Uptake, Degradation, and Release of Fibrillar and Soluble Forms of Alzheimer's Amyloid β-Peptide by Microglial Cells
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32301
ISSN1083-351X
AutoresHaeyong Chung, Melanie Brazil, Thwe Thwe Soe, Frederick R. Maxfield,
Tópico(s)Neurological Disease Mechanisms and Treatments
ResumoMicroglia are phagocytic cells that are the main inflammatory response cells of the central nervous system. In Alzheimer's disease brain, activated microglia are concentrated in regions of compact amyloid deposits that contain the 39–43-amino acid Aβ peptide. We examined the uptake, degradation, and release of small aggregates of fibrillar Aβ (fAβ) or soluble Aβ (sAβ) by microglia. We found that although some degradation of fAβ was observed over 3 days, no further degradation was observed over the next 9 days. Instead, there was a slow release of intact Aβ. The poor degradation was not due to inhibition of lysosomal function, since the rate of α2-macroglobulin degradation was not affected by the presence of fAβ in the late endosomes/lysosomes. In contrast to fAβ, internalization of sAβ was not saturable. After internalization, sAβ was released rapidly from microglia, and very little was degraded. These data show that fAβ and sAβ interact differently with microglia but that after internalization a large fraction of both are released without degradation. Microglia are phagocytic cells that are the main inflammatory response cells of the central nervous system. In Alzheimer's disease brain, activated microglia are concentrated in regions of compact amyloid deposits that contain the 39–43-amino acid Aβ peptide. We examined the uptake, degradation, and release of small aggregates of fibrillar Aβ (fAβ) or soluble Aβ (sAβ) by microglia. We found that although some degradation of fAβ was observed over 3 days, no further degradation was observed over the next 9 days. Instead, there was a slow release of intact Aβ. The poor degradation was not due to inhibition of lysosomal function, since the rate of α2-macroglobulin degradation was not affected by the presence of fAβ in the late endosomes/lysosomes. In contrast to fAβ, internalization of sAβ was not saturable. After internalization, sAβ was released rapidly from microglia, and very little was degraded. These data show that fAβ and sAβ interact differently with microglia but that after internalization a large fraction of both are released without degradation. Alzheimer's disease β-amyloid precursor protein fibrillar Aβ soluble Aβ α2-macroglobulin fluorescein isothiocyanate low density lipoprotein Alzheimer's disease (AD)1 represents the most frequent cause of dementia in the elderly, accounting for more than half of all cases (1Terry R.D. Katzman R. Bick K.L. Alzheimer Disease. 1994; (eds), pp., Raven Press, New York: 9-25-305-326Google Scholar). The characteristic histological features of AD are senile plaques, neurofibrillary tangles, congophilic angiopathy of the vessels, neuropil threads, and neuronal cell loss (1Terry R.D. Katzman R. Bick K.L. Alzheimer Disease. 1994; (eds), pp., Raven Press, New York: 9-25-305-326Google Scholar, 2Selkoe D.J. Neuron. 1991; 6: 487-498Abstract Full Text PDF PubMed Scopus (2259) Google Scholar). Senile plaques are classified into two major types: the classical (neuritic) and the diffuse (preamyloid) plaques. The classical plaque is a complex lesion of the cortical neuropil containing several abnormal elements: a central deposit of extracellular amyloid fibrils ("the core" composed of β-amyloid or Aβ peptide (3Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4379) Google Scholar, 4Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3791) Google Scholar)) surrounded by dystrophic neurites (both dendrites and axonal terminals), activated microglia, and reactive astrocytes (1Terry R.D. Katzman R. Bick K.L. Alzheimer Disease. 1994; (eds), pp., Raven Press, New York: 9-25-305-326Google Scholar, 2Selkoe D.J. Neuron. 