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Adult mouse astrocytes degrade amyloid-β in vitro and in situ TONY WYSS-CORAY1,2, JOHN D. LOIKE3, THOMAS C. BRIONNE2, EMILY LU3, ROMAN ANANKOV3, FENGRONG YAN4, SAMUEL C. SILVERSTEIN3 & JENS HUSEMANN3 1
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2
Geriatric Research, Education and Clinical Center, VA Palo Alto Health Care System, Palo Alto, California, USA Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA 3 Department of Physiology and Cellular Biophysics, Columbia University, New York, New York, USA 4 Gladstone Institute of Neurological Disease, San Francisco, California, USA Correspondence should be addressed to J.H.; e-mail:
[email protected] Published online 3 March 2003; doi:10.1038/nm838
Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-β (Aβ) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Aβ deposition1, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions1, and cease migration upon interaction with immobilized Aβ1–42. We also show that astrocytes bind and degrade Aβ1–42. Astrocytes plated on Aβ-laden brain sections from a mouse model of AD associate with the Aβ deposits and reduce overall Aβ levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Aβ deposits, indicate a direct role for astrocytes in degradation of Aβ and implicate deficits in astroglial clearance of Aβ in the pathogenesis of AD. Treatments that increase removal of Aβ by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD. The presence of large numbers of astrocytes associated with Aβ deposits in AD suggests that these lesions generate chemotactic molecules that mediate astrocyte recruitment. In fact, AD lesions contain MCP-1 (ref. 1), a potent chemoattractant for neonatal astrocytes in vitro2, but the cellular source of MCP-1 in the AD brain is unclear. Whereas neonatal astrocytes from various species produce MCP-1 after stimulation with Aβ1–42 in vitro3–5, astrocytes surrounding Aβ plaques in the AD brain and in a mouse model for AD do not express detectable levels of MCP-1 (refs. 6, 7). These findings prompted us to compare Aβ1–42-stimulated release of MCP-1 by astrocytes cultured from neonatal and adult mouse brains. We found that Aβ1–42-stimulated neonatal mouse astrocytes produced significantly (P = 0.05) higher amounts of MCP-1 compared with non-stimulated controls, whereas similarly stimulated adult astrocytes do not (Table 1). However, both neonatal and adult astrocytes significantly (P = 0.0001 and 0.01, respectively) increased MCP-1 release in response to lipopolysaccharide (LPS; Table 1). Because cultured adult mouse astrocytes seem to be refractory to Aβ-induced MCP-1 production, similar to astrocytes surrounding Aβ deposits in AD, we used these cells for most of our experiments. We used cell culture inserts to test the ability of chemoattractants MCP-1 and LPS8 to stimulate migration of adult astrocytes across a porous membrane coated with collagen IV (CIV), an extracellular matrix protein found in Aβ plaques (Fig. 1a). In the NATURE MEDICINE • VOLUME 9 • NUMBER 4 • APRIL 2003
presence of 10–8 M MCP-1, 2-fold more astrocytes migrated across CIV-coated membranes than in the absence of MCP-1. Control experiments with LPS produced similar results (Fig. 1a). Migration of astrocytes in response to either chemoattractant was reduced to non-stimulated levels when the membranes were coated with CIV and Aβ1–42 (Fig. 1a). These studies indicate that astrocytes can be recruited to sites of Aβ deposition by locally released MCP-1 or other chemoattractants, and become immobilized when they contact Aβ in the extracellular matrix. Immobilization of adult astrocytes upon interaction with Aβ indicates that these cells adhere to Aβ-coated surfaces. To examine astrocyte adhesion, we took advantage of the fact that adhesion of astrocytes to CIV-coated surfaces is mediated by integrins9 and is therefore dependent on divalent cations. In the absence of Ca2+ and Mg2+, astrocyte adhesion to CIV-coated multi-spot glass slides was 99%, as determined by immunofluorescence using antibodies specific for glial fibrillary acidic protein (Santa Cruz Biotechnology, Santa Cruz, California) and S100β (Sigma, St. Louis, Missouri). Antibodies specific to CD11b (Serotec, Raleigh, North Carolina) and galactocerebroside (Sigma) were used to identify microglia or oligodendrocytes, respectively. Aβ. Human (Bachem, Torrance, California) and mouse (Anaspec, San Jose, California) Aβ1–42 were dissolved in double-distilled water at 1 mg/ml and incubated at 37 °C (for details, see ref. 22). Human Cy3-Aβ1–42 was prepared as described23. MCP-1 production. Astrocytes in 100 µl DMEM/F12 containing N2 supplements, penicillin and streptomycin were plated into 96-well plates at 5 × 104 cells/well. After 24 h, cultures were incubated in 100 µl fresh medium with or without 25 µM human Aβ1–42 or 10 µg/ml LPS (Sigma) for an additional 24 h. Mouse MCP-1 in the supernatants was measured by ELISA (Pharmingen, San Diego, California). Adhesion. Multi-spot glass slides (Shandon, Pittsburgh, Pennsylvania) were coated with CIV (Fluka, Milwaukee, Wisconsin; 50 µg/ml in water for 1 h), air-dried and overlaid with various amounts of human or mouse Aβ1–42, as described11. Astrocytes (5 × 104 cells/spot) suspended in HBSS with or without Ca2+ and Mg2+ were plated on peptide-coated spots, incubated for 45 min at 37 °C and washed. Adherent cells were quantitated using CyQuantGR (Molecular Probes, Eugene, Oregon) as described11. Migration. Cell culture inserts (8-µm pore size; Becton Dickinson, Franklin Lakes, New Jersey) were coated with CIV and overlaid with 8 µg human Aβ1–42. Astrocytes (105 cells) suspended in RPMI 1640 with 25 mM HEPES and 0.1% BSA were added to the upper chamber of the inserts; LPS (100 456
Fig. 3 Degradation of Aβ1–42 by adult mouse astrocytes as measured by western blot and ELISA. a and b, Astrocytes were incubated with synthetic Aβ1–42, and supernatant or adherent cells (pellet) were collected after the indicated time points. Monomeric Aβ is detected in the cell pellet (a, left panel; b, ) after 3 h and disappears at 48 h in both pellet and supernatant (a, right panel; b, ). *, non-specific band. Higher molecular weight bands in pellet and supernatant may be Aβ polymers or aggregates with other proteins that are also degraded. One representative experiment is shown (n = 3 experiments).
