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GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration

Nature Neuroscience volume 18, pages 521–530 (2015)Cite this article

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Abstract

The glucose transporter GLUT1 at the blood-brain barrier (BBB) mediates glucose transport into the brain. Alzheimer's disease is characterized by early reductions in glucose transport associated with diminished GLUT1 expression at the BBB. Whether GLUT1 reduction influences disease pathogenesis remains, however, elusive. Here we show that GLUT1 deficiency in mice overexpressing amyloid β-peptide (Aβ) precursor protein leads to early cerebral microvascular degeneration, blood flow reductions and dysregulation and BBB breakdown, and to accelerated amyloid β-peptide (Aβ) pathology, reduced Aβ clearance, diminished neuronal activity, behavioral deficits, and progressive neuronal loss and neurodegeneration that develop after initial cerebrovascular degenerative changes. We also show that GLUT1 deficiency in endothelium, but not in astrocytes, initiates the vascular phenotype as shown by BBB breakdown. Thus, reduced BBB GLUT1 expression worsens Alzheimer's disease cerebrovascular degeneration, neuropathology and cognitive function, suggesting that GLUT1 may represent a therapeutic target for Alzheimer's disease vasculo-neuronal dysfunction and degeneration.

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Figure 1: Microvascular and cerebral blood flow reductions and diminished glucose uptake in GLUT1-deficient APPSw/0 mice.
Figure 2: GLUT1 deficiency leads to early BBB breakdown in Slc2a1+/− and Slc2a1+/−APPSw/0 mice.
Figure 3: Aβ pathology in GLUT1-deficient APPSw/0 mice and reversal by LRP1 and GLUT1 re-expression.
Figure 4: Neuronal dysfunction in GLUT1-deficient APPSw/0 mice.
Figure 5: Accelerated cognitive impairment in GLUT1-deficient APPSw/0 mice.
Figure 6: Accelerated neurodegenerative changes in GLUT1-deficient APPSw/0 mice.
Figure 7: GLUT1 deficiency in endothelial cells (Slc2a1loxP/+Tie2-Cre+/0 mice), but not astrocytes (Slc2a1loxP/+GFAP-Cre+/0 mice) leads to early BBB breakdown.

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Acknowledgements

We thank E. Schauwecker for most helpful discussion. This research was supported by US National Institutes of Health grants AG039452, AG023084 and NS034467 to B.V.Z. and U01 HL087947 and R01DK092065 to E.D.A.

Author information

Author notes

  1. Ethan A Winkler, Yoichiro Nishida and Abhay P Sagare: These authors contributed equally to this work.

Authors and Affiliations

  1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Ethan A Winkler, Abhay P Sagare, Sanket V Rege, Jesse D Sengillo, Pan Kong, Amy R Nelson, John S Sullivan, Zhen Zhao, Jacob Makshanoff, Edward Zuniga & Berislav V Zlokovic

  2. Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA

    Ethan A Winkler

  3. Center for Neurodegenerative and Vascular Brain Disorders, University of Rochester School of Medicine & Dentistry, Rochester, New York, USA

    Yoichiro Nishida, Robert D Bell, David Perlmutter, Jesse D Sengillo & Sara Hillman

  4. Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

    Yoichiro Nishida

  5. Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Herbert J Meiselman, Rosalinda B Wenby & Berislav V Zlokovic

  6. Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, Iowa, USA

    Jamie Soto & E Dale Abel

  7. Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Jamie Soto & E Dale Abel

  8. Colleen Giblin Laboratories for Pediatric Neurology Research, Columbia University, New York, New York, USA

    Darryl C De Vivo

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  1. Ethan A Winkler
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Contributions

E.A.W. contributed to manuscript preparation, experimental design and analysis, and conducted experiments. Y.N. and A.P.S. contributed to experimental design, data analysis and interpretation, and conducted experiments. S.V.R., R.D.B., D.P., J.D.S., S.H., J.S.S., P.K., A.R.N., R.B.W., J.M., E.Z. and Z.Z. conducted and analyzed experiments. H.J.M. contributed to hematological analysis and data interpretation. E.D.A. provided Slc2a1loxP/loxP mice and contributed to project design. J.S. generated Slc2a1loxP/+Tie2-Cre mice. D.C.D.V. provided Slc2a1+/− mice and contributed to project design. B.V.Z. supervised and designed all experiments and analysis, and wrote the manuscript.