1991; 6: 487-498Abstract Full Text PDF PubMed Scopus (2259) Google Scholar). The diffuse plaques are devoid of amyloid core, with very few or no surrounding dystrophic neurites or glia and contain nonfibrillar (amorphous) Aβ. Despite intense investigation, the sequence of events that lead to neuronal pathology in AD is still largely unknown. Many studies have supported the hypothesis that Aβ deposition may play an early and, in some cases, causative role in the disease. Aβ is a 39–43-amino acid fragment of a larger membrane-spanning glycoprotein called β-amyloid precursor protein (βAPP) (3Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4379) Google Scholar, 5Kang J. Lemaire H.G. Unterbeck A. Slabaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (4067) Google Scholar). The different forms of Aβ have varying solubilities. Aβ is constitutively produced in cultured cells (6Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1779) Google Scholar, 7Shoji M. Golde T.E. Ghiso J. Cheung T.T. Estus S. Shaffer L.M. Cai X.-D. McKay D.M. Tintner R. Frangione B. Younkin S.G. Science. 1992; 258: 126-129Crossref PubMed Scopus (1334) Google Scholar), and soluble Aβ (sAβ) is present at similar concentrations (10−8 to 10−10m) in normal and AD cerebrospinal fluid (7Shoji M. Golde T.E. Ghiso J. Cheung T.T. Estus S. Shaffer L.M. Cai X.-D. McKay D.M. Tintner R. Frangione B. Younkin S.G. Science. 1992; 258: 126-129Crossref PubMed Scopus (1334) Google Scholar, 8Seubert P. Vigo-Pelfrey C. Esch F. Lee M. Dovey H. Davis D. Sinha S., M., S. McCormack R. Wolfert R. Selkoe D. Lieberburg I. Schenk D. Nature. 1992; 359: 325-327Crossref PubMed Scopus (1624) Google Scholar). These observations indicate that Aβ production is a normal physiological process in vivo and in vitro. sAβ can spontaneously form insoluble assemblies of β-pleated sheet (fibrillar) conformation similar to amyloid fibrils found in AD brain. The formation of fibrils in vitro depends on concentration, time, pH, temperature, and ionic strength (9Kirschner D.A. Inouye H. Duffy L.K. Sinclair A. Lind M. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6953-6957Crossref PubMed Scopus (494) Google Scholar, 10Halverson K. Fraser P.E. Kirschner D.A. Lansbury Jr., P.T. Biochemistry. 1990; 29: 2639-2644Crossref PubMed Scopus (298) Google Scholar, 11Jarrett J.T. Berger E.P. Lansbury P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1790) Google Scholar, 12Lomakin A. Teplow D.B. Kirschner D.A. Benedek G.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7942-7947Crossref PubMed Scopus (496) Google Scholar, 13Kusumoto Y. Lomakin A. Teplow D.A. Benedek G.B. Proc. Natl. Acad. Sci. 1998; 95: 12277-12282Crossref PubMed Scopus (240) Google Scholar). The Aβ that accumulates in AD brains is heterogeneous at its C terminus, resulting in peptides 39–43 amino acids in length. It has been shown that diffuse plaques almost exclusively consist of Aβ-(1–42/43) (14Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1590) Google Scholar). In contrast, neuritic plaques contain both Aβ-(1–40) (Aβ40) and Aβ-(1–42) (Aβ42) and also shorter Aβ peptides that are truncated at the N terminus (14Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1590) Google Scholar, 15Iwatsubo T. Mann D.M.A. Odaka A. Suzuki N. Ihara Y. Ann. Neurol. 1995; 37: 294-299Crossref PubMed Scopus (342) Google Scholar, 16Iwatsubo T. Saido T.C. Mann D.M.A. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 1996; 143: 1823-1830Google Scholar, 17Savage M.J. Kawooja J.K. Pinsker L.R. Emmons T.L. Mistretta S. Siman R. Greenberg B.D. J. Exp. Clin. Invest. 1995; 2: 234-240Google Scholar). Aβ40 is believed to be the major isoform generated by cultured cells (8Seubert P. Vigo-Pelfrey C. Esch F. Lee M. Dovey H. Davis D. Sinha S., M., S. McCormack R. Wolfert R. Selkoe D. Lieberburg I. Schenk D. Nature. 1992; 359: 325-327Crossref PubMed Scopus (1624) Google Scholar, 18Dovey H.F. Suomensaari-Chrysler S. Lieberburg I. Sinha S. Keim P.S. Neuroreport. 1993; 4: 1039-1042Crossref PubMed Scopus (75) Google Scholar,19Suzuki N. Cheung T.T. Cai X.-D. Odaka A. Otvos L.J. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1369) Google Scholar) as well as in cerebrospinal fluid (20Vigo-Pelfrey C. Lee D. Keim P. Lieberburg I. Schenk D.B. J. Neurochem. 1993; 61: 1965-1968Crossref PubMed Scopus (376) Google Scholar), while Aβ42 is the predominant species accumulated in senile plaques in AD brains as well as in the brains of nondemented aged individuals and appears to be the initially deposited species (14Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1590) Google Scholar, 16Iwatsubo T. Saido T.C. Mann D.M.A. Lee V.M.-Y. Trojanowski J.Q. Am. J. Pathol. 1996; 143: 1823-1830Google Scholar, 21Lemere C.A. Blusztajn J.K. Yamaguchi H. Wisniewski T. Saido T.C. Selkoe D.J. Neurobiol. Dis. 1996; 3: 16-32Crossref PubMed Scopus (471) Google Scholar). Differences in the deposited and soluble forms of Aβ may be significant in β-amyloidogenesis. In vitro, Aβ42 is less soluble and forms fibrils at a greater rate than shorter isoforms (11Jarrett J.T. Berger E.P. Lansbury P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1790) Google Scholar). Since AD involves the net accumulation of Aβ, it is important to understand both the synthesis and clearance of Aβ. It has been long recognized that reactive microglia are closely associated with neuritic plaques in AD, and these cells could be involved in either the removal or the production of amyloid fibrils (22Giulian D. Haverkamp L.J., Yu, J.H. Karshin W. Tom D. Li J. Kirkpatrick J. Kuo Y.-M. Roher A.E. J. Neurosci. 1996; 16: 6021-6037Crossref PubMed Google Scholar). Microglia are macrophage-like phagocytic cells. They are the main inflammatory response cells of the brain. Microglia found in normal adult brain are highly ramified, quiescent cells that retract their processes and become reactive during CNS injury (22Giulian D. Haverkamp L.J., Yu, J.H. Karshin W. Tom D. Li J. Kirkpatrick J. Kuo Y.-M. Roher A.E. J. Neurosci. 1996; 16: 6021-6037Crossref PubMed Google Scholar, 23del Rio-Hortega P. Penfield W. Cytology and Cellular Pathology of the Nervous System. Paul P. Hocker, Inc., New York1932: 481-584Google Scholar). In AD, quantitative histopathology has been used to show that clusters of reactive microglia are frequently associated with the amyloid core and/or within the plaques, whereas diffuse plaques are generally not associated with microglia (24Giulian D. Haverkamp L.J. Li J. Karshin W.L., Yu, J. Tom D. Li X. Kirkpatrick J.B. Neurochem. Int. 1995; 27: 119-137Crossref PubMed Scopus (210) Google Scholar, 25Itagaki S. McGeer P.L. Akiyama H. Zhu S. Selkoe D.J. J. Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (787) Google Scholar, 26Rozemuller J.M. Bots G.T. Roos R.A. Eikelenboom P. Neurosci. Lett. 1992; 140: 137-140Crossref PubMed Scopus (27) Google Scholar). Reactive microglia are also found associated with plaques that develop in a transgenic mouse model expressing mutant βAPP (27Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Guido T. Hagopian S. Johnson-Wood K. Khan K. Lee M. Leibowitz P. Lieberburg I. Little S. Masliah E. McConlogue L. Montoya-Zavala M. Mucke L. Paganini L. Penniman E. Power M. Schenk D. Seubert P. Snyder B. Soriano F. Tan H. Vitale S. Wadsworth B. Wolozin B. Zhao J. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2272) Google Scholar, 28Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3740) Google Scholar, 29Frautschy S.A. Yang F. Irizarry M.C. Hyman B.T. Saido T.C. Hsiao K. Cole G.M. Am. J. Pathol. 1998; 152: 307-317PubMed Google Scholar). These observations suggest that brain inflammatory responses may be directed specifically against the constituents of neuritic plaques and that one pathway for Aβ-induced neuron damage may involve inflammatory microglia. Activated microglia are capable of releasing cytotoxic agents such as proteolytic enzymes, cytokines, complement proteins, reactive oxygen intermediates, and nitric oxide (30Giulian D. Li J. Bartel S. Broker J. Li X. Kirkpatrick J.B. J. Neurosci. 