Evidence of a Role for Lactadherin in Alzheimer's Disease
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060664
ISSN1525-2191
AutoresJacques Boddaert, Kiyoka Kinugawa, Jean‐Charles Lambert, Fatiha Boukhtouche, Joffrey Zoll, Régine Merval, Olivier Blanc‐Brude, David M. A. Mann, Claudine Berr, José Vilar, Béatrice Vernet-der Garabedian, Nathalie Journiac, Dominique Charue, Jean‐Sébastien Silvestre, Charles Duyckaerts, Philippe Amouyel, Jean Mariani, Alain Tedgui, Ziad Mallat,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoLactadherin is a secreted extracellular matrix protein expressed in phagocytes and contributes to the removal of apoptotic cells. We examined lactadherin expression in brain sections of patients with or without Alzheimer's disease and studied its role in the phagocytosis of amyloid β-peptide (Aβ). Cells involved in Alzheimer's disease, including vascular smooth muscle cells, astrocytes, and microglia, showed a time-related increase in lactadherin production in culture. Quantitative analysis of the level of lactadherin showed a 35% reduction in lactadherin mRNA expression in the brains of patients with Alzheimer's disease (n = 52) compared with age-matched controls (n = 58; P = 0.003). Interestingly, lactadherin protein was detected in the brains of patients with Alzheimer's disease and controls, with low expression in areas rich in senile plaques and marked expression in areas without Aβ deposition. Using surface plasmon resonance, we observed a direct protein-protein interaction between recombinant lactadherin and Aβ 1-42 peptide in vitro. Lactadherin deficiency or its neutralization using specific antibodies significantly prevented Aβ 1-42 phagocytosis by murine and human macrophages. In conclusion, lactadherin plays an important role in the phagocytosis of Aβ 1-42 peptide, and its expression is reduced in Alzheimer's disease. Alterations in lactadherin production/function may contribute to the initiation and/or progression of Alzheimer's disease. Lactadherin is a secreted extracellular matrix protein expressed in phagocytes and contributes to the removal of apoptotic cells. We examined lactadherin expression in brain sections of patients with or without Alzheimer's disease and studied its role in the phagocytosis of amyloid β-peptide (Aβ). Cells involved in Alzheimer's disease, including vascular smooth muscle cells, astrocytes, and microglia, showed a time-related increase in lactadherin production in culture. Quantitative analysis of the level of lactadherin showed a 35% reduction in lactadherin mRNA expression in the brains of patients with Alzheimer's disease (n = 52) compared with age-matched controls (n = 58; P = 0.003). Interestingly, lactadherin protein was detected in the brains of patients with Alzheimer's disease and controls, with low expression in areas rich in senile plaques and marked expression in areas without Aβ deposition. Using surface plasmon resonance, we observed a direct protein-protein interaction between recombinant lactadherin and Aβ 1-42 peptide in vitro. Lactadherin deficiency or its neutralization using specific antibodies significantly prevented Aβ 1-42 phagocytosis by murine and human macrophages. In conclusion, lactadherin plays an important role in the phagocytosis of Aβ 1-42 peptide, and its expression is reduced in Alzheimer's disease. Alterations in lactadherin production/function may contribute to the initiation and/or progression of Alzheimer's disease. Alzheimer's disease (AD) is the leading cause of dementia in the elderly, possibly affecting 20 to 40% of the population older than age 85.1Small GW Rabins PV Barry PP Buckholtz NS DeKosky ST Ferris SH Finkel SI Gwyther LP Khachaturian ZS Lebowitz BD McRae TD Morris JC Oakley F Schneider LS Streim JE Sunderland T Teri LA Tune LE Diagnosis and treatment of Alzheimer disease and related disorders. 