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Correspondence to Berislav V Zlokovic.

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Integrated supplementary information

Supplementary Figure 1 Slc2a1 haploinsufficiency leads to reductions in GLUT1 transporter and hypoglycorrhachia.

(a) Representative confocal microscopy analysis showing GLUT1 immunodetection (red) and lectin-positive capillaries (green) in 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Colocalization, yellow. Scale bar, 100 µm. (b) Quantification of GLUT1-positive immunofluorescent signal normalized to vascular density in cortex and hippocampus in 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01. (c) Representative immunoblotting of GLUT1 protein levels in isolated cerebral microvessels from 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. (d) Quantification of GLUT1 protein expression in isolated cerebral microvessels from 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01. (e) Quantification of fasting glucose values in blood plasma and cerebrospinal fluid (CSF) from 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01. (f) Quantification of CSF-to-blood plasma ratios in 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01.

Supplementary Figure 2 Blood-brain barrier GLUT1 reductions do not alter systemic hemodynamic or physiologic parameters in Slc2a1+/–, Slc2a1+/+APPSw/0 and Slc2a1+/–APPSw/0 mice.

(a-f) Mean arterial blood pressure (a), respiratory rate (b), heart rate (c), body temperature (d), hematocrit (e) and bodyweight (f) in 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01. (g-i) Arterial blood gas values showing arterial pH (g), PCO2 (h), and PO2 (i) in 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01.

Supplementary Figure 3 GLUT1 reductions lead to progressive breakdown of the blood-brain barrier (BBB) with aging.

(a) Representative immunoblotting of extravascular IgG in brain tissue of 6-month-old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. β-actin was used as loading control. (b) Quantification of relative protein abundance of extravascular IgG (c) Representative confocal microscopy analysis of plasma-derived IgG (red) and lectin-positive capillaries (white) in 6-month-old Slc2a1+/+,Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Scale bar, 20 µm. Mean ± SEM, n=3-5 mice per group; *p<0.05 or **p<0.01.

Supplementary Figure 4 GLUT1 reductions in Slc2a1+/–APPSw/0 mice do not lead to enhanced APP production and/or processing or reduced Aβ enzymatic degradation.

(a) Represenative immunoblotting of human amyloid precursor protein (APP) and β-secretase (BACE1) in 16-month old Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as loading control. (b-c) Relative protein abundance of human APP (b) and BACE1 (c) using densitometry analysis from immunoblots in 16-month old Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; ns, non-significant. (d-g) Represenative immunoblotting of Aβ degradative enzymes neprilysin (d) and insulin degrading enzyme (IDE) (f) in 16-month old Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as loading control. Relative protein abundance of neprilysin (e) and IDE (g) using densitometry analysis from immunoblots in 16-month old Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-5 mice per group; ns, non-significant.

Supplementary Figure 5 Re-expression of LRP1 and GLUT1 in the hippocampus of Slc2a1+/–APPSw/0 mice using adenovirus-mediated transfer with Ad.mLRP1 and Ad.Slc2a1.

(a) Representative immunoblotting analysis of LRP-515 and mLRP1 levels in 10-month old Slc2a1+/−APPSw/0 mouse hippocampus injected with Ad.Ctrl and Ad.mLRP1. (b) Representative confocal microscopy analysis of LRP1 (green) and lectin-positive (red) microvessels in 10-month old Slc2a1+/−APPSw/0 mouse hippocampus injected with Ad.Ctrl and Ad.mLRP1. Co-localization (yellow) denotes vascular LRP1. Scale bar, 20 µm. (c) Quantification of microvascular LRP1-positive area in 10-month old Slc2a1+/−APPSw/0 mice after hippocampal injection with Ad.Ctrl and Ad.mLRP1. Mean ± SEM, n=3 mice; *p<0.05. (d) Representative confocal microscopy analysis of GLUT1 (green) and lectin-positive (red) microvessels in 10-month old Slc2a1+/−APPSw/0 mouse hippocampus injected with Ad.Ctrl and Ad.Slc2a1. Co-localization (yellow) denotes vascular GLUT1. Scale bar, 20 µm. (e) Quantification of microvascular GLUT1-positive microvessels in 10-month old Slc2a1+/−APPSw/0 mice after hippocampal injection with Ad.Ctrl and Ad.Slc2a1. Mean ± SEM, n=3 mice; **p<0.01. (f) Representative confocal microscopy analysis of LRP1 (green) and lectin-positive (red) microvessels in 10-month old Slc2a1+/−APPSw/0 mouse hippocampus injected with Ad.Ctrl and Ad.Slc2a1. Co-localization (yellow) denotes vascular LRP1. Scale bar, 20 µm. (g) Quantification of microvascular LRP1-positive microvessels in 10-month old Slc2a1+/−APPSw/0 mice after hippocampal injection with Ad.Ctrl and Ad.Slc2a1. Mean ± SEM, n=3 mice; *p<0.05.