1995; 15: 7712-7726Crossref PubMed Google Scholar). Although these considerations suggest that the involvement of microglia in AD is mainly harmful, these cells could also play a protective role by digesting amyloid fibrils. It has been suggested that clearance of amyloid deposits by immunization against Aβ in a mouse model for AD may occur via microglial cells (31Schenk D. Barbour R. Dunn W. Gordon G. Grajeda H. Guido T. Hu K. Huang J. Johnson-Wood K. Khan K. Kholodenko D. Lee M. Liao Z. Lieberburg I. Motter R. Mutter L. Soriano F. Shopp G. Vasquez N. Vandevert C. Walker S. Wogulls M. Yednock T. Games D. Seubert P. Nature. 1999; 400: 173-177Crossref PubMed Scopus (2991) Google Scholar). While amyloid is present in the intracellular organelles of microglia (32Frackowiak J. Wisniewski H.M. Wegiel J. Merz G.S. Iqbal K. Wang K.C. Acta Neuropathol. ( Berlin ). 1992; 84: 225-233Crossref PubMed Scopus (239) Google Scholar, 33Wisniewski H.M. Wegiel J. Wang K.C. Lach B. Acta Neuropathol. ( Berlin ). 1992; 84: 117-127Crossref PubMed Scopus (205) Google Scholar), it is not known if this results from phagocytosis of previously deposited amyloid by microglia or from microglial processing of βAPP to Aβ. In culture, microglia can synthesize βAPP (34Bauer J. Konig G. Strauss S. Jonas U. Ganter U. Weidemann A. Monning U. Masters C.L. Volk B. Berger M. Beyreuther K. FEBS Lett. 1991; 282: 335-340Crossref PubMed Scopus (58) Google Scholar, 35Haass C. Hung A.Y. Selkoe D.J. J. Neurosci. 1991; 11: 3783-3793Crossref PubMed Google Scholar, 36LeBlanc A.C. Chen H.Y. Autilio G.L. Gambetti P. FEBS Lett. 1991; 292: 171-178Crossref PubMed Scopus (64) Google Scholar, 37Banati R.B. Gehrmann J. Czech C. Monning U. Jones L.L. Konig G. Beyreuther K. Kreutzberg G.W. Glia. 1993; 9: 199-210Crossref PubMed Scopus (125) Google Scholar). Also, some investigators have observed amyloid precursor protein mRNA in microglia in AD brain (38Goldgaber D. Schmechel D.E. Adv. Neurol. 1990; 51: 163-169PubMed Google Scholar), but others have not (39Goedart M. EMBO J. 1987; 6: 3627-3632Crossref PubMed Scopus (191) Google Scholar, 40Scott S.A. Johnson S.A. Zarow C. Perlmutter L.S. Exp. Neurol. 1993; 121: 113-118Crossref PubMed Scopus (34) Google Scholar). It is likely that some, if not all, of the intracellular Aβ observed in microglia is extracellular in origin. It is possible that microglia facilitate the formation of plaques upon ingestion of fibrillar or soluble Aβ and delivery to acidic endosomes. We and others have identified receptors on microglia that bind purified Aβ (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 42El-Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Crossref PubMed Scopus (682) Google Scholar, 43Yan S.D. Chen X. Fu J. Chen M. Zhu H. Roher A. Slattery T. Zhao L. Nagashima M. Morser J. Migheli A. Nawroth P. Stern D. Schmidt A.M. Nature. 1996; 382: 685-691Crossref PubMed Scopus (1848) Google Scholar). These include members of the scavenger receptor family (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 42El-Khoury J. Hickman S.E. Thomas C.A. Cao L. Silverstein S.C. Loike J.D. Nature. 1996; 382: 716-719Crossref PubMed Scopus (682) Google Scholar). We have found that two forms of the scavenger receptor, SRA and SRB, could mediate the uptake of fAβ in transfected Chinese hamster ovary cells (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). We examined the degradation of fAβ up to 3 days after internalization, both immunocytochemically and biochemically, and found that microglia showed intracellular accumulation of fAβ and slow, incomplete degradation of fAβ (44Paresce D.M. Chung H. Maxfield F.R. J. Biol. Chem. 1997; 272: 29390-29397Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). If microglia do not degrade fAβ after a certain time point, this might mean that microglia accumulate fAβ and participate in the formation of amyloid. The existence of undegraded fAβ could be due to slow degradation of fAβ in all cells or to a population of cells that cannot degrade fAβ. To distinguish between these possibilities, we followed the degradation for a longer period