LRP and senile plaques in Alzheimer’s disease: colocalization with apolipoprotein E and with activated astrocytes

Molecular Brain Research 104 (2002) 38–46 www.elsevier.com / locate / bres

Research report

LRP and senile plaques in Alzheimer’s disease: colocalization with apolipoprotein E and with activated astrocytes ´ a , Ayae Kinoshita a , Christa M. Whelan a , Michael C. Irizarry a , Katrin Arelin G. William Rebeck a , Dudley K. Strickland b , Bradley T. Hyman a , * a

Alzheimer Research Unit, Department of Neurology, Massachusetts General Hospital, 114 16 th Street, Room 2009, Charlestown, MA 02129, USA b Department of Vascular Biology, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA Accepted 22 March 2002

Abstract The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer’s disease (AD). It is suggested to play an important role in the balance between amyloid beta (Ab) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Ab. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Ab deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Ab. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Ab /ApoE complexes.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s—other Keywords: Alzheimer; LRP; Astrocyte; apoE; PDAPP mice

1. Introduction Senile plaques (SPs), a major pathologic hallmark of AD, consist primarily of the amyloid b-peptide which is derived from the amyloid precursor protein (APP) by proteolytic processing. In addition to Ab, several diverse proteins have been found to be associated with senile plaques. These include a variety of proteinases (such as trypsin [38]), proteinase inhibitors (such as a 2 -macroglobulin (a2M) [4,45] and a 1 -antichymotrypsin [1]), complement (C3c) [10], apolipoprotein E (apoE), and its receptor LRP [29,34,35,37,43,47]. ApoE is strongly related to Alzheimer’s disease patho*Corresponding author. Tel.: 11-617-726-2299; fax: 11-617-7241480. E-mail address: b [email protected] (B.T. Hyman). ]

genesis from several perspectives. A role for apoE in promoting amyloid deposition is suggested by the genetic predisposition for AD and the increased amyloid burden in AD brains of patients carrying the APOE ´4 allele [13,35,37]. Further data suggest that apoE can modulate Ab fibrillogenesis [7,11,25,41] and LRP-mediated Ab clearance [5,19,46]. Additional evidence of a role for apoE in Ab deposition comes from studies of APP V717F transgenic mice in wild-type and APOE-knockout backgrounds. APOE-knockout mice showed a reduction of cortical Ab deposits and elimination of fibrillar Ab-deposits. The diffuse Ab deposits in the APOE null mice also showed a different distribution from the deposits in wild-type mice [3,16,17]. These data suggest that apoE is critical for the development of fibrillar, compact amyloid plaques. The apoE receptor LRP has also been implicated in AD pathophysiology (see Ref. [15] for review). LRP is a

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member of the LDL-receptor family. These receptors each possess clusters of ligand binding repeats for binding of multiple ligands. Ligand–receptor interactions can be antagonized by the receptor associated protein (RAP). LRP binds more than 20 identified ligands, including ApoE, APP, a2M and tissue plasminogen activator [40]. After receptor binding, these ligands undergo endocytosis and degradation. In the brain, LRP is expressed in neurons, activated astrocytes, choroid plexus and microglia [26,28,35,48]. A shed form of the receptor (lacking the C-terminal portion that anchors LRP to the membrane) is also present in the cerebrospinal fluid [32,33]. In AD, LRP is upregulated in activated astrocytes and associated with senile plaques [6,14,35,43]. In fact, many LRP ligands are also found on senile plaques [34]. The origin of the LRP associated with plaques is unknown and the relationship between LRP and plaque type has not been examined closely. In order to address these questions, we carried out immunohistochemical studies of LRP and Ab deposits in both transgenic mice and human AD using confocal microscopy. We examined colocalization of LRP with Ab, apoE, microglia and activated astrocytes. Furthermore we used transgenic mouse brains of different ages to study the chronology of deposition of LRP on senile plaques.