Supplementary Figure 6 GLUT1, LRP1 and SREBP2 protein levels in brain endothelial cells isolated from Slc2a1+/+ and Slc2a1+/– mice.

(a) Representative immunoblotting (upper panel) and LRP1 (LRP-85) and GLUT1 protein abundance relative to β-actin determined by scanning densitometry (lower panel) in Slc2a1+/+ brain endothelial cells (BEC) transfected with siControl or Slc2a1 siRNA (siSlc2a1). (b) Real-time quantitative PCR (qPCR) for Lrp1 mRNA relative to GAPDH in Slc2a1+/+ BEC transfected with siControl or siSlc2a1. (c) Representative immunoblotting (upper panel) and quantification of LRP1 and GLUT1 protein abundance relative to β-actin (lower panel) in Slc2a1+/− BEC after transduction with either Ad.Ctrl or Ad.Slc2a1 to re-express GLUT1. (d) Representative immunoblotting (upper panel) and quantification of LRP1 and GLUT1 protein abundance relative to β-actin (lower panel) in Slc2a1+/+ BEC transfected with siControl or Lrp1 siRNA (siLRP1). (e) Representative immunoblotting for GLUT1, SREBP2 and LRP1 (LRP-85) in Slc2a1+/+ BEC transfected with siControl, siSlc2a1 or Srebp2 siRNA (siSrebp2). (f-h) Quantification of GLUT1 (f) SREBP2 (g) and LRP1 (h) protein abundance relative to β-actin from experiments as shown in e. (i-k) Representative immunoblotting (upper panels) and quantification (lower panels) of GLUT1 (i), SREBP2 (j) and LRP1 (k) protein abundance relative to β-actin in Slc2a1+/+ and Slc2a1+/− BEC. Mean ± SEM, n=3 independent cultures per group; *p<0.05, **p<0.01.

Supplementary Figure 7 GLUT1 isoforms in brain microvessels, capillary-depleted brain homogenates, brain endothelial cells and astrocytes in Slc2a1+/+ and Slc2a1+/– mice.

Representative immunoblotting of GLUT1 endothelial 55 kDa isoform in brain microvessels and GLUT1 astrocytic 45 kDa isoform in capillary-depleted brain homogenates from Slc2a1+/+ control mice determined at (a) short 15 s and (b) longer 3 min exposure times (left panel) with quantification relative to β-actin (right panel). Of note, the levels of 45 kDa isoform in capillary-depleted brain homogenates are low and undetectable over short exposure times as shown in a vs. b. (c) Representative immunoblotting of CD31 (endothelial marker), Tuj-1 (neuronal marker) and GFAP (astrocytic marker) in brain microvessels and capillary-depleted brains from Slc2a1+/+ mice. (d-e) Representative immunoblotting (upper panel) and quantification of GLUT1 levels (lower panel) in brain microvessels (d) and capillary-depleted brains (e) from Slc2a1+/+ and Slc2a1+/− mice. (f-g) Representative immunoblotting (upper panel) and quantification of GLUT2 (f) and GLUT3 (g) protein abundance relative to β-actin (lower panel) in capillary-depleted brains from Slc2a1+/+ and Slc2a1+/− mice. Notably, GLUT2 and GLUT3 were not found in brain microvessels (not shown), as reported1,3,52. (h) Representative immunoblotting (upper panel) and quantification of GLUT1 55 kDa and 45 kDa isoforms abundance (lower panel) relative to β-actin in brain endothelial cells (BEC) and astrocytes isolated from Slc2a1+/+ mice. (i) Representative immunoblotting (upper panel) and quantification of GLUT1 55 kDa protein abundance (lower panel) relative to β-actin in BEC from Slc2a1+/+ and Slc2a1+/− mice. (j) Representative immunoblotting (upper panel) and quantification of GLUT1 45 kDa protein abundance and GLUT2 protein abundance (lower panel) relative to β-actin in astrocytes from Slc2a1+/+ and Slc2a1+/− mice. GLUT2 was not present in BEC (not shown), as reported1,3,52. Mean ± SEM, n=3-4 mice per group (b, d-g) and 3 independent cultures per group (h-j); **p<0.01.