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Primary normal human adult astrocytes were obtained from Clonetics (Cambrex BioScience, Saint-Beauzire, France) and were cultured in Clonetics astrocyte medium (CC-3186). Glial fibrillary acid protein (GFAP) positivity (>80%) was unchanged during the period of the culture experiments (data not shown). Primary cultures of mouse neonatal astrocytes were generated (∼99% positivity for GFAP) after purification of astrocytes as described.30Pousset F Cremona S Dantzer R Kelley K Parnet P Interleukin-4 and interleukin-10 regulate IL1-beta induced mouse primary astrocyte activation: a comparative study.Glia. 1999; 26: 12-21Crossref PubMed Scopus (65) Google Scholar Primary cultures of mixed glia were prepared from newborn mice as described.31Nakajima K Tsuzaki N Shimojo M Hamanoue M Kohsaka S Microglia isolated from rat brain secrete a urokinase-type plasminogen activator.Brain Res. 1992; 577: 285-292Crossref PubMed Scopus (115) Google Scholar Pure microglia were magnetically isolated from primary cultures of mixed glia using CD11b microbeads from Miltenyi Biotech (Bergisch Gladbach, Germany), according to the manufacturer's instructions. Exosomes were prepared from cell supernatants.32Théry C Boussac M Veron P Ricciardi-Castagnoli P Raposo G Garin J Amigorena S Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.J Immunol. 2001; 166: 7309-7318PubMed Google Scholar Protein extracts from cells and exosomes were examined for the presence of lactadherin and other exosomal-specific proteins by Western blotting using specific antibodies.32Théry C Boussac M Veron P Ricciardi-Castagnoli P Raposo G Garin J Amigorena S Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.J Immunol. 2001; 166: 7309-7318PubMed Google Scholar We previously described the generation and specificity of the anti-mouse and anti-human lactadherin antibodies.33Silvestre JS Thery C Hamard G Boddaert J Aguilar B Delcayre A Houbron C Tamarat R Blanc-Brude O Heeneman S Clergue M Duriez M Merval R Levy B Tedgui A Amigorena S Mallat Z Lactadherin promotes VEGF-dependent neovascularization.Nat Med. 2005; 11: 499-506Crossref PubMed Scopus (249) Google Scholar Other antibodies were commercially available: goat anti-mouse or anti-human Lamp-2 (Santa Cruz Biotechnology, Santa Cruz, CA), goat or mouse anti-mouse or anti-human tsg101 (Santa Cruz Biotechnology) and rabbit anti-mouse or anti-human MHC class I (Santa Cruz Biotechnology). Fifty-two AD brains were obtained at autopsy from patients with sporadic AD accessioned from the Greater Manchester region of United Kingdom during the years 1986 to 2001 (mean age at onset, 65.9 ± 10.3 years; mean age at death, 73.6 ± 9.7 years; 55% of males). All patients were at Braak stages 5 or 6 at time of death. All pathological diagnoses were made in accordance with the Consortum to Establish a Registry to Alzheimer's Disease neuropathological criteria for AD.34Thaker U McDonagh AM Iwatsubo T Lendon CL Pickering-Brown SM Mann DM Tau load is associated with apolipoprotein E genotype and the amount of amyloid beta protein, Abeta40, in sporadic and familial Alzheimer's disease.Neuropathol Appl Neurobiol. 2003; 29: 35-44Crossref PubMed Scopus (31) Google Scholar Fifty-eight control brains presenting Braak stages less than 2 were obtained from routine autopsies performed at the Hospices Civils de Strasbourg (Strasbourg, France) (mean age at death, 79.3 ± 6.5 years, 38% of males).35Berr C Lambert JC Sazdovitch V Amouyel P Chartier-Harlin MC Mohr M Heldt N Kiesmann M Hauw JJ Neuropathological epidemiology of cerebral aging: a study of two genetic polymorphisms.Neurobiol Aging. 2001; 22: 227-235Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Total RNA from frontal areas was extracted from frozen brain tissue using phenol/chloroform protocol (TRIzol reagent; Invitrogen, Cergy, France). The quality of total RNA was assessed using the Agilent 2100 Bioanalyzer (Palo Alto, CA) and the ratio of ribosomal RNA 28S/18S systematically estimated using the Agilent 2100 Bioanalyzer bio-sizing software.36Lambert JC Mann D Richard F Tian J Shi J Thaker U Merrot S Harris J Frigard B Iwatsubo T Lendon C Amouyel P Is there a relation between APOE expression and brain amyloid load in Alzheimer's disease?.J Neurol Neurosurg Psychiatry. 