2. Materials and methods

2.1. Human tissue Brain tissue from five patients with the diagnosis and neuropathological confirmation of AD was provided by the Alzheimer’s Disease Research Center Brain Bank (Dr E.T. Hedley-Whyte, Director). The age of patients ranged from 73 to 83 years. Brain tissues from three age-matched individuals without any neurological diseases (postmortem interval for all tissues ,24 h) were used as controls. All brains were fixed in paraformaldehyde-lysine-metaperiodate for 24–36 h and transferred to 15% glycerol in tris(hydroxymethyl)aminomethane-buffered saline solution (TBS). Sections were prepared at 50 mm on a freezing sledge microtome and stored in a solution of 15% glycerol

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in TBS, pH 7.4 at 220 8C. Immunohistochemistry was carried out on free-floating sections.

2.2. Transgenic mice The PDAPP transgenic mouse overexpresses human amyloid precursor protein with the V717F mutation. These mice develop age-related Ab deposits in a characteristic and discrete anatomical pattern similar to senile plaques in Alzheimer’s disease [8,12,18]. For our studies we used 9-, 15- and 22-month-old PDAPP mice (courtesy of Dr D. Schenk, Elan Pharmaceuticals). We also evaluated tissue from a 12-month-old APP V717F1 / 1 APOE 2 / 2 mouse [3,16] (courtesy of Dr K. Bales and Dr S. Paul, Lilly Pharmaceuticals). Transgenic mouse brains were immersion-fixed in 4% paraformaldehyde overnight at 4 8C and immersed in 30% sucrose solution. Then, 30-mm-thick slices were cut on a freezing microtome and immunostained as described below.

2.3. Immunostaining The antibodies used in this study are shown in Table 1. For analysis by immunoperoxidase-based methods, brain tissue was washed in TBS and treated for 30 min with 0.3% H 2 O 2 . Sections were blocked in 1.59% normal goat serum, followed by incubation with the primary antibody at 4 8C overnight. After washing with TBS, biotinylated anti-rabbit or anti-mouse IgG secondary antibodies were added for 1 h at room temperature followed by avidin– biotin–peroxidase complex treatment for 1 h (ABC kit 1:100 Vector Laboratories, Inc. Burlingame, CA). Staining was visualized with diaminobenzidine (DAB). For immunofluorescent studies of mouse tissue, sections were blocked in 1.5% normal goat serum for 1 h at room temperature. Primary antibodies were incubated overnight at 4 8C in TBS-solution containing 0.1% Triton X-100, 1.5% normal goat serum. Sections were washed in TBS three times and labeled with appropriate secondary antibodies for 1 h at room temperature (fluorescein anti-rabbit

Table 1 Antibodies Antibody

Antigen

Concentration

Source

3d6 (ms) 10d5 (ms) R829 (rb) R704 (rb) GFAP-Cy3 (ms) Bi-tomato-lectin 3H1 (ms) 1D7 (ms) AT8 (ms)

Ab Ab LRP LRP (b-chain) GFAP Microglia ApoE C-term. ApoE Phospho-tau

1:500 1:100 1:1000 1:1000 1:400 1:2000 1:500 1:200 1:100

Elan Pharmaceuticals Elan Pharmaceuticals D. Strickland [21] D. Strickland [39] Sigma Vector Laboratories Ottawa Heart Inst. Research Corp. [9] Ottawa Heart Inst. Research Corp. [9] Innogenetics

