Evidence of a Role for Lactadherin in Alzheimer??s Disease

【摘要】  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 ß-peptide (Aß). 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 Aß deposition. Using surface plasmon resonance, we observed a direct protein-protein interaction between recombinant lactadherin and Aß 1-42 peptide in vitro. Lactadherin deficiency or its neutralization using specific antibodies significantly prevented Aß 1-42 phagocytosis by murine and human macrophages. In conclusion, lactadherin plays an important role in the phagocytosis of Aß 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.
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Alzheimer??s disease (AD) is the leading cause of dementia in the elderly, possibly affecting 20 to 40% of the population older than age 85.1-3 The hallmarks of AD are neuronal loss, extracellular accumulation of senile plaques, the major component of which is the amyloid ß-peptide (Aß), and the presence of neurofibrillary tangles.3,4 Excess deposition of Aß is involved in neur-onal cell death and is thought to result from an imbal-ance between Aß production, aggregation, and/or clearance.5,6
The Aß peptides result from the processing of the ß-amyloid precursor protein (APP) by the ß- and -secretases.7-10 Pathogenic mutations in the APP and presenilin 1 and 2 genes (involved in the -secretase activity) have clearly demonstrated the major role of Aß overproduction in the AD process.3,8,11,12 Recently reinforcing this role, genetic variability in the ubiquilin gene, which regulates endoproteolysis of presenilin and modulates -secretase components, has been associated with familial forms of AD.13,14
The most consistent genetic variability associated with the nonfamilial, sporadic form of the disease is that at the apolipoprotein (apo)E locus, which encodes a protein involved in lipid transport.3,15 The molecular mechanisms by which the apoE 4 allele alters the lifetime risk of AD are currently unknown. ApoE 4 may modulate Aß oligomerization into fibrils.16 In addition, recent studies have suggested that apoE may be involved in Aß clearance, in part through its receptor, the low-density lipoprotein receptor-related protein.17 Understanding how the Aß peptide is removed from the brain could lead to the identification of critical molecular pathways that could be targeted to reduce Aß burden.18 However, apart from lipoprotein receptor-related protein- or P-glycoprotein-mediated Aß efflux,19-21 little is known about the mechanisms responsible for Aß clearance from the brain.18
Lactadherin or Mfge8 (milk fat globule-epidermal growth factor-like-factor VIII) is a glycoprotein originally identified as a component of the membrane of milk fat globules, with an RGD motif that binds to integrins vß3 and vß5, and a domain involved in binding to phosphatidylserine.22-24 As a component of the exosomes of dendritic cells or macrophages,25,26 lactadherin forms a bridge between phosphatidylserine on apoptotic cells and integrins on phagocytes, leading to engulfment and phagocytosis of apoptotic cells.26 This function is critical to tissue homeostasis as total deficiency in lactadherin leads to autoimmune diseases.27,28 Interestingly, apoE has been involved in phagocytosis of both apoptotic cells and Aß peptide.17,29 We therefore hypothesized that in a similar way, lactadherin may also contribute to clearance of the Aß peptide.

【关键词】  evidence lactadherin alzheimer??s

Materials and Methods

Expression of Lactadherin in Cultured Cells and Supernatants

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.30 Primary cultures of mixed glia were prepared from newborn mice as described.31 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.32 Protein extracts from cells and exosomes were examined for the presence of lactadherin and other exosomal-specific proteins by Western blotting using specific antibodies.32 We previously described the generation and specificity of the anti-mouse and anti-human lactadherin antibodies.33 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).

