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Cited by 114Alzheimer's disease research and treatmentsNeuroscience and Neuropharmacology ResearchNeuroinflammation and Neurodegeneration Mechanisms

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Hippocampal disruptions of synaptic and astrocyte metabolism are primary events of early amyloid pathology in the 5xFAD mouse model of Alzheimer’s disease

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Andersen, Jens V., Skotte, Niels H., Christensen, Sofie K., Polli, Filip S., Shabani, Mohammad, Markussen, Kia H., Haukedal, Henriette, Westi, Emil W., Diaz-delCastillo, Marta, Sun, Ramon C., Kohlmeier, Kristi A., Schousboe, Arne, Gentry, Matthew S., Tanila, Heikki, Freude, Kristine K., Aldana, Blanca I., Mann, Matthias, Waagepetersen, Helle S.·2021·Cell Death & Disease
DOI 10.1038/s41419-021-04237-yPMC PMC8520528
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Abstract

Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal tricarboxylic acid (TCA) cycle metabolism mapped by stable 13 C isotope tracing. Particularly, reduced astrocyte TCA cycle activity and decreased glutamine synthesis led to hampered neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism, which may suggest a metabolic compensation in this brain region. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was selectively impaired in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunctions in AD.

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Fig. 1 Hippocampal and cerebral cortical amyloid-β (Aβ) burden, proteome and metabolome of 5xFAD mice. A Experimental overview of the study: male 5xFAD and control mice were utilized at 2 and 4 months (mo) of age. Representative Aβ staining of the cerebral cortex (B) and the hippocampus (C) of 2 and 4 mo old 5xFAD mice. Scale bars: B: 100 µm, C: 250 µm. D Quantification of regional Aβ immunoreactive areas in the cerebral cortex and hippocampus of the 5xFAD mice, mean ± SEM, n = 3-4, Student’s paired t-test. E Principal component analysis (PCA) plot of the 5xFAD proteome shows distinct clustering of samples based on brain region and age, n = 4. F Volcano plots displaying significant alterations in protein expression in the 5xFAD hippocampus and cerebral cortex. Proteins in the upper left quadrant signify decreased expression, whereas protein in the upper right quadrant signify increased expression. Statistical analysis of the proteome and metabolome is described in detail in the ‘Materials and methods’ section. G Heatmap displaying normalized metabolite abundance of glycolytic and TCA cycle intermediates, amino acids and other metabolites in microwave fixated brain tissue from 5xFAD mice, n = 6–7, Student’s paired t-test. Blue tones denote reduced abundance, whereas red tones denote elevated abundance. CTX: cerebral cortex, HP: hippocampus.
Fig. 1 Hippocampal and cerebral cortical amyloid-β (Aβ) burden, proteome and metabolome of 5xFAD mice. A Experimental overview of the study: male 5xFAD and control mice were utilized at 2 and 4 months (mo) of age. Representative Aβ staining of the cerebral cortex (B) and the hippocampus (C) of 2 and 4 mo old 5xFAD mice. Scale bars: B: 100 µm, C: 250 µm. D Quantification of regional Aβ immunoreactive areas in the cerebral cortex and hippocampus of the 5xFAD mice, mean ± SEM, n = 3-4, Student’s paired t-test. E Principal component analysis (PCA) plot of the 5xFAD proteome shows distinct clustering of samples based on brain region and age, n = 4. F Volcano plots displaying significant alterations in protein expression in the 5xFAD hippocampus and cerebral cortex. Proteins in the upper left quadrant signify decreased expression, whereas protein in the upper right quadrant signify increased expression. Statistical analysis of the proteome and metabolome is described in detail in the ‘Materials and methods’ section. G Heatmap displaying normalized metabolite abundance of glycolytic and TCA cycle intermediates, amino acids and other metabolites in microwave fixated brain tissue from 5xFAD mice, n = 6–7, Student’s paired t-test. Blue tones denote reduced abundance, whereas red tones denote elevated abundance. CTX: cerebral cortex, HP: hippocampus.CC BY · PubMed Central
Fig. 2 Elevated spontaneous excitatory synaptic activity in 5xFAD hippocampal neurons. Neuronal signaling was assessed by whole-cell patch-clamp electrophysiology in brain slices of 2 and 4 months (mo) old 5xFAD and control mice. A Representative recordings of spontaneous excitatory post-synaptic currents (sEPSCs) (black traces: control, gray traces: 5xFAD). Quantification of amplitude (B) and frequency (C) in cerebral cortical and hippocampal slices of 2 and 4 mo old 5xFAD and control mice. sEPSCs were obtained in voltage-clamp, gap-free mode, with a voltage command set to −60 mV. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 10–24 from 4–6 pairs of animals, Student’s unpaired t-test or Mann-Whitney test.