Supplementary Figure 8 GLUT1 expression in Slc2a1loxP/+;Tie2-Cre+/0 mice and Slc2a1loxP/+;GFAP-Cre+/0 mice with specific GLUT1 partial deletions from endothelium and astrocytes, respectively.

(a) Representative confocal microscopy analysis showing GLUT1 immunodetection (red) and lectin-positive endothelial vascular profiles (green) in 2-week-old Slc2a1lox/+, Slc2a1lox/+Tie2-Cre+/0, and Slc2a1lox/+GFAP-Cre+/0 mice. Colocalization (yellow) denotes endothelial GLUT1. Scale bar, 25 µm. (b) Quantification of GLUT1-positive immunofluorescent endothelial signal expressed as GLUT1-positive area (percentage) occupying lectin-positive endothelial capillary profiles in the cortex and hippocampus in 2-week-old Slc2a1lox/+, Slc2a1lox/+Tie2-Cre+/0, and Slc2a1lox/+GFAP-Cre+/0 mice. Mean ± SEM, n=4-5 mice per group; **p<0.01. (c-d) Representative immunoblotting of GLUT1 protein abundance relative to β-actin in brain microvessels after 15 s short exposure (c) and capillary-depleted brain homogenates after 3 min longer exposure (d) in 2-week-old Slc2a1lox/+, Slc2a1lox/+Tie2-Cre+/0, and Slc2a1lox/+GFAP-Cre+/0 mice. Mean ± SEM, n=3-4 mice per group; *p<0.05.

Supplementary Figure 9 Slc2a1 haplosufficiency does not affect red blood cell (RBC) indices and hemoglobin (Hb) oxygen saturation or dissociation in Slc2a1+/–, Slc2a1+/+APPSw/0 and Slc2a1+/–APPSw/0 mice.

(a) Quantification of Glut1 membrane protein levels in circulating RBCs from 2 month old Slc2a1+/− mice. (b) Representative bright field microscopy showing RBC morphology in 2 month old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. (c) Graph showing RBC deformability profiles, a measure of mechanical properties, in 2 month old Slc2a1+/+ (green), Slc2a1+/− (blue), Slc2a1+/+APPSw/0 (red) and Slc2a1+/−APPSw/0 (purple) mice. (d-j) Quantification of RBC hemoglobin (d), mean corpuscular volume (e), mean corpuscular hemoglobin (MCH) (f), mean corpuscular hemoglobin concentration (MCHC) (g), red cell distribution width (RDW) (h), mean arterial oxygen saturation (SaO2) (i), and mean P50 value of oxygen dissociation (j), in 2 month old Slc2a1+/+, Slc2a1+/−, Slc2a1+/+APPSw/0 and Slc2a1+/−APPSw/0 mice. Mean ± SEM, n=3-4 mice per group. ns, nonsignificant.

Supplementary Figure 10 GLUT1 deficiency leads to early cerebral microvascular degeneration, BBB breakdown, hypoperfusion, metabolic stress and accelerated accumulation of Aβ preceding neuronal functional and degenerative changes.

GLUT1 deficiency in endothelium leads to early cerebral microvascular degeneration (blue), limited BBB glucose uptake into the brain (blue), and disrupted Aβ metabolism (red) resulting in progressive BBB breakdown (blue), perfusion deficit (blue), potentially hypometabolic state (blue) and impaired vascular clearance of Aβ (red) via disruption of LRP1 (red). Ultimately, early microvascular degenerative changes resulting in accumulation of blood-borne toxins through a disrupted BBB, chronic perfusion and metabolic stress during limited glucose availability, as well as accumulation of neurotoxic Aβ species converge at the neuronal interface leading to neuronal dysfunction (blue), neuronal injury and neurodegeneration. Importantly, vascular damage precedes and helps drive neuronal changes as can be seen on the time scale.

Supplementary Figure 11 Full scans of Western blots presented in the main figures.

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Winkler, E., Nishida, Y., Sagare, A. et al. GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration. Nat Neurosci 18, 521–530 (2015). https://doi.org/10.1038/nn.3966

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