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and anti-mouse, Cy3 anti-rabbit and anti-mouse, Cy5 antimouse, Jackson ImmunoResearch, West Grove, PA; Alexa 488 anti-rabbit, Molecular Probes, Inc., Eugene, OR). Sections were placed on gelatin-coated slides and mounted with Vectashield mounting medium for fluorescence (Vector Laboratories, Inc. Burlingame, CA). For immunofluorescence studies of human tissue, freefloating sections were washed in TBS three times and then treated with 0.3% H 2 O 2 in TBS for 30 min. For immunostaining of apoE, pretreatment with 70% formic acid was carried out for 10 min at room temperature to maximize epitope recognition [9]. After washing in TBS, sections were blocked in normal goat serum for 1 h at room temperature and incubated overnight at 4 8C with primary antibodies in TBS-solution containing 0.1% Triton X-100, 1.5% normal goat serum. Washing in TBS was followed by labeling with appropriate secondary antibodies for 1 h at room temperature and sections were mounted with Vectashield. Immunostaining was observed by confocal microscopy with appropriate filters (Bio-Rad 1024 confocal microscope mounted on a Nikon Eclipse TE300 inverted microscope) and Bioquant Image Analysis System.

3. Results

3.1. LRP immunostains the periphery of senile plaques in PDAPP mice and in human AD Previous studies showed that LRP is strongly associated with b-amyloid deposits in Alzheimer’s disease. Ab clearance mediated through LRP has been demonstrated in several systems in vitro [5,19,31,44,49]. It is not yet known whether LRP is deposited on senile plaques or whether LRP is associated with cellular structures around senile plaques. We used two different rabbit polyclonal LRP antibodies: R829 was raised against the holoprotein [20], whereas R704 recognizes only the intracellular part of LRP (Cterminus) [21,39]. LRP immunostaining of PDAPP transgenic mice and five human AD brains revealed strong neuronal and some astrocytic staining, as well as a subset of senile plaques. LRP was present exclusively on the periphery of the plaques (Fig. 1). The ‘empty core’ pattern of staining was found using both the antibody against holo-LRP (R829) and the antibody against the intracellular part of the b-chain (antibody R704). R704 would not label shed forms of LRP implying that full-length LRP is present in these amyloid deposits. The empty core pattern of LRP immunostaining was particularly noticeable in PDAPP transgenic mice (panels A and B) but less pronounced in human brain (panels C and D). In the three control cases we detected neuronal and some astrocytic staining, but no LRP positive senile plaques.

3.2. LRP labels only a subset of Ab deposits We examined colocalization of LRP and Ab to determine the extent of LRP staining of senile plaques. Double labeling with antibodies against the Ab peptide and LRP showed that a large fraction of Ab-positive plaques did not stain for LRP. We assessed about 500 plaques in five AD cases and found that only one-third to one-half of plaques were positive for both Ab and LRP. Similar results were obtained in analysis of Ab deposits in the brain of a 22-month-old PDAPP mouse. The double labeling for Ab and LRP showed that positive staining for LRP occurs in larger, denser Ab deposits rather than in diffuse Ab deposits. To confirm the impression that dense plaques, rather than diffuse plaques, were LRP positive, we examined the brains of PDAPP transgenic mice at two ages, 9 and 15 months old. In 9-month-old mice, many Ab-labeled diffuse plaques were found, but no LRP staining of plaques was observed. In contrast, 15-month-old mice showed dense-core Ab deposits that were LRP-positive, as well as diffuse Ab deposits that were LRP-negative. These results demonstrate that LRP is a component of cored senile plaques but is not a component of diffuse deposits.

3.3. LRP on senile plaques colocalizes with activated astrocytes Microglia, astrocytes and neurons all express LRP in normal brain [26,28,35,48]. We hypothesized that LRP around amyloid deposits was associated with processes from one or more of these cell types. Therefore we examined whether LRP colocalized with microglia (using tomato-lectin), activated astrocytes (anti-GFAP antibody) or dystrophic neurites (AT8 antibody). Double-labeling of senile plaques showed strong colocalization of LRP with distal processes of activated astrocytes (Fig. 2). In the PDAPP mice, GFAP-positive activated astrocytes surrounding plaques showed fine distal processes that form a pattern like that seen in the LRP staining. In human tissue the staining was similar, although resolving these fine astrocytic processes was more difficult. LRP was far less prominent in proximal processes and the astrocyte soma. To confirm that the colocalization did not occur with the shed form of the extracellular domain of LRP, we carried out double labeling with antibody R704, which recognizes the intracellular domain of LRP, and GFAP. As shown in Fig. 3, R704 also revealed staining in the distal astrocytic processes, suggesting that the LRP is present in its full length, membrane associated form. Double labeling of LRP with microglia showed an association of microglia with LRP-positive plaques, but microglial staining did not appear to account for the pattern of LRP staining in plaques (Fig. 4). Similarly, antibody AT8, which identifies a subset of dystrophic neurites, showed no clear association with LRP-positive