µg/ml) or MCP-1 (10–8 M; Leinco Technologies, St. Louis, Missouri) was added to the bottom chamber. After incubation at 37 °C for 6 h, non-migrated cells on the upper surface of the membrane were removed by scraping. Cells that migrated to the lower surface of the membrane were stained with hematoxylin and counted with an inverted microscope.
Aβ1–42 (ng/ml)
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Time (h)
Supernatant
Binding inhibition studies. Astrocyte cultures maintained in Krebs-Ringer buffer containing 1 mM glucose and 0.1% bovine serum albumin (KRBGA) were pretreated for 30 min at 37 °C with or without fucoidan, polyinosinic acid (100 and 500 µg/ml) or 200 µg/ml SRBI-specific rabbit antibody (Novus Biologicals, Littleton, Colorado), before adding 10 µg/ml Cy3-Aβ1–42 in KRBGA as described11. After 1 h, cells were washed with KRBGA, fixed in 5% formalin and photographed. Cy3 fluorescence intensity was quantified using NIH Image software (NIH, freeware at http://rsb.info.nih.gov/nihimage). Aβ removal. Multi-spot glass slides were overlaid with DMEM/F12 containing 10 µg/ml Cy3-Aβ1–42 for 1 h at 37 °C, washed with sterile water and airdried. Astrocytes suspended in DMEM/F12 complete were plated on Cy3-Aβ1–42–coated spots (5 × 103 cells per 50 µl per spot) and incubated for up to 2 d at 37 °C. Cells were fixed in 5% formalin, nuclei were stained with DAPI, and fluorescence and phase images were taken. Clearance of Aβ from APP mouse brain slices. Brains of saline-perfused 22-month-old mice expressing human APP under control of the plateletderived growth factor B chain promoter25 (line J20; L. Mucke, Gladstone Institute of Neurological Disease) were divided sagittally and snap frozen. Cryosections (10 µm) were mounted on poly-L-lysine-coated coverslips, transferred to 12-well plates and incubated with or without adult astrocytes (6 × 105 /well) for 24 h at 37 °C. Sections were then fixed in 4% paraformaldehyde, treated with 0.1% Triton X-100, immunostained with 3D6 antibody (1:1000) and developed with diaminobenzidine and hydrogen peroxide. Relative areas of the hippocampus occupied by 3D6 immunoreactivity were measured for 8–15 sections per condition with Bioquant 98 software (R&M Biometrics, Nashville, Tennessee). For confocal microscopy, sections similarly incubated with astrocytes were fixed in acetone and immunolabeled with rabbit antibody 1280 against human Aβ (1:1000; D. Selkoe, Harvard University, Boston, Massachusetts) and goat antibody against S100β (1:1000). After washing, sections were incubated with FITC- or Texas Red–conjugated secondary antibodies (Vector Laboratories, Burlingame, California), mounted and imaged by scanning confocal microscopy. All animal experiments were performed in accordance with institutional guidelines. Acknowledgments This work was supported by National Institutes of Health grants AG-15871 (to T.W.-C.) and AG-19772 (to S.C.S.), the Alzheimer’s Association (T.W.-C. and NATURE MEDICINE • VOLUME 9 • NUMBER 4 • APRIL 2003
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ARTICLES J.H.) and a Pilot Grant Award (to J.H.) from Columbia University’s Alzheimer Disease Research Center (through National Institutes of Health grant AG08702). Competing interests statement The authors declare that they have no competing financial interests.
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RECEIVED 20 NOVEMBER 2002; ACCEPTED 5 FEBRUARY 2003
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15. 1. Role of inflammatory processes and microglial activation in Alzheimer’s disease. In Alzheimer’s Disease (ed. Terry, R.D.) chap. 26, 390 (Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 1999). 2. Heesen, M. et al. Mouse astrocytes respond to the chemokines MCP-1 and KC, but reverse transcriptase-polymerase chain reaction does not detect mRNA for the KC or new MCP-1 receptor. J. Neurosci. Res. 45, 382–391 (1996). 3. Hayashi, M., Luo, Y., Laning, J., Strieter, R.M. & Dorf, M.E. Production and function of monocyte chemoattractant protein-1 and other β-chemokines in murine glial cells. J. Neuroimmunol. 60, 143–150 (1995). 4. Johnstone, M., Gearing, A.J. & Miller, K.M. A central role for astrocytes in the inflammatory response to β-amyloid; chemokines, cytokines and reactive oxygen species are produced. J. Neuroimmunol. 93, 182–193 (1999). 5. Rezaie, P., Trillo-Pazos, G., Everall, I.P. & Male, D.K. Expression of β-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS. Glia 37, 64–75 (2002). 6. Ishizuka, K. et al. Identification of monocyte chemoattractant protein-1 in senile plaques and reactive microglia of Alzheimer’s disease. Psychiat. Clin. Neurosci. 51, 135–138 (1997). 7. Mehlhorn, G., Hollborn, M. & Schliebs, R. Induction of cytokines in glial cells surrounding cortical β-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology. Int. J. Dev. Neurosci. 18, 423–431 (2000). 8. Tezel, G., Hernandez, M.R. & Wax, M.B. In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head. Glia 34, 178–189 (2001). 9. Tawil, N.J. et al. α 1 β 1 integrin heterodimer functions as a dual laminin/collagen receptor in neural cells. Biochemistry 29, 6540–6544 (1990). 10. Husemann, J., Loike, J.D., Anankov, R., Febbraio, M. & Silverstein, S.C. Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia 40, 195–205 (2002). 11. Husemann, J., Loike, J.D., Kodama, T. & Silverstein, S.C. Scavenger receptor class B
NATURE MEDICINE • VOLUME 9 • NUMBER 4 • APRIL 2003
16. 17. 18.
19.
20.
21.
22.
23.
24. 25.
type I (SR-BI) mediates adhesion of neonatal murine microglia to fibrillar β-amyloid. J. Neuroimmunol. 114, 142–150 (2001). Wegiel, J., Wang, K.C., Tarnawski, M. & Lach, B. Microglia cells are the driving force in fibrillar plaque formation, whereas astrocytes are a leading factor in plague degradation. Acta Neuropathol. (Berl.) 100, 356–364 (2000). Kurt, M.A., Davies, D.C. & Kidd, M. β-amyloid immunoreactivity in astrocytes in Alzheimer’s disease brain biopsies: an electron microscope study. Exp. Neurol. 158, 221–228 (1999). Thal, D.R. et al. Amyloid β-protein (Aβ)-containing astrocytes are located preferentially near N-terminal-truncated Aβ deposits in the human entorhinal cortex. Acta Neuropathol. (Berl.) 100, 608–617 (2000). Yamaguchi, H., Sugihara, S., Ogawa, A., Saido, T.C. & Ihara, Y. Diffuse plaques associated with astroglial amyloid β protein, possibly showing a disappearing stage of senile plaques. Acta Neuropathol. (Berl.) 95, 217–222 (1998). Shaffer, L.M. et al. Amyloid β protein (A β) removal by neuroglial cells in culture. Neurobiol. Aging 16, 737–745 (1995). Wyss-Coray, T. & Mucke, L. Inflammation in neurodegenerative disease—a doubleedged sword. Neuron 35, 419–432 (2002). DeWitt, D.A., Perry, G., Cohen, M., Doller, C. & Silver, J. Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer’s disease. Exp. Neurol. 149, 329–340 (1998). Bard, F. et al. Peripherally administered antibodies against amyloid β-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat. Med. 6, 916–919 (2000). Savchenko, V.L., McKanna, J.A., Nikonenko, I.R. & Skibo, G.G. Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity. Neuroscience 96, 195–203 (2000). De Groot, C.J. et al. Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization. J. Neurosci. Res. 49, 342–354 (1997). Coraci, I.S. et al. CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer’s disease brains and can mediate production of reactive oxygen species in response to β-amyloid fibrils. Am. J. Pathol. 160, 101–112 (2002). Paresce, D.M., Ghosh, R.N. & Maxfield, F.R. Microglial cells internalize aggregates of the Alzheimer’s disease amyloid β-protein via a scavenger receptor. Neuron 17, 553–565 (1996). Wyss-Coray, T. et al. TGF-β1 promotes microglial amyloid-β clearance and reduces plaque burden in transgenic mice. Nat. Med. 7, 612–618 (2001). Mucke, L. et al. High-level neuronal expression of aβ1–42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J. Neurosci. 20, 4050–4058 (2000).
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