Fig. 2 Elevated spontaneous excitatory synaptic activity in 5xFAD hippocampal neurons. Neuronal signaling was assessed by whole-cell patch-clamp electrophysiology in brain slices of 2 and 4 months (mo) old 5xFAD and control mice. A Representative recordings of spontaneous excitatory post-synaptic currents (sEPSCs) (black traces: control, gray traces: 5xFAD). Quantification of amplitude (B) and frequency (C) in cerebral cortical and hippocampal slices of 2 and 4 mo old 5xFAD and control mice. sEPSCs were obtained in voltage-clamp, gap-free mode, with a voltage command set to −60 mV. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 10–24 from 4–6 pairs of animals, Student’s unpaired t-test or Mann-Whitney test.CC BY · PubMed Central
Fig. 3 Disrupted glucose metabolism in cerebral cortical and hippocampal brain slices of 5xFAD mice. Cycling ratios, reflecting the rate of the TCA cycle, calculated from metabolism of [U-13C]glucose in acutely isolated cerebral cortical and hippocampal brain slices of 2 and 4 months (mo) old 5xFAD mice. GABA and glutamine are selectively synthesized in neurons and astrocytes, respectively. AAT: aspartate aminotransferase, CTX: cerebral cortex, GAD: glutamate decarboxylase, GDH: glutamate dehydrogenase, GS: glutamine synthetase, HP: hippocampus, PAG: phosphate-activated glutaminase. Mean ± SEM, n = 6–8, Student’s unpaired t-test with Benjamini-Hochberg correction.
Fig. 3 Disrupted glucose metabolism in cerebral cortical and hippocampal brain slices of 5xFAD mice. Cycling ratios, reflecting the rate of the TCA cycle, calculated from metabolism of [U-13C]glucose in acutely isolated cerebral cortical and hippocampal brain slices of 2 and 4 months (mo) old 5xFAD mice. GABA and glutamine are selectively synthesized in neurons and astrocytes, respectively. AAT: aspartate aminotransferase, CTX: cerebral cortex, GAD: glutamate decarboxylase, GDH: glutamate dehydrogenase, GS: glutamine synthetase, HP: hippocampus, PAG: phosphate-activated glutaminase. Mean ± SEM, n = 6–8, Student’s unpaired t-test with Benjamini-Hochberg correction.CC BY · PubMed Central
Fig. 4 Hampered astrocyte metabolism and glutamine synthesis in 5xFAD hippocampal brain slices. Cycling ratios, reflecting the rate of the TCA cycle, calculated from metabolism of [1,2-13C]acetate in acutely isolated cerebral cortical and hippocampal brain slices of 2 and 4 months (mo) old 5xFAD mice. [1,2-13C]Acetate is predominantly metabolized in astrocytes. Astrocytes synthesize glutamine, which is essential for the replenishment of the neuronal GABA pools. AAT: aspartate aminotransferase, CTX: cerebral cortex, GAD: glutamate decarboxylase, GDH: glutamate dehydrogenase, GS: glutamine synthetase, HP: hippocampus, PAG: phosphate-activated glutaminase. Mean ± SEM, n = 7–8, Student’s unpaired t-test with Benjamini-Hochberg correction.
