Astrocytic gap junctional communication is reduced in amyloid-β-treated cultured astrocytes, but not in Alzheimer's disease transgenic mice

Abstract

Alzheimer's disease is characterized by accumulation of amyloid deposits in brain, progressive cognitive deficits and reduced glucose utilization. Many consequences of the disease are attributed to neuronal dysfunction, but roles of astrocytes in its pathogenesis are not well understood. Astrocytes are extensively coupled via gap junctions, and abnormal trafficking of metabolites and signalling molecules within astrocytic syncytia could alter functional interactions among cells comprising the neurovascular unit. To evaluate the influence of amyloid-beta on astrocyte gap junctional communication, cultured astrocytes were treated with monomerized amyloid-β(1-40) (1 μmol/l) for intervals ranging from 2 h to 5 days, and the areas labelled by test compounds were determined by impaling a single astrocyte with a micropipette and diffusion of material into coupled cells. Amyloid-β-treated astrocytes had rapid, sustained 50-70% reductions in the area labelled by Lucifer Yellow, anionic Alexa Fluor® dyes and energy-related compounds, 6-NBDG (a fluorescent glucose analogue), NADH and NADPH. Amyloid-β treatment also caused a transient increase in oxidative stress. In striking contrast with these results, spreading of Lucifer Yellow within astrocytic networks in brain slices from three regions of 8.5-14-month-old control and transgenic Alzheimer's model mice was variable, labelling 10-2000 cells; there were no statistically significant differences in the number of dye-labelled cells among the groups or with age. Thus amyloid-induced dysfunction of gap junctional communication in cultured astrocytes does not reflect the maintenance of dye transfer through astrocytic syncytial networks in transgenic mice; the pathophysiology of Alzheimer's disease is not appropriately represented by the cell culture system.

Keywords: 6-NBDG, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose; A350, Alexa Fluor® 350; A568, Alexa Fluor® 568; APP, amyloid-β precursor protein; Cx, connexin; DCF, dichlorofluorescein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; H2DCF-DA, carboxydihydrodichlorofluorescein diacetate; L-LME, L-leucine methyl ester hydrochloride; ROS, reactive oxygen species; SR101, sulforhodamine 101; STZ, streptozotocin; aCSF, artificial cerebral spinal fluid; amyloid protein; astrocyte; connexin; dBcAMP, dibutyryl cAMP; dye transfer; gap junction; metabolite trafficking.

Figure 1. Representative images of reduced Lucifer Yellow spread among astrocytes after brief exposure to amyloid-β

Cultured astrocytes grown in (A) low (5.5 mmol/l) or (B) high (25 mmol/l) glucose-containing medium for 2–3 weeks. Cells were visualized and impaled with a micropipette (a and d), and Lucifer Yellow was diffused for 2 min into a single astrocyte in a culture treated with vehicle (b and e) or with 1 μmol/l of amyloid-β_1–40 for 2 h prior to analysis (c and f). Scale bars indicated apply to all panels.

Figure 2. Amyloid-β treatment reduces gap junctional spreading of Lucifer Yellow in low- and high-glucose astrocyte cultures

Cultured astrocytes grown in low (A) or high (B) glucose were treated with 1 μmol/l monomerized amyloid-β_1–40 or vehicle for up to 5 days prior to assay of the Lucifer Yellow-labelled area (see Figure 1). The number of injected cells for the control and amyloid-β-treated groups respectively, at each time point are as follows: (A) 2 h, n = 6, 5; 5 days, n = 5, 6; (B) 2 h, n = 9, 9; 1 day, n = 10, 11; 2 days, n = 14, 14.

Figure 3. DCF fluorescence in amyloid-β-treated astrocytes

Cultured astrocytes grown in low-glucose medium for 2–3 weeks were treated with 1 μmol/l monomerized amyloid-β_1–40 or vehicle for up to 5 days. The number of injected cells for the control and amyloid-treated groups respectively at each time point are as follows: 2 h, n = 16, 16; 1 day, n = 11, 12; 5 days, n = 10, 10.

Figure 4. Representative images of impaired Alexa Fluor® dye spread among astrocytes after brief exposure to amyloid-β

Cultured astrocytes grown in low-glucose medium for 2–3 weeks were treated with vehicle (a and c) or 1 μmol/l monomerized amyloid-β_1–40 for 48 h (b and d) prior to assay of dye spreading after diffusion of tracer into a single cell. Scale bars shown apply to all panels.