of time. Since soluble Aβ production is a normal physiological process, we also studied the uptake and degradation of sAβ, and we found that the uptake and degradation kinetics of fibrillar Aβ were quite different from those of soluble Aβ. In the present study, we measured the degradation of fAβ over the course of 12 days. After a short pulse, there was a slow, partial degradation for 3 days. After that time, degradation stopped, but the release of trichloroacetic acid-insoluble material from microglia continued throughout the 12 days. The degradative function of late endosomes/lysosomes was not affected by the presence of fAβ in these compartments. In contrast to fAβ, sAβ was not internalized by scavenger receptors in microglia, and very little degradation was observed. Microglia were isolated from mixed glial cultures prepared from newborn mice as described previously (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar,44Paresce D.M. Chung H. Maxfield F.R. J. Biol. Chem. 1997; 272: 29390-29397Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The mixed glial cultures were grown in bicarbonate-buffered Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a 5% CO2 humidified air atmosphere. Microglia were harvested by orbital shaking for 20–45 min at 150 × g in growth medium. Cells were centrifuged for 10 min at 350 × gand plated onto 24-well tissue culture plates at 105cells/well or onto 35-mm diameter plastic tissue culture dishes in which a 7-mm diameter hole was punched in the bottom, and a polylysine-coated number 1 glass coverslip was attached beneath the hole (45Salzman N.H. Maxfield F.R. J. Cell Biol. 1988; 106: 1083-1091Crossref PubMed Scopus (53) Google Scholar) at a density of 104 to 105cells/coverslip. Cell viability was assayed with a commercial kit (LIVE/DEAD; Molecular Probes, Inc., Eugene, OR). 1–40 and 1–42 Aβ peptides were purchased from Bachem (Torrance, CA). 125I-Labeled Aβ was prepared using the chloramine T method (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 46Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (431) Google Scholar). Purified α2-macroglobulin (α2M) was converted to the receptor-binding form by dialyzing in 25 mm methylamine (pH 8.0) for 4 h at room temperature, followed by dialysis against phosphate-buffered saline for 3 days with four buffer changes at 4 °C. This procedure cleaves the internal thioester bond and converts α2M to a high affinity ligand for its receptor (47Tycko B. DiPaola M. Yamashiro D.J. Fluss S. Maxfield F.R. Ann. N.Y. Acad. Sci. 1983; 421: 424-433Crossref PubMed Scopus (14) Google Scholar). 125I-Labeled α2M was prepared using the chloramine T method (46Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (431) Google Scholar, 48Yamashiro D.J. Borden L.A. Maxfield F.R. J. Cell. Physiol. 1989; 139: 377-382Crossref PubMed Scopus (32) Google Scholar). Iodinated Aβ and α2M retain the ability to bind specifically to their receptors (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 46Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (431) Google Scholar,49Tabas I. Zha X. Beatini N. Myers J.N. Maxfield F.R. J. Biol. Chem. 1994; 269: 22547-22556Abstract Full Text PDF PubMed Google Scholar). The specific activities of 125I-labeled Aβ40, Aβ42, and α2M in the representative experiments shown in the figures ranged between 1.2 × 10−3 and 3.4 × 10−3 μCi/ng. Fluorescently labeled Aβ was prepared by conjugation with Cy3, a carbocyanine dye (Biological Detection Systems Inc., Pittsburgh, PA), as described previously (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 44Paresce D.M. Chung H. Maxfield F.R. J. Biol. Chem. 1997; 272: 29390-29397Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). α2M was conjugated to fluorescein isothiocyanate (FITC) as described previously (50Salzman N.H. Maxfield F.R. J. Cell Biol. 1989; 109: 2067-2072Crossref PubMed Scopus (90) Google Scholar). For all of our studies on uptake and degradation of labeled and unlabeled fAβ, Aβ was preaggregated before being added to microglia. Aβ was first diluted in labeling medium (Dulbecco's modified Eagle's medium with 10 mg/ml bovine serum albumin) or Dulbecco's modified Eagle's medium with 10% fetal bovine serum and vortexed. The mixture was then pH-adjusted to 5.0 with 1 mHCl and incubated at 37 °C for 4–16 h. For studies using Cy3 and125I-labeled soluble Aβ, Aβ was diluted in labeling medium with 10 mg/ml bovine serum albumin, slowly mixed by pipetting, and then subjected to ultracentrifugation at 100,000 ×g for 1 h at 25 °C. Only the resulting supernatant was added to microglia to ensure that no aggregated Aβ was added to the cells. Since Aβ is known to spontaneously aggregate in vitro, for each experiment using 125I-labeled sAβ, we incubated the supernatant as above in 37 °C in a Petri dish in parallel with the experimental dishes for the same amount of time as the pulse time. The mixture in the Petri dish was centrifuged at 100,000 × g for 1 h at 4 °C. We found that all of the 125I-labeled sAβ remained soluble. Cells were grown in 24-well plates for 1–2 days before the start of the experiment. The cells were washed twice with labeling medium and then incubated with the radiolabeled fibrillar or soluble peptides for 1 h at 37 °C in a 5% CO2 humidified air atmosphere. The cells were rinsed three times with labeling medium and then incubated at 37 °C for 15 min to allow the release of nonspecifically bound Aβ. The cells were rinsed further, twice with medium without serum and three times with chase medium, which was the microglia growth medium, and then incubated with 1 ml of chase medium for varying times. At the end of the chase, the medium bathing the cells was removed, and the radioactivity was measured. The cells were rinsed and solubilized with 1 m NaOH. The chase medium was precipitated with 10% trichloroacetic acid on ice for 1 h and centrifuged at 14,000 × g for 10 min to separate the trichloroacetic acid-soluble fraction from the insoluble fraction. Specific binding was determined by adding unlabeled Ac-LDL, fucoidan, or α2M as competitive inhibitors at a molar concentration 100–200-fold greater than the radiolabeled ligands. Where indicated, the excess unlabeled ligands were added to the incubation medium along with the radiolabeled peptides and were maintained in the labeling medium for the entire incubation. The radioactivity of these background dishes was subtracted from the values for the control experiments. The binding of 125I-labeled fAβ in the presence of excess Ac-LDL or fucoidan was less than 10% of control binding, whereas α2M uptake in the presence of excess α2M was about 25%. All radioactive experiments were conducted with three wells per condition and repeated at least five times on separate days. Cy3-Aβ (1 μg/ml) was diluted in pH 7.4 labeling medium, allowed to aggregate at 37 °C for 4 h, and then added to microglia in coverslip dishes. After 1 h of incubation, cells were washed extensively, three times in labeling medium, twice in medium without serum, and three times in chase medium. The cells were incubated in chase medium with no added peptides and chased for varying times. After chase, cells were rinsed twice with medium 1 (150 mm NaCl, 20 mm HEPES, 1 mm CaCl2, 5 mm KCl, 1 mm MgCl2, pH 7.4) and fixed with 3.3% paraformaldehyde diluted in medium 1 for 10 min at room temperature. Microglia plated on coverslip dishes were loaded with Cy3-labeled fAβ42 (3 μg/ml) diluted in growth medium for 3 days. Cells were rinsed twice with labeling medium and pulsed with FITC-α2M (5 μg/ml) in labeling medium for 45 min. Cells were washed extensively as described previously and then chased for 2, 3, and 6 h. After each chase time, cells were rinsed with medium 1 and fixed with 3.3% paraformaldehyde. 