Fig. 4 Hampered astrocyte metabolism and glutamine synthesis in 5xFAD hippocampal brain slices. Cycling ratios, reflecting the rate of the TCA cycle, calculated from metabolism of [1,2-13C]acetate in acutely isolated cerebral cortical and hippocampal brain slices of 2 and 4 months (mo) old 5xFAD mice. [1,2-13C]Acetate is predominantly metabolized in astrocytes. Astrocytes synthesize glutamine, which is essential for the replenishment of the neuronal GABA pools. AAT: aspartate aminotransferase, CTX: cerebral cortex, GAD: glutamate decarboxylase, GDH: glutamate dehydrogenase, GS: glutamine synthetase, HP: hippocampus, PAG: phosphate-activated glutaminase. Mean ± SEM, n = 7–8, Student’s unpaired t-test with Benjamini-Hochberg correction.CC BY · PubMed Central
Fig. 5 Non-synaptic mitochondrial function is maintained in the 5xFAD brain. Analysis of oxygen consumption rate (OCR) of isolated non-synaptic cortical and hippocampal mitochondria of 4 months old 5xFAD mice. A The mitochondria were provided with two different substrate combinations: pyruvate in combination with malate (B, D) or succinate (C, E). The mitochondria were stimulated with ADP, inducing coupled respiration and the mitochondrial uncoupler FCCP leading to maximal uncoupled respiration. Basal refers to non-stimulated OCR. The respiratory control ratio (RCR) is a general measure of mitochondrial function. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 5, Student’s unpaired t-test with Benjamini-Hochberg correction.
Fig. 5 Non-synaptic mitochondrial function is maintained in the 5xFAD brain. Analysis of oxygen consumption rate (OCR) of isolated non-synaptic cortical and hippocampal mitochondria of 4 months old 5xFAD mice. A The mitochondria were provided with two different substrate combinations: pyruvate in combination with malate (B, D) or succinate (C, E). The mitochondria were stimulated with ADP, inducing coupled respiration and the mitochondrial uncoupler FCCP leading to maximal uncoupled respiration. Basal refers to non-stimulated OCR. The respiratory control ratio (RCR) is a general measure of mitochondrial function. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 5, Student’s unpaired t-test with Benjamini-Hochberg correction.CC BY · PubMed Central
Fig. 6 Impaired oxidative and glycolytic capacity of hippocampal synaptosomes of 5xFAD mice. Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated cortical and hippocampal synaptosomes of 4 months old 5xFAD mice. A The synaptosomes were provided with two different substrate combinations: glucose (B, D) or glucose in combination with pyruvate (C, E). The synaptosomes were stimulated with veratridine, a neurotoxin inhibiting closure of voltage-gated sodium channels leading to depolarization, and with the mitochondrial uncoupler FCCP inducing maximal uncoupled respiration. Basal refers to non-stimulated OCR and ECAR. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 7–8, Student’s unpaired t-test with Benjamini-Hochberg correction.
Fig. 6 Impaired oxidative and glycolytic capacity of hippocampal synaptosomes of 5xFAD mice. Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of isolated cortical and hippocampal synaptosomes of 4 months old 5xFAD mice. A The synaptosomes were provided with two different substrate combinations: glucose (B, D) or glucose in combination with pyruvate (C, E). The synaptosomes were stimulated with veratridine, a neurotoxin inhibiting closure of voltage-gated sodium channels leading to depolarization, and with the mitochondrial uncoupler FCCP inducing maximal uncoupled respiration. Basal refers to non-stimulated OCR and ECAR. CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 7–8, Student’s unpaired t-test with Benjamini-Hochberg correction.CC BY · PubMed Central
Fig. 7 Synaptic and mitochondrial changes in the 5xFAD brain. Ultrastructural analyses by transmission electron microscopy (TEM) evaluating mitochondria, synapses, and synaptic vesicles in fixated cerebral cortical and hippocampal tissues of 4 months old 5xFAD mice. A Number of synapses relative to cytoplasm. B Number of vesicles per synapse. C Representative images of hippocampal synapses of control and 5xFAD. Scale bars: 2 µm. D Representative images of mitochondrial abundance and structure in cerebral cortex and hippocampus of control and 5xFAD mice. Scale bars: 2 µm. E Relative mitochondrial size. F, G Analysis of cytosolic (F) and synaptic (G) mitochondria. Both number of total and number of cristaeless mitochondria were assessed relative to cytoplasm (F) or synapses (G). CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 4–5, Student’s unpaired t-test with Benjamini-Hochberg correction.
