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, 290 (20), 12504-13

Bioenergetic Mechanisms in Astrocytes May Contribute to Amyloid Plaque Deposition and Toxicity

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Bioenergetic Mechanisms in Astrocytes May Contribute to Amyloid Plaque Deposition and Toxicity

Wen Fu et al. J Biol Chem.

Abstract

Alzheimer disease (AD) is characterized neuropathologically by synaptic disruption, neuronal loss, and deposition of amyloid β (Aβ) protein in brain structures that are critical for memory and cognition. There is increasing appreciation, however, that astrocytes, which are the major non-neuronal glial cells, may play an important role in AD pathogenesis. Unlike neurons, astrocytes are resistant to Aβ cytotoxicity, which may, in part, be related to their greater reliance on glycolytic metabolism. Here we show that, in cultures of human fetal astrocytes, pharmacological inhibition or molecular down-regulation of a main enzymatic regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3), results in increased accumulation of Aβ within and around astrocytes and greater vulnerability of these cells to Aβ toxicity. We further investigated age-dependent changes in PFKFB3 and astrocytes in AD transgenic mice (TgCRND8) that overexpress human Aβ. Using a combination of Western blotting and immunohistochemistry, we identified an increase in glial fibrillary acidic protein expression in astrocytes that paralleled the escalation of the Aβ plaque burden in TgCRND8 mice in an age-dependent manner. Furthermore, PFKFB3 expression also demonstrated an increase in these mice, although at a later age (9 months) than GFAP and Aβ. Immunohistochemical staining showed significant reactive astrogliosis surrounding Aβ plaques with increased PFKFB3 activity in 12-month-old TgCRND8 mice, an age when AD pathology and behavioral deficits are fully manifested. These studies shed light on the unique bioenergetic mechanisms within astrocytes that may contribute to the development of AD pathology.

Keywords: Alzheimer disease; amyloid precursor protein (APP); amyloid β (Aβ); astrocyte; glial cell; glycolysis; phosphofructokinase; reactive astrogliosis.