Brain Samples

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.34 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).35 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.36

Real-Time Polymerase Chain Reaction (PCR) on Brain Samples

Real-time quantitative PCR was performed as described.37 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 on Brain Samples

Immunohistochemical studies were performed on brain temporal sections using specific anti-lactadherin antibody,33 mouse anti-CD68 antibody (DAKO), mouse anti-GFAP antibody, and/or anti-human Aß 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

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 Aß 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.33

In Vitro Phagocytosis Studies

Lactadherin-deficient mice and control littermates were obtained as we previously described.33 Macrophages were generated from bone marrow progenitors (from C57BL6 mice, lactadherin-deficient mice, and littermates controls), as described.38 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 Aß 1-42 peptide (rPeptide, Athens, GA) as previously described,39 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 Aß 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).

In Vivo Phagocytosis Studies

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 Aß 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 Analysis

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.36 Results are expressed as means ?? SD. The Mann-Whitney rank-sum test was used for quantitative data. All reported P values are two-sided.

Results

Age- and Time-Related Increase in the Production of Lactadherin

Lactadherin is expressed in vessels of aged persons.40 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 (<2 years old) or adults (>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) .

Figure 1. Lactadherin expression in cultured cells. Shown are the results of Western blot analyses. In each panel, equal amounts of proteins were loaded. A: Western blot analysis was performed on protein extracts of confluent human aortic smooth muscle cells (SMCs) obtained at different time points (d indicates day) after the beginning of culture. SMCs were from either newborn (N; <2 years old) or adult (A; >18 years old) humans. Lactadherin expression is higher in adult SMCs and increases as a function of time in culture in both newborn and adult SMCs. B: Lactadherin is enriched in supernatant membrane fractions presenting the characteristics of exosomes, ie, membrane fractions rich in Lamp-2, MHC-I, and tsg101 (pellet 3). Pellets 1, 2, and 3 were obtained after serial centrifugation of cell supernatants at 2,500 x g for 20 minutes (pellet 1; cell debris), then 10,000 x g for 45 minutes (pellet 2, cell microparticles), then 100,000 x g for 90 minutes (pellet 3, exosomes). C: Lactadherin expression in confluent adult human astrocytes. D: Lactadherin expression in confluent murine neonatal astrocytes. Cell-associated lactadherin expression increases with time in culture.

Lactadherin Expression and AD

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, ACC) . 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 Aß accumulation, whereas it was frequently absent at close vicinity from senile plaques (Figure 3, DCF) .

Figure 2. Lactadherin expression in normal and AD brains. A: Representative immunohistochemical staining of lactadherin protein expression (green) in brains of patients with AD or controls. B: Brain mRNA levels of lactadherin/GAPDH ratio (expressed as percentage of controls), determined using quantitative RT-PCR. Results are expressed as mean ?? SD (n = 52 AD brains and 58 control brains).

Figure 3. Lactadherin expression in brain temporal sections of patients with or without AD. ACE: Representative immunohistochemical staining of lactadherin protein expression in brain specimens of patients with AD (BCE) 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. CCE: Lactadherin appears in brown (arrows) and Aß staining in pink/red (arrowheads). Note that lactadherin-expressing cells are negative for Aß 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 Aß (n = 6 patients). Three Aß-positive and three Aß-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 Aß-free areas.

Lactadherin Contributes to Phagocytosis of the Amyloid ß-Peptide

The inverse association between lactadherin expression and Aß deposits in AD brain suggested a potential role for lactadherin in removal of the Aß peptide. In addition, using surface plasmon resonance, we observed a dose-dependent increase in direct protein-protein interaction between Aß 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 Aß 1-42.

Figure 4. Direct protein-protein interaction between recombinant human lactadherin and Aß 1-42 peptide using surface plasmon resonance. Top: Recombinant human lactadherin or an RGE lactadherin mutant was injected at 25??C at a flow rate of 10 µl/minute over the active CM5 surface on which the Aß 1-42 peptide had been immobilized. The RGE mutant of lactadherin does not bind Aß 1-42 peptide. Bottom: Increased lactadherin-Aß interaction with increasing amounts of lactadherin. The figure also shows partial but significant inhibition of lactadherin-Aß interaction using anti-human lactadherin antibody (anti-lac, 45 µg/ml) directed against the RGD sequence of human lactadherin.