Figure 5. Amyloid-β impairs trafficking of large and small Alexa Fluor® dyes

Cultured astrocytes grown in low-glucose medium for 2–3 weeks were treated with 1 μmol/l monomerized amyloid-β_1–40 or vehicle for up to 2 days prior to assay of the labelled area by (A) A568 or (B) A350 that was diffused for 2 min into a single cell. The number of injected cells for the control and amyloid-treated groups respectively, at each time point are as follows: (A) 2 h, n = 37, 41; 1 day, n = 61, 61; 2 days, 31, 48; (B) 2 h, n = 13, 16; 2 days, n = 16, 19.

Figure 6. Amyloid-β reduces syncytial distribution of energy-related molecules

Cultured astrocytes grown in low-glucose medium for 2–3 weeks were treated with 1 μmol/l monomerized amyloid-β_1–40 or vehicle for up to 2 days prior to assay of the labelled area by (A) 6-NBDG, a fluorescent glucose analogue (molecular mass, 342 Da) or (B) NADH (molecular mass, 663 Da) which was diffused into a single astrocyte. The number of injected cells for the control and amyloid-treated groups respectively, at each time point are as follows: (A) 1 day, n = 12, 16; 2 days, n = 16, 19; (B) 2 h, n = 37, 41; 1 day, 61, 61; 2 days, n = 30, 48.

Figure 7. Amyloid deposits in parenchyma and perivascular space of Alzheimer's transgenic mice

Brain sections from anterior dorsal hippocampus (A), anterior cerebral cortex (B) and inferior colliculus (C) from each of the aged control and transgenic mice were stained with haematoxylin and Congo Red after dye-labelled cells were counted. Control mice (a, d and g) had no amyloid plaques, whereas plaques (vertical arrows: b, c, d and f) were present in hippocampus and cerebral cortex of all transgenic mice. Perivascular amyloid deposits (horizontal arrows) are visible in cross-sectional views of blood vessels (b, e and h). The horizontal arrow in (c) indicates an artefact owing to folded tissue. Two representative images from each brain region from different transgenic mice are illustrated for each brain region. Panel (e) is close to the midline of the brain and perivascular labelling is in the meninges between the two hemispheres; in hippocampus panels (a) and (c), ‘p’ denotes the pyramidal neuronal layer. The scale bar shown applies to all panels.

Figure 8. Representative images of Lucifer Yellow diffusion among astrocytes in slices from three brain regions of aged Alzheimer's transgenic mice

Brain slices (250 μm thick) were prepared from hippocampus (a–b), cerebral cortex (e) and inferior colliculus (f) of aged Alzheimer's transgenic mice. Astrocytes were identified as sulforhodamine-positive cells (a), then impaled with a micropipette (b) containing Lucifer Yellow that was allowed to diffuse into the astrocyte for 5 min (c). Then the sections were immediately fixed, cut into 7 μm-thick serial sections and dye-labelled cells counted in each section from each slice. Fluorescence images of typical sections are shown for each brain region to illustrate the different patterns of dye-labelled cells (bright spots) derived from impaling an astrocyte near the pyramidal neuronal layer in the hippocampus (d), or an astrocyte in cerebral cortex (e) and inferior colliculus (f). Arrowheads in (a), (c) and (d) denote dark spaces corresponding to unlabelled neuronal cell bodies. The scale bar in (a) also applies to (c).

Figure 9. Dye transfer in brain regions in slices from aged control and Alzheimer's mice

Total number of Lucifer-Yellow-labelled cells in three brain regions (A, B and C) from control and Alzheimer's transgenic mice ranging in age from 8.5 to 14 months. Each point represent one brain slice; 1–3 slices were obtained from each region of six control and six transgenic mice. The number of slices for inferior colliculus, cerebral cortex and hippocampus respectively, were: controls, 10, 5, 6; transgenic, 10, 5, 5. Note that two of the control cortical slices had similar numbers of labelled cells and the values in the Figure overlap. There were no statistically significant differences between the control and experimental groups in each region.

Figure 10. Dye transfer as function of age in control and Alzheimer's mice

Values from all structures and all slices in Figure 9 are plotted as a function of age of the animal.