Fig. 7 Synaptic and mitochondrial changes in the 5xFAD brain. Ultrastructural analyses by transmission electron microscopy (TEM) evaluating mitochondria, synapses, and synaptic vesicles in fixated cerebral cortical and hippocampal tissues of 4 months old 5xFAD mice. A Number of synapses relative to cytoplasm. B Number of vesicles per synapse. C Representative images of hippocampal synapses of control and 5xFAD. Scale bars: 2 µm. D Representative images of mitochondrial abundance and structure in cerebral cortex and hippocampus of control and 5xFAD mice. Scale bars: 2 µm. E Relative mitochondrial size. F, G Analysis of cytosolic (F) and synaptic (G) mitochondria. Both number of total and number of cristaeless mitochondria were assessed relative to cytoplasm (F) or synapses (G). CTX: cerebral cortex, HP: hippocampus. Mean ± SEM, n = 4–5, Student’s unpaired t-test with Benjamini-Hochberg correction.CC BY · PubMed Central

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Authors
Andersen, J. V., Skotte, N. H., Christensen, S. K., Polli, F. S., Shabani, M., Markussen, K. H., Haukedal, H., Westi, E. W., Diaz-delCastillo, M., Sun, R. C., Kohlmeier, K. A., Schousboe, A., Gentry, M. S., Tanila, H., Freude, K. K., Aldana, B. I., Mann, M., & Waagepetersen, H. S.
Affiliations
  • University of Copenhagen (DK)
  • Scripps Research Institute (US)
  • Kerman University of Medical Sciences (IR)
  • University of Kentucky (US)
  • University of Eastern Finland (FI)
Corresponding author
  • Andersen, Jens V.
  • Waagepetersen, Helle S.
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2021-10-16
Volume
12
Issue
11
Pages
954-954
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/s41419-021-04237-y
PMC
PMC8520528
OpenAlex
W3206998885
Cited by
104
References
82
Citations by year
2026: 15 · 2025: 28 · 2024: 21 · 2023: 30 · 2022: 10
FWCI
11.15
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • SSRN Electronic Journal (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • University of Southern Denmark Research Portal (University of Southern Denmark) (acceptedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Hørslev-Fonden (203866)
  • Lundbeckfonden (R333‐2019‐1244, NNF14CC0001, R211-2015-3062, R338-2019-2997, R208‐2015‐2859)
  • Novo Nordisk (NNF14CC0001, NNF15CC0001)
  • Augustinus Fonden (18-1624)
  • Novo Nordisk Fonden (NNF 14CC0001, NNF18OC0052369, NNF15CC0001)
  • Grosserer L. F. Foghts Fond
  • H. Lundbeck A/S
  • National Institutes of Health
  • National Institute on Aging (R01AG066653)
  • National Institute of Neurological Disorders and Stroke (R35NS116824)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
Alzheimer's disease, 5xFAD mice, amyloid-beta deposition, hippocampus, astrocytes, neuronal metabolism, TCA cycle, glutamine synthesis, GABA synthesis, glucose oxidation, mitochondrial dysfunction, glycolysis, synaptic dysfunction, neuronal hyperexcitability, cerebral cortex, brain energy metabolism, 13C isotope tracing
MeSH terms
Alzheimer Disease, Amyloid, Animals, Astrocytes, Cerebral Cortex, Citric Acid Cycle, Disease Models, Animal, Energy Metabolism, Glucose, Glutamine, Glycolysis, Hippocampus, Male, Mice, Transgenic, Mitochondria, Synapses, Signal Transduction, Neurotransmitter Agents, Proteome, Metabolome

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