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Fig. 1. LRP immunostaining of senile plaques in PDAPP mice and human AD. Antibodies against the entire LRP molecule (panel A) and the intracellular part of LRP (b-chain) (panel B) stain exclusively the peripheral part of senile plaques in PDAPP mice. Antibodies R829 (holo-LRP) and R704 (intracellular domain) were detected with Cy3-labeled secondary antibodies. LRP staining of plaques was also seen in human brain: holo-LRP (panel C), and b-chain (panel D) (antibodies detected using peroxidase-labeled secondaries, developed with DAB). Scale bar: A523 mm, B528 mm, C522 mm, D520 mm.

structures, suggesting that dystrophic neurites also do not contribute directly to the LRP staining of plaques.

3.4. LRP and apoE: all LRP-positive plaques are also stained for apoE ApoE is an important ligand for LRP and is known to be present on amyloid deposits in AD. Many in vitro studies have suggested its involvement in lipid and Ab-clearance [5,19,27,46]. We analyzed apoE and LRP around Ab deposits to determine if there was in vivo data to support

this model. We immunostained five AD cases and three controls with the apoE antibodies 3H1 (which identifies the C-terminus of apoE, residues 243–272) and 1D7 (which identifies the apoE ligand binding side, residues 140–160). All plaques that were labeled for LRP were also positive for apoE (Fig. 5). In addition, there were apoE-positive plaques that did not stain for LRP. We did not detect any LRP-stained plaques that were apoE-negative. In contrast to LRP staining of plaques, which is located in the periphery, apoE was present throughout plaques with a concentration in the central part (Fig. 5). Manual counting

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Fig. 2. LRP colocalizes with fine astrocytic processes. PDAPP mouse brain was triple-stained for GFAP (direct-labeled anti-GFAP-Cy3 in red, panel A), LRP (R829 labeled with secondary antibody Alexa 488 in green, panel B) and Ab (3d6 labeled with secondary antibody Cy5 in blue, panel C). Panel D (combined panels A, B and C) showed colocalization of LRP and surrounding astrocytes. Scale bar520 mm.

of plaques in the five human AD cases showed that about 50% of apoE-positive plaques were also positive for LRP. From these data we hypothesized that apoE is necessary for the accumulation of LRP around plaques. We tested this hypothesis by analyzing LRP expression around Ab deposits in an APOE knockout mouse [16]. We found prominent LRP staining of neurons, but no staining of plaques. Importantly, APOE knockout mice do not develop cored plaques, exhibiting only diffuse Ab-deposits. These data show that, in the absence of apoE, neither cored plaques nor LRP-positive plaques are developed.

4. Discussion ApoE and its receptor LRP are both closely associated with senile plaques in the AD brain [29,34,35,37,43,47]. In this study we used LRP immunohistochemistry to closely examine the localization of LRP in plaques and associated cell types. We found that LRP is a prominent component of compact senile plaques, but is not present in diffuse plaques. In AD brain, only about one-third to one-half of plaques are LRP-positive. LRP-immunostaining of plaques

