 due to reduced oxidative phosphorylation from mitochondrial dysfunction. Considering aging is a risk factor for AD, mitochondrial dysregulation and the subsequent increase in cerebral lactate is postulated to instigate disease onset 40,41 . For example, CSF lactate levels are increased in AD patients 25 . While elevated hippocampal lactate levels are observed at 12 months and correspond with cognitive deficits in the APP/PS1 amyloidogenic AD mouse model 27 . At this same age in APP/PS1 mice, we reported elevated tonic levels of hippocampal glutamate 8,42 that would contribute to these observed lactate levels. www.nature.com/scientificreports/ Although basal and transient lactate were similar between sexes of C57BL/6J mice, we did observe increased transient amplitude in the CA1 and CA3 compared to the DG. A couple of factors may be responsible for these differences. While histological analysis regarding the number of cells within a hippocampal subregion varies, the DG is known to have a higher density of neurons 43 . The increased MCT density reduces the amount of lactate present in the extracellular space. Additionally, pyramidal neurons require significantly more energy substrates to maintain synaptic integrity than granule cells resulting in larger lactate transient amplitude in the CA3 and CA1. This suggests that bioenergetic disruptions do not affect all neuronal populations equally particularly during AD progression.
In male mice, Aβ 42 -evoked lactate release coupled to glutamate clearance was observed in all subregions, while this was only prominent in the CA1 of female mice. This suggests sexually dimorphic bioenergetics in the DG and CA3 with female mice relying on greater glucose utilization due to decreased glutamate coupled lactate release. The enhanced gene expression of glial LDHA and SLC16A1 as well as neuronal LDHB could be a compensatory mechanism to increase lactate production, transfer, and utilization to meet hippocampal bioenergetic demands. The upregulation of CHRNA7 gene expression in conjunction with soluble Aβ 42 accumulation causes excessive activation during AD progression. These factors coupled with the concomitant glucose hypometabolism in AD challenges the ANLS to maintain the electrochemical gradient and leaves these regions particularly vulnerable to neurodegeneration. The translational implications provides a better understanding as to the increased AD prevalence in females.
Accumulation and deposition of Aβ 42 containing senile plaques is the earliest known pathological feature associated with AD. But, therapies targeting clearance of aggregated amyloid are only purported to modify disease progression in a subset of clinical trial participants 44 . This may be a consequence of how amyloid accumulation alters bioenergetics during AD progression. We have previously demonstrated soluble Aβ 42 elicits glutamate release through sodium dependent presynaptic activation of the α7nAChR 7 . This mechanism contributes to elevated hippocampal tonic and phasic glutamate levels prior to onset of cognitive deficits in APP/PS1 mice 8 . The current study demonstrates Aβ 42 also elicits lactate release coupled to glutamate uptake as a possible means to maintain energy demands for enhanced synaptic signaling based on the ANLS. Furthermore, neuronal activity is known to modulate Aβ 42 accumulation and aggregation suggestive of a vicious pathophysiological cascade 45 . During the initial phases of Aβ 42 accumulation, astroglia might be able to sustain the enhanced energy substrate needs. However, amyloid deposition alters the morphology and function of astroglia. These reactive astrocytes have decreased expression of EAAT1 and 2 as well as glucose transporter 1 46 . This would diminish their bioenergetic capacity resulting in glucose hypometabolism and neuronal loss typified in later stages of AD progression more akin to cognitive deficits 47 .
The main source of oxidative substrates for neuronal energy demands is contested throughout the literature. Neurons can readily utilize both glucose and lactate as an energy substrate. But, vascular dysfunction and reduced cerebral blood flow are observed in AD 48 . The subsequent decrease in blood derived glucose forces neurons to rely on lactate. Additionally, synaptic glutamate release is heightened by Aβ 42 activation thereby increasing ANLS as a means to meet the elevated neuronal energy demands. As soluble Aβ 42 levels increase during AD progression, the elevated hippocampal activity 49 makes larger pyramidal neurons particularly vulnerable to damage and is why they constitute a larger percentage of cell loss observed in post-mortem AD brains 50 . Prior to cell death, though, these damaged neurons undergo energetic inefficiency requiring additional substrates and creating competition for available resources with healthy neurons 41 . Eventually, the overutilization of energy substrates propagates neuronal dysfunction affecting neighboring brain regions. These changes in glutamate coupled lactate release may serve as an early biomarker for bioenergetic perturbations driving AD progression.
Limitations of the study are related to the specificity of the EAAT and MCT inhibitors. At the concentrations used, TBOA inhibits clearance of glutamate through both the glial EAAT1 and 2 and the neuronal EAAT3. Although > 90% of extracellular glutamate clearance is mediated by glial EAAT2 in the forebrain 10 , neuronal uptake is a contributing factor. Likewise, CHC at the concentrations studied inhibits transport of lactate across either glial MCT1 or 4 and neuronal MCT2. Thus, the nonspecificity of the inhibitors used in the present study suggests a role for glutamate coupled lactate release from either neurons or glia. Future experiments with selective inhibitors or knockdown of individual MCT are needed to definitely conclude the cellular localization Aβ 42 stimulated lactate release.

Conclusion
The present study provides evidence that soluble Aβ 42 alters hippocampal lactate dynamics through mechanisms associated with glutamate release and clearance. Considering pathophysiological changes occur decades prior to dementia onset, the hyperexcitable neuronal networks would eventually culminate in bioenergetic dysfunction, neuronal death, and dementia progression. Hippocampal lactate dynamic differences may account for the increased incidence of dementia in females and further indicates a need for personalized patient care. Additional experiments are needed to dissect sexually dimorphic effects of lactate release in hippocampal subregions and their resulting impact on AD progression.