20 mm methylamine in medium 1 was added to cells to collapse intracellular pH gradients. Cells were imaged by confocal microscopy. Fluorescence microscopy and digital image collection were performed using a Leica DMIRB microscope (Leica Mikroscopie und Systeme GmbH, Germany) equipped with a Princeton Instruments cooled CCD camera driven by Image-1/MetaMorph Imaging System software (Universal Imaging Corporation) as described previously (51Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 52Mukherjee S. Soe T.T. Maxfield F.R. J. Cell. Biol. 1999; 144: 1271-1284Crossref PubMed Scopus (311) Google Scholar). Fluorescence quantification was carried out with a × 25, NA 0.75 objective to obtain a large number of cells per field, whereas the images for visualization purposes were obtained with a × 63, NA 1.32 objective (51Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 52Mukherjee S. Soe T.T. Maxfield F.R. J. Cell. Biol. 1999; 144: 1271-1284Crossref PubMed Scopus (311) Google Scholar, 53Mayor S. Presley J.F. Maxfield F.R. J. Cell Biol. 1993; 121: 1257-1269Crossref PubMed Scopus (425) Google Scholar, 54Ghosh R. Maxfield F. J. Cell Biol. 1995; 128: 549-561Crossref PubMed Scopus (97) Google Scholar, 55Mayor S. Maxfield F.R. Mol. Biol. Cell. 1995; 6: 929-944Crossref PubMed Scopus (252) Google Scholar). Fields used for quantification were selected at random throughout the dish and focused using phase contrast optics before viewing the fluorescence. Digital fluorescence images were obtained, and the background signal was removed from the images as described previously (41Paresce D.M. Ghosh R. Maxfield F.R. Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 44Paresce D.M. Chung H. Maxfield F.R. J. Biol. Chem. 1997; 272: 29390-29397Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 56Dunn K.W. McGraw T.E. Maxfield F.R. J. Cell Biol. 1989; 109: 3303-3314Crossref PubMed Scopus (272) Google Scholar). Cells were identified in the image using an intensity threshold set at the mean pixel intensity for the entire image plus the S.D. of the pixel intensity. Objects of the size of cells and above the threshold intensity were identified using MetaMorph software routines (Universal Imaging, West Chester, PA). Total fluorescence power per cell was taken as the sum of all intensity in each object identified as a cell. The total fluorescence power per cell remaining after various chase times was determined for Cy3-fAβ. Fluorescent beads were used to normalize the variations in the intensities that result from taking images on different chase days. For every field analyzed, the phase contrast images were juxtaposed to the corresponding fluorescence images to ensure that any noncell objects or partially viewed cells were eliminated from the analysis. An axial series of fluorescence images was obtained with a Zeiss LSM 510 laser-scanning confocal microscope equipped with a × 63, NA 1.4 plan Apochromat objective (Carl Zeiss, Inc., Jena, Germany). Red and green images were acquired sequentially using the 543-nm (exciting the Cy3) and 488-nm (exciting the fluorescein) lines from a 1.0-milliwatt HeNe laser, and a 25 milliwatt argon laser, respectively. For images acquired in this manner, cross-talk of FITC fluorescence into the Cy3 channel was not detectable. 0.5-μm vertical steps were used, with a vertical optical resolution of less than 1.0 μm. In our previous study, adherent microglia were incubated with either Cy3-labeled fAβ42 or 125I-labeled fAβ42 for a short period of time and kept in label-free growth medium for up to 3 days. We found that microglia retained Cy3-labeled fAβ42 for up to 3 days, and only about 30% of internalized 125I-labeled fAβ42 was degraded in that time. We wanted to look at the degradation kinetics of fAβ for a longer period of time to see if the cells carried out progressive, slow degradation. We used both fAβ40 and fAβ42, and we observed no difference in the uptake, degradation, or release between fAβ40 and fAβ42. We found that about 60% of internalized 125I-labeled fAβ was released after 12 days, while 40% of the fAβ still remained cell-associated (Fig.1 A). Only a fraction of the released Aβ was degraded to trichloroacetic acid-soluble fragments. The degradation of fAβ, as observed by the amount of trichloroacetic acid-soluble material released from
Referência(s)