Figures

FIGURE 1.
FIGURE 1.
Uptake, accumulation, and amyloid plaque formation in HFAs exposed to Aβ. a, immunofluorescent staining of human fetal astrocytes cultured with fluorescent Aβ1–42 (1 μm, 24 h) and stained with Aβ (6E10) and GFAP antibody showing that Aβ1–42 is taken up into astrocytes and forms plaques that build up on the reactive astrocyte surface. b, in-cell-Western blot stained with Aβ (6E10) showing increased Aβ plaque formation followed by increasing extracellular Aβ1–42 concentration (Conc.). The Western blot shows that human fetal astrocytes exposed to Aβ1–42 become reactive, as characterized by increased GFAP protein expression (c, 1 μm, 24 h, n = 3).
FIGURE 2.
FIGURE 2.
Glycolytic activity affects Aβ uptake and plaque formation in human astrocytes. a, Western blot analysis showing significantly different basal levels of the glycolytic enzyme PFKFB3 in HFNs and HFAs (n = 6). b, HFAs increase PFKFB3 protein expression after exposure to Aβ1–42 (1 μm) or human amylin (hAmylin, another amyloidogenic peptide, 1 μm) for 24 h. c, HFAs become “reactive”; that is, characterized by increased GFAP protein expression after metabolic stress, such as inhibition of glycolysis with the PFKFB3 inhibitor PFK15 (20 μm, 24 h, n = 3). d, representative photographs showing that HFA cultures become reactive and demonstrate morphological changes and an increase in GFAP staining after exposure to Aβ or an impairment in glucose metabolism with application of the glycolytic inhibitors 3PO (20 μm) or PFK15 (10 μm), which further increased Aβ buildup and plaque formation (e, f, and g). The results shown are from five independent experiments. h, the glycolytic metabolic stress not only increases Aβ buildup but also results in the formation of larger Aβ plaques. *, p < 0.05; **, p < 0.01. Scale bars = 20 μm.
FIGURE 3.
FIGURE 3.
Aβ plaque formation in human fetal astrocytes is correlated with PFKFB3 activity. a, in-cell Western blot stained with Aβ (6E10) showing that deteriorated astrocyte metabolism enhances Aβ plaque formation, followed by glycolysis inhibition with the PFKFB3 inhibitors 3PO (20 μm) or PFK15 (10 μm). Conc, concentration. b and c, Aβ plaque formation increases with increasing concentrations of glycolysis inhibitor 3PO (b) or PFK15 (c) (Aβ1–42 concentration, 1 μm). d, using confocal microscopy quantification, Aβ fluorescence increased significantly after glycolysis inhibition with 3PO (20 μm, n = 9). *, p < 0.05.
FIGURE 4.
FIGURE 4.
PFKFB3 activity affects Aβ cytotoxicity in human fetal astrocytes. a, in-cell analysis imaging the live/dead assay with calcein (green, live cells) and ethidium homodimer I (red, dead cells) with human fetal astrocytes cultured with 3PO (20 μm) and Aβ1–42 (1 μm) for 48 h. b, using the live/dead assay and quantifying with InCell analyzer, resting human astrocytes are not sensitive to Aβ cytotoxicity. However, astrocytes had increased sensitivity to Aβ cytotoxicity after PFKFB3 activity was inhibited with 3PO. c, using an MTT proliferation assay, increased Aβ induced astrocyte cell death after metabolic stress when PFKFB3 activity was inhibited with PFK15 or 3PO. d, another proliferation assay (WST-1) showed similar increased Aβ toxicity following glycolysis inhibition with 3PO (20 μm, 24 h) but not the reverse sequence peptide Aβ42–1. e and f, the increased Aβ cytotoxicity after PFKFB3 inhibition is PFKFB3-specific because it is down-regulated with PFKFB3 siRNA transfection in these cells. *, p < 0.05.
FIGURE 5.
FIGURE 5.
PFKFB3 activity is important for Aβ cytotoxicity in a mixed culture of human fetal neurons and astrocytes. a, in cocultured HFNs and HFAs, HFAs maintain a homeostatic environment and keep HFNs healthy. b and c, after b and c) and have decreased capability to maintain the microenvironment, resulting in a gradual loss of HFNs. d, when these stressed HFAs are exposed to Aβ, they become more sensitive to Aβ cytotoxicity, resulting in a further loss of their capability to support neurons and, hence, increased neuronal cell death (3PO, 20 μm; PFK15, 10 μm; Aβ1–42, 1 μm; 24 h). *, p < 0.05; **, p < 0.01. Scale bar = 20 μm).
FIGURE 6.
FIGURE 6.
Temporal changes in Aβ, GFAP, and PFKFB3 expression in AD transgenic mice (TgCRND8). a, Western blot analyses for GFAP, PFKFB3, and Aβ protein expression from the cortex of transgenic AD mouse brain (TgCRND8) compared with the age-matched wild type (n = 4 for each group). Quantitative changes in GFAP, PFKFB3, and Aβ protein expression in wild-type and TgCRND8 mice over time (in months) are depicted in the left panels. b, immunofluorescence staining of 12-month-old AD mouse brain with Aβ (6E10) and GFAP antibodies showing Aβ plaque surrounded closely by enlarged and stronger GFAP-stained astrocytes (right panels), whereas the age-matched wild-type mouse brain shows no significant plaque formation and reactive astrogliosis (left panels). c, in AD mouse brain (right panels), increased phospho-PFKFB3 (green) surrounds and is colocalized with Aβ plaques (red), shown as yellow, whereas the age-matched wild-type mouse brain shows weak PFKFB3 activity (n = 4 for each group). *, p < 0.05; **, p < 0.01.
FIGURE 7.
FIGURE 7.
Delayed glycolysis inhibition further enhances plaque formation in HFAs. a, HFAs cultured with 200 nm Fluor-Aβ1–42 for 12 h forms Aβ plaques. b and c, with continued application of Fluor-Aβ1–42, delayed inhibition of glycolysis with the PFKFB3 inhibitor PFK15 (1 or 5 μm) for another 24 h results in accelerated and increased amyloid plaque formation. The increase in the plaque-covered area (d) and plaque size and distribution (e and f) appear to be correlated with the concentration of PFK15 that is applied. Results from three separate experiments with different batches of cultured HFAs were analyzed. *, p < 0.05 compared with the Aβ control group. Scale bar = 100 μm.
FIGURE 8.
FIGURE 8.
Astrocytic bioenergetic mechanisms contribute to amyloid accumulation and neuronal death. The schematic depicts the transformation of astrocytes to a reactive state when exposed to Aβ protein, a process that is augmented by loss of glycolytic function (PFKFB3 inhibition), accelerated aging, or cellular stress. The homeostatic disturbance that ensues in reactive astrocytes (identified by an increase in GFAP) results in a loss of neuronal metabolic support and an inability to clear Aβ protein. These changes lead to accumulation of Aβ and adversely impact neuronal function, resulting in neurodegeneration.

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