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Fig. 3. Distal astrocytic processes colocalize with the membrane-bound form of LRP. PDAPP mouse brain was stained for activated astrocytes (direct labeled anti-GFAP-Cy3, panel A) and the C-terminal part of LRP (membrane-bound form) (R704 labeled with secondary antibody Alexa 488, panel B). The staining showed colocalization similar to antibody R829. Scale bar520 mm.

is identical for N-terminal and C-terminal epitopes of LRP, suggesting that the full molecule is present. In contrast, Thal et al. [42] reported a difference in staining using other antibodies specific for the a- and b-subunits of LRP. The

Fig. 4. Double labeling of LRP and microglia. PDAPP mouse brain was stained for LRP (R829 labeled with secondary antibody Cy3, in red) and microglia (bi-tomato-lectin labeled with secondary antibody FITC, in green). Microglia and LRP were both present on some Ab deposits, but LRP did not significantly colocalize to microglial processes (colocalization in yellow). Scale bar520 mm.

difference between our results may well be due to the sensitivity of the reagents used to detect the b-subunit carboxyl terminus of LRP. In addition to diverse proteins that are found on senile plaques, reactive astrocytes and microglia have been observed in association with amyloid deposits in AD brain and may act as mechanisms of Ab-clearance [2,30,35,36]. In vitro, LRP can clear soluble forms of Ab in the presence of the LRP ligands apoE and a2M [5,31,44,49]. These ligands, which are prominently associated with amyloid deposits, can induce LRP expression in astrocytes in vitro [32]. This induction in the vicinity of plaques would allow increased clearance of LRP ligands and perhaps the Ab-deposits themselves. In this study we found that membrane-bound LRP around plaques colocalized with fine surrounding astrocyte processes. The strong expression of LRP in astrocytes around plaques supports the hypothesis that it could act as an Ab clearance mechanism into activated astrocytes. We found that LRP is only observed around plaques that are apoE immunoreactive. ApoE is not found in a subset of small or diffuse Ab deposits [9]. These deposits also do not have elevated astrocytic LRP. In vitro, Ab can activate glia directly through LRP [22], and this activation can be inhibited by exogenous apoE [23]. A role for apoE in inhibiting glial activation is supported by studies of glia from ApoE knock-out mice, which show enhanced secretion of several cytokines after activation by lipopolysaccharide [24] or Ab [23]. Together these data suggest that a complex between Ab, apoE, and LRP forms on glia, resulting in a reduction in the inflammatory response; this model is supported by the colocalization in vivo of these

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Fig. 5. LRP and ApoE on senile plaques. Human AD brains were stained with an antibody against LRP (R829 labeled with secondary antibody Cy3, panel A) and an antibody against the receptor binding site of ApoE (1d7 labeled with secondary antibody FITC, panel B). In contrast to the peripheral localization of LRP, apoE was more concentrated in the central part of plaques. Scale bar520 mm.

three molecules on the surface of astrocytes around Ab deposits. In this study, we made several observations from close analysis of LRP expression in senile plaques in AD brain. First, LRP is a prominent component of compact senile plaques and is located exclusively in the periphery of plaques. Second, membrane-bound LRP colocalizes with surrounding astrocytes. Third, there is a high degree of colocalization of LRP with the LRP-ligand, apoE. Together, these data suggest that apoE induces LRP expression around plaques, and may act as a local scaffolding protein for LRP and Ab, perhaps promoting astrocytic clearance of Ab. Continued in vivo and in vitro analysis of apoE and its receptors may lead to a better understanding of the mechanisms affecting Ab deposition in the brain.

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Acknowledgements Supported by NIH AG12406 (B.T. Hyman), NIH AG14473 (G.W. Rebeck) and the Uehara Foundation (A. ´ Kinoshita). Katrin Arelin was supported by the German National Scholarship Foundation (Studienstiftung des deutschen Volkes). We thank Drs Dale Schenk and Dora Games (Elan Pharmaceuticals) and Kelly Bales and Steven Paul (Lilly Pharmaceuticals) for access to tissue from transgenic mice.

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