2005; 76: 928-933Crossref PubMed Scopus (45) Google Scholar Real-time quantitative PCR was performed as described.37Gutala RV Reddy PH The use of real-time PCR analysis in a gene expression study of Alzheimer's disease post-mortem brains.J Neurosci Methods. 2004; 132: 101-107Crossref PubMed Scopus (94) Google Scholar The primer sequences were as follows: lactadherin forward, 5′-GACAAGCAGGGCAACTTCAAC-3′; lactadherin reverse, 5′-CAGGATGGGCGTCTCAAAC-AA-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-GAAGGTGAAGGTCGGAGTC-3′; and GAPDH reverse, 5′-GAAGATGGTGATGGGATTTC-3′. Immunohistochemical studies were performed on brain temporal sections using specific anti-lactadherin antibody,33Silvestre JS Thery C Hamard G Boddaert J Aguilar B Delcayre A Houbron C Tamarat R Blanc-Brude O Heeneman S Clergue M Duriez M Merval R Levy B Tedgui A Amigorena S Mallat Z Lactadherin promotes VEGF-dependent neovascularization.Nat Med. 2005; 11: 499-506Crossref PubMed Scopus (249) Google Scholar mouse anti-CD68 antibody (DAKO), mouse anti-GFAP antibody, and/or anti-human Aβ antibody (clone 4G8; Sigma, St. Louis, MO) after pretreatment of the section with 70% formic acid. Sudan Black was used to prevent nonspecific background fluorescence. Surface plasmon resonance, a technique for quantitating protein-protein interaction, was measured by a BIAcore X instrument (BIAcore International AB, Uppsala, Sweden) using CM5 sensor chips. Lactadherin was diluted in Hanks’ balanced salt solution buffer. Samples were injected at 25°C at a flow rate of 10 μl/minute over the active CM5 surface on which the Aβ 1-42 peptide had been immobilized to 7000 resonance units. An RGE lactadherin mutant was also used. It contains an Asp69 to glutamic acid point mutation in the RGD motif of lactadherin.33Silvestre JS Thery C Hamard G Boddaert J Aguilar B Delcayre A Houbron C Tamarat R Blanc-Brude O Heeneman S Clergue M Duriez M Merval R Levy B Tedgui A Amigorena S Mallat Z Lactadherin promotes VEGF-dependent neovascularization.Nat Med. 2005; 11: 499-506Crossref PubMed Scopus (249) Google Scholar Lactadherin-deficient mice and control littermates were obtained as we previously described.33Silvestre JS Thery C Hamard G Boddaert J Aguilar B Delcayre A Houbron C Tamarat R Blanc-Brude O Heeneman S Clergue M Duriez M Merval R Levy B Tedgui A Amigorena S Mallat Z Lactadherin promotes VEGF-dependent neovascularization.Nat Med. 2005; 11: 499-506Crossref PubMed Scopus (249) Google Scholar Macrophages were generated from bone marrow progenitors (from C57BL6 mice, lactadherin-deficient mice, and littermates controls), as described.38Lake FR Noble PW Henson PM Riches DW Functional switching of macrophage responses to tumor necrosis factor-alpha (TNF alpha) by interferons. Implications for the pleiotropic activities of TNF alpha.J Clin Invest. 1994; 93: 1661-1669Crossref PubMed Scopus (90) Google Scholar The cells were seeded in uncoated 24-well culture plates with RPMI medium containing 20% fetal calf serum and 20% L929 cell-conditioned medium. Human peripheral blood mononuclear cells were isolated using Ficoll gradient from whole blood (50 ml) of healthy donors (two men, one woman; mean age, 32 ± 5 years) who gave informed consent. The cells were allowed to adhere for 2 hours on uncoated 24-well culture plates, washed, and used for the following experiments. Cell preparations were incubated with microaggre-gates of 1 μg/ml fluorescein isothiocyanate (FITC)-con-jugated Aβ 1-42 peptide (rPeptide, Athens, GA) as previously described,39Paresce DM Ghosh RN Maxfield FR Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor.Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar with or without neutralizing anti-mouse or anti-human lactadherin antibody (45 μg/ml), or with an isotype-matched control antibody. The cells were washed, stained with 4,6-diamidino-2-phenylindole, and fixed with 1% paraformaldehyde. Phagocytosed FITC-conjugated Aβ appeared as punctuate fluorescent vesicles under a fluorescence microscope. The percentage of FITC-positive cells, as well as FITC intensity, was quantified using Histolab software (Microvisions, Louisville, KY). Lactadherin-deficient mice and control littermates received an intraperitoneal injection of thioglycollate (7%, 2 ml) (Sigma). Three days later, mice were injected intraperitoneally with either microaggregates of FITC-conjugated Aβ 1-42 peptide (500 μl at 5 μg/ml) or saline. Phagocytosis was allowed to proceed for 1 hour in vivo. Cells were collected from the peritoneal cavities, suspended in culture medium, pelleted, extensively washed, counted, and fixed with 1% paraformaldehyde. FITC-labeled macrophages were quantified by flow cytometry (Epics XL; Coulter, Hialeah, FL). Statistical analyses were performed using SAS statistical software, v.8.2 (SAS Institute Inc., Cary, NC). Values of lactadherin mRNA were log-transformed to normalize their distributions. The level of total RNA degradation (estimated by rRNA 28S/18S ratio) was identified as a confounder for lactadherin/GAPDH ratio using linear regression model (nonparametric Spearman's test, P = 0.047) and subsequently included (as well as age and gender) in a multivariate analysis of covariance using a general linear model for comparison of mRNA amount between AD and control cases.36Lambert JC Mann D Richard F Tian J Shi J Thaker U Merrot S Harris J Frigard B Iwatsubo T Lendon C Amouyel P Is there a relation between APOE expression and brain amyloid load in Alzheimer's disease?.J Neurol Neurosurg Psychiatry. 2005; 76: 928-933Crossref PubMed Scopus (45) Google Scholar Results are expressed as means ± SD. The Mann-Whitney rank-sum test was used for quantitative data. All reported P values are two-sided. Lactadherin is expressed in vessels of aged persons.40Häggqvist B Naslund J Sletten K Westermark GT Mucchiano G Tjernberg LO Nordstedt C Engstrom U Westermark P Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid.Proc Natl Acad Sci USA. 1999; 96: 8669-8674Crossref PubMed Scopus (179) Google Scholar However, the relation between lactadherin expression and age had not been explored. We examined lactadherin expression in cultured smooth muscle cells obtained from either newborns ( 18 years old). We observed higher lactadherin expression in adult smooth muscle cells compared with those from newborns (Figure 1). Interestingly, lactadherin expression increased in both newborn and adult smooth muscle cells as a function of time (Figure 1) and was secreted in the culture supernatants in exosomes (Figure 1). We obtained similar results in murine smooth muscle cells (data not shown), suggesting a species-independent process. Because AD is a prototype of age-associated disease, we examined lactadherin expression in brain cells. Lactadherin was undetectable in neonatal murine neuronal cells but was readily expressed in neonatal murine astrocytes, as well as in human adult astrocytes (Figure 1, C and D). Lactadherin expression in astrocytes also increased as a function of time in culture and was greatly enriched in astrocyte-derived exosomes (Figure 1, C and D). To examine the clinical relevance of these findings, we looked at lactadherin expression in brain specimens of AD patients and controls. Interestingly, we observed a 35% decrease of lactadherin mRNA expression in the brain of AD cases compared with controls (Figure 2). Immunohistochemical analysis of the protein also showed reduced expression in AD sections (Figure 2). Lactadherin expression was mostly detected in astrocytes (Figure 3, A–C). Expression was also