First, we observed a time-dependent increase in the phagocytosis of FITC-conjugated Aß 1-42 peptide by bone marrow-derived (Supplemental Figure 3 , see http://ajp.amjpathol.org) or peritoneal-derived (not shown) murine macrophages. FITC-conjugated Aß 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.39 Interestingly, uptake of FITC-Aß 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 (Figures 5 and 6) . The anti-lactadherin antibody also inhibited the uptake of Aß 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 Aß 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-Aß 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 Aß 1-42 peptide by peritoneal macrophages in vivo compared with control littermate mice (Figure 8) .

Figure 5. Lactadherin and phagocytosis of Aß microaggregates. A: Representative example of uptake of FITC-conjugated Aß 1-42 peptide by cultured bone marrow-derived murine macrophages obtained from lactadherin wild-type mice with (+anti-lac) or without (+IgG) in vitro pretreatment with a neutralizing anti-mouse lactadherin antibody (n = 3 to 5 per condition). B: Representative example of uptake of FITC-conjugated Aß 1-42 peptide by primary murine microglial cells from C57BL/6 mice with (+anti-lac) or without (+IgG) in vitro pretreatment with a neutralizing anti-mouse lactadherin antibody (n = 3 per condition).

Figure 6. Lactadherin and phagocytosis of Aß microaggregates. A: Example of uptake of FITC-conjugated Aß 1-42 peptide by cultured bone marrow-derived murine macrophages obtained from either lactadherin-deficient mice (lac KO) or wild-type (lac WT) littermate controls. B: Quantitative analysis (Histolab software; Microvision) of the percentage of FITC-positive cells (percent positive cells) and the mean fluorescence intensity (expressed in arbitrary units, AU) in the murine macrophage cultures from lactadherin-deficient mice or from wild-type mice with or without in vitro pretreatment with a neutralizing anti-mouse lactadherin antibody (anti-lac) (n = 3 to 5 per condition). Control IgG antibody was added in lac WT and lac KO conditions.

Figure 7. Lactadherin and phagocytosis of Aß microaggregates by blood-derived human macrophages in vitro. A: Two examples of uptake of FITC-conjugated Aß 1-42 peptide by cultured blood-derived human macrophages in the presence (+anti-Lac) or absence (+IgG) of a neutralizing anti-human lactadherin antibody. B: Quantitative analysis (Histolab software; Microvision) of the percentage of FITC-positive cells (percent positive cells) and the mean fluorescence intensity (expressed in arbitrary units, AU) in the above-mentioned cultures (n = 3 per condition). Values are means ?? SD. P values are versus IgG.

Figure 8. Lactadherin and phagocytosis of Aß microaggregates by murine peritoneal macrophages in vivo. Quantification using flow cytometry of the uptake of FITC-conjugated Aß 1-42 peptide by peritoneal macrophages in vivo. Macrophages of lactadherin-deficient mice (lac KO, n = 10) showed reduced Aß uptake compared with littermate controls (lac WT, n = 11; P = 0.0009).

Discussion

Aß accumulation results from an imbalance between production and clearance.3 Although overproduction of Aß is mostly involved in familial forms of AD, sporadic, late-onset AD is thought to result from impaired clearance, leading to Aß accumulation and precipitating its conversion into toxic forms. Several mechanisms account for Aß clearance, including proteolytic degradation,41,42 elimination by passive bulk flow,43 active transport across the blood-brain barrier,20,21,44 and phagocytosis by astrocytes and microglia.45,46

In this study, we have provided a series of data that implicate lactadherin in a major process related to the pathophysiology of AD, namely Aß-peptide phagocytosis. The age- or time-related expression of lactadherin, its vascular and brain parenchymal expression (astrocytic and microglial), further highlight the potential role of this glycoprotein in the protection against excess Aß accumulation with aging.