Methods
All methods were conducted in accordance with relevant guidelines and regulations.

Animals. Protocols for animal use were approved by the Institutional Animal Care and Use Committee at
Southern Illinois University School of Medicine and in compliance with the ARRIVE guidelines. Male and female 3-6 month old C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME; RRID:IMSR_ JAX:000664) and allowed at least 1 week to acclimate to our animal facility before electrochemical measure-  Enzyme-based MEA. MEA with platinum (Pt) recording surfaces were obtained from Quanteon LLC (Nicholasville, KY; Fig. 4A) and made selective for l-lactate recordings as previously described 28,29 . One microliter of lactate oxidase stock solution (1 IU/µl) was added to 9 µl of a 1.0% BSA and 0.125% glutaraldehyde w/v solution and applied dropwise to a Pt recording surface. Lactate oxidase enzymatically degrades lactate to pyruvate and H 2 O 2 , the electroactive reporter molecule (Fig. 4B). The other Pt recording site (sentinel site) was coated with the BSA/glutaraldehyde solution that does not enzymatically generate H 2 O 2 . When a potential of + 0.7 V vs a Ag/AgCl reference electrode was applied to the recording surfaces, H 2 O 2 is oxidized and the current generated from the two electron transfer is amplified and digitized by the Fast Analytical Sensing Technology (FAST) 16mkIII (Quanteon LLC) electrochemistry instrument. Reference electrode. A Ag/AgCl reference wire was prepared by stripping the teflon coating from both ends of a silver wire (A-M Systems, Carlsberg, WA). One end was soldered to a gold-plated connector (Newark element14 Chicago, IL), while the other was placed (cathode) into a 1 M HCl bath saturated with NaCl that also contained a stainless steel counter wire (anode). Passing a + 9 V DC to the cathode versus the anode for 15 min deposited Ag/Cl onto the Ag wire.
In vivo anesthetized recordings. Mice were anesthetized using 1.5-2.0% isoflurane (Abbott Lab, North Constant voltage amperometry (4 Hz) was performed with a potential of + 0.7 V vs the Ag/AgCl reference electrode applied by the FAST16mkIII. MEAs reached a stable baseline for 60 min before basal and transient lactate analysis followed by pressure ejection studies. The FAST software saved amperometric data, time, and pressure ejection events. Calibration data, in conjunction with a MATLAB (MathWorks, Natick, MA; RRID:SCR_001622) graphic user interface program was used to calculate basal, transient, and stimulus-evoked lactate. Five evoked lactate signals in each hippocampal subfield were averaged into a representative signal for comparison. Only one compound was pressure-ejected per hemisphere for each mouse.
Cresyl violet staining. After lactate recordings, mice were euthanized with an overdose of isoflurane followed by rapid decapitation with sharp scissors. The brains were removed and placed in 4% paraformaldehyde for 24-48 h then stored in 30% sucrose. Twenty micron coronal sections through the hippocampus were obtained using a cryostat (Model HM525 NX, Thermo Fisher Scientific) and mounted on a glass slide. Slices were stained with cresyl violet and coverslipped. MEA placement was verified for each mouse (Fig. 4D).

RT-PCR.
A separate group of 3-6 month male and female C57BL/6J mice were euthanized according to the above procedure. The brain was extracted and the hippocampus was dissected on wet ice and stored at − 80 °C until processing. RNA was extracted from tissue by homogenization in Trizol Reagent and separated by centrifugation at 12,000×g for 15 min at 4 °C with chloroform. RNA was isolated by centrifugation at 12,000×g for 25 min at 4 °C in 100% isopropanol. The pellet was resuspended in RNAse free water and quantified using a NanoDrop Spectrophotometer. cDNA was synthesized using candidate primers (Integrated DNA Technologies; Table 2) and an iScript cDNA Synthesis Kit (Bio-Rad). Relative mRNA expression was analyzed by quantitative RT-PCR as previously described 54 using the StepOne Real-Time PCR System (Thermo Fisher Scientific) and SYBR Green MasterMix (Bio-Rad) and using Ubiquitin-conjugating enzyme E2D2 (UBE2D2) as the internal housekeeping gene. Statistical analysis. Prism (GraphPad Software Version 9, Inc., La Jolla, CA; RRID:SCR_002798) was used for all statistical analyses. For basal lactate and transient lactate release analysis, sexes and hippocampal brain  -CAC CAT GGC AGC CCT CTG  GCCC-3′   5′-ATA GAT AAA GAT GAT GGT AAC  CAA CA-3′   CHRNA7  Cholinergic receptor nicotinic alpha 7  subunit  5′-CCT GCA AGG CGA GTTCC-3′  5′-CTC AGG GAG AAG TAC ACG  GTGA-3′   LDHA  Lactate dehydrogenase A  5′-ATG CAC CCG CCT AAG GTT CTT-3′ 5′-GCC TAC GAG GTG ATC AAG CT-3′   LDHB  Lactate dehydrogenase B  5′-AGT CTC CCG TGC ATC CTC AA-3′  5′-AGG GTG TCC GCA CTC TTC CT-3′   SLC1A2  Excitatory amino acid transporter 2  5′-CTG GTG CAA GCC TGT TTC C-3′  5′-GCC TGT TCA CCC ATC TTC C- www.nature.com/scientificreports/