detected occasionally in CD68-positive cells (Supplemental Figure 1, see http://ajp.amjpathol.org) and in smooth muscle cells of large arteries (Supplemental Figure 2, see http://ajp.amjpathol.org). Of note, lactadherin expression in AD sections was frequently observed in areas showing little or no Aβ accumulation, whereas it was frequently absent at close vicinity from senile plaques (Figure 3, D–F).Figure 3Lactadherin expression in brain temporal sections of patients with or without AD. A–E: Representative immunohistochemical staining of lactadherin protein expression in brain specimens of patients with AD (B–E) or controls (A). A: Staining for lactadherin (green) showing expression in cells with astrocyte morphology. B: Lactadherin appears in green and GFAP staining in red. C–E: Lactadherin appears in brown (arrows) and Aβ staining in pink/red (arrowheads). Note that lactadherin-expressing cells are negative for Aβ staining. F: Relation between lactadherin expression and the occurrence of senile plaques. Brain temporal sections of patients with AD were double-stained with lactadherin and Aβ (n = 6 patients). Three Aβ-positive and three Aβ-negative areas were identified in each patient, in which the mean intensity of lactadherin staining was estimated using a semiquantitative immunohistochemical score (0, no staining; 1, weak staining; 2, moderate; 3, strong). The histograms show the mean ± SD scores from six patients. Clearly, lactadherin expression occurred preferentially in Aβ-free areas.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The inverse association between lactadherin expression and Aβ deposits in AD brain suggested a potential role for lactadherin in removal of the Aβ peptide. In addition, using surface plasmon resonance, we observed a dose-dependent increase in direct protein-protein interaction between Aβ 1-42 peptide and recombinant human lactadherin (Figure 4), which was not observed with an RGE mutant of lactadherin. Thus, we examined the role of lactadherin in the phagocytosis of microaggregates of Aβ 1-42. First, we observed a time-dependent increase in the phagocytosis of FITC-conjugated Aβ 1-42 peptide by bone marrow-derived (Supplemental Figure 3, see http://ajp.amjpathol.org) or peritoneal-derived (not shown) murine macrophages. FITC-conjugated Aβ peptide was seen in vesicular structures within the macrophages (Supplemental Figure 3, see http://ajp.amjpathol.org); its uptake was saturable and was almost totally inhibited by excess of the scavenger receptor ligands, fucoidan and acetylated low-density lipoprotein (not shown), as previously described.39Paresce DM Ghosh RN Maxfield FR Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor.Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar Interestingly, uptake of FITC-Aβ showed a marked decrease (∼40% reduction, P = 0.03) in cell preparations treated with a neutralizing anti-lactadherin antibody in comparison with preparations treated with a control antibody (Figure 5, Figure 6). The anti-lactadherin antibody also inhibited the uptake of Aβ microaggregates by cultured murine microglial cells (Figure 5). The results were reproduced in mice deficient for lactadherin, whose macrophage preparations showed a marked reduction in the uptake of Aβ in comparison with control macrophages (Figure 6). Importantly, treatment of blood-derived human macrophages by a neutralizing anti-human lactadherin antibody significantly inhibited the uptake of FITC-Aβ microaggregates in comparison with a control antibody (55% reduction in fluorescence intensity, P = 0.007) (Figure 7). Finally, lactadherin-deficient mice showed significant reduction in the phagocytosis of Aβ 1-42 peptide by peritoneal macrophages in vivo compared with control littermate mice (Figure 8).Figure 6Lactadherin and phagocytosis of Aβ microaggregates. A: Example of uptake of FITC-conjugated Aβ 1
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