We found a significant reduction in lactadherin mRNA expression in AD brains compared with controls. The difference of lactadherin expression between AD and control brains was independent of RNA degradation and unlikely to be attributable to differences in genetic background between UK and French populations.47-50 However, at this stage of the analysis, it is not possible to take into account environmental factors such as dietary habits.

The present studies conducted in human brain tissue showing decreased mRNA expression of lactadherin and altered protein expression/distribution in patients with AD are highly suggestive of a role for this glycoprotein in the disease process. The finding that a neutralizing antibody against lactadherin or a complete deficiency of this glycoprotein in mice inhibits Aß 1-42 phagocytosis by macrophages/microglia, provides an interesting mechanistic insight into the direct role of lactadherin in a critical process contributing to the neuropathology of AD.

A recent study showed that a small fraction of Aß (<1%) produced by neurons may be secreted in exosomes.51 Thus, it could be argued that association of lactadherin and Aß on exosomes would rather enhance Aß secretion and facilitate its extracellular accumulation. However, we were unable to detect significant lactadherin expression in neurons. Thus, exosomal Aß secreted by neurons would not associate with lactadherin before secretion. In contrast, secreted Aß could encounter exosomal lactadherin (produced by astrocytes and microglia) in the extracellular milieu, leading to its phagocytosis and clearance. Our observations in vitro and in vivo clearly suggest that when lactadherin is expressed, Aß phagocytosis is enhanced, impeding its extracellular accumulation. This could explain, at least in part, why Aß fibrils accumulate in areas with no or low lactadherin expression. In addition to reduced mRNA expression of lactadherin, alterations in vesicular transport, as reported in AD,52 may impair exosome secretion in several cell types, potentially leading to a reduction in lactadherin-mediated Aß phagocytosis.

It is noteworthy that Aß peptides, particularly Aß 1-42, show high affinity for phosphatidylserine,53 the aminophospholipid that interacts with lactadherin for apoptotic cell phagocytosis.26,27 Thus, Aß peptides may concentrate in phosphatidylserine-rich membranes and become targets for removal by lactadherin (ultimately leading to their degradation). This could be particularly relevant if, as widely reported,3 Aß peptides induce cell activation or apoptosis, exposing phosphatidylserine at the external membrane leaflet. Our findings that lactadherin contributes to Aß phagocytosis suggest that Aß would tend to accumulate and exert its neurotoxic effects in areas with altered lactadherin production. Reduced expression of lactadherin in patients with AD would also lead to accumulation of apoptotic debris. We speculate that increased cell death and defective phagocytosis of apoptotic cells could alter the anti-inflammatory/tolerogenic arm of the immune response and set the stage for exaggerated pathogenic responses,27,54 leading to disease aggravation. Finally, lactadherin deficiency might aggravate tissue response to ischemia,33 magnifying the vascular dysfunction of AD.55

However, it remains to be established that lactadherin deficiency directly leads or contributes to Alzheimer??s neuropathology in experimental models of the disease. Future studies should examine the effect of lactadherin deficiency on the accumulation of senile plaques in animal models of AD. Nevertheless, we believe that the data presented here point to a potentially important and new role for lactadherin in AD and should pave the way for future studies aiming at the comprehension of the precise mechanisms involved in these processes.

Acknowledgements

We thank Clotilde Thery and Sebastian Amigorena (Inserm U653) for sharing the polyclonal anti-lactadherin antibodies and the lactadherin-deficient mice and Alain Delcayre (Anosys) for providing the recombinant mouse lactadherin.

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作者单位：From the Institut National de la Sant? et de la Recherche M?dicale (INSERM),* Unit 689, Centre de Recherche Cardiovasculaire Lariboisire, Paris, France; INSERM U508, Epid?miologie des Maladies Chroniques D?g?n?ratives, Institut Pasteur de Lille, Lille, France; the Laboratoire D?veloppement et Vieill