Changes in Brain β-Amyloid Deposition and Aquaporin 4 Levels in Response to Altered Agrin Expression in Mice

Changes in Brain β-Amyloid Deposition and Aquaporin 4 Levelsin Response to Altered Agrin Expression in Mice

Steven M. Rauch, MD1, Kathy Huen, BSc2, Miles C. Miller, MSc2, Hira Chaudry, BSc2,Melissa Lau, AB2, Joshua R. Sanes, PhD3, Conrad E. Johanson, PhD2, Edward G. Stopa,MD2, and Robert W. Burgess, PhD1

1The Jackson Laboratory, Bar Harbor, Maine 2Department of Pathology and Laboratory Medicine,Brown University, Providence, Rhode Island; 3Department of Molecular and Cellular Biology,Harvard University, Cambridge, Massachusetts

AbstractConditions that compromise the blood-brain barrier (BBB) have been increasingly implicated inthe pathogenesis of Alzheimer disease (AD). AGRIN is a heparan sulfate proteoglycan (HSPG)found abundantly in basement membranes of the cerebral vasculature, where it has been proposedto serve a functional role in the BBB. Furthermore, AGRIN is the major HSPG associated withamyloid plaques in AD brains. To examine the relationship of AGRIN, the BBB and AD-relatedpathologies, we generated mice in which the Agrn gene was deleted from either endothelial cellsor neurons using gene-targeting or was overexpressed using a genomic transgene construct. Thesemice were combined with a transgenic model of AD that overexpresses disease-associated formsof amyloid precursor protein and presenilin1. In mice lacking endothelial cell expression of Agrn,the BBB remained intact but aquaporin 4 levels were reduced, indicating that the loss of AGRINaffects BBB-associated components. This change in Agrn resulted in an increase in β-amyloid(Aβ) in the brain. Conversely, overexpression of Agrn decreased Aβ deposition, whereaselimination of Agrn from neurons did not change Aβ levels. These results indicate that AGRIN isimportant for maintaining BBB composition and that changes in Agrn expression (particularlyvessel-associated AGRIN) influence Aβ homeostasis in mouse models of AD.

KeywordsAlzheimer disease; Aquaporin 4; Blood-brain barrier; Conditional knockout; Fibrillogenesis;Isoform-specific knockout; Orthogonal arrays; Transmembrane agrin

INTRODUCTIONAlzheimer disease (AD) pathology is characterized by the accumulation of intracellulartangles containing tau, and extracellular plaques containing β-amyloid (Aβ) (1). Therelationship between these pathological features and the progression of neuronal loss anddementia is unclear (2), although several molecular and physiological pathways that

Send correspondence and reprint requests to: Robert W. Burgess, The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609.Phone: 207-288-6706; Fax: 207-288-6077; [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptJ Neuropathol Exp Neurol. Author manuscript; available in PMC 2012 December 1.

Published in final edited form as:J Neuropathol Exp Neurol. 2011 December ; 70(12): 1124–1137. doi:10.1097/NEN.0b013e31823b0b12.

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contribute to the neurodegeneration are emerging. A longstanding observation is thatheparan sulfate proteoglycans (HSPGs) are associated with plaques in AD brains (3). Thereis a direct interaction between Aβ and heparan sulfate moieties that hastens Aβ fibrilformation, suggesting that the presence of HSPGs in plaques may result in a positivefeedback mechanism and increased plaque pathology (4–6).

HSPGs may affect AD progression in other ways as well. HSPGs are key components of thebasement membranes that surround cerebral vessels and may, therefore, be required for theintegrity of the blood-brain barrier (BBB). Compromised function of the BBB has beenincreasingly implicated in AD pathogenesis (7–9). The BBB is comprised of cerebralendothelial cells, astrocytes, and pericytes. It physically consists of specialized tightjunctions between endothelial cells, a basement membrane of extracellular matrix (ECM)surrounding the endothelium, and extensive abluminal basement membrane contact,primarily with endfeet of pericytes and astrocytes (10). Changes in the function of the BBBmay result in altered permeability and active transport across the barrier, with resultingdefects in cerebral extracellular fluid Aβ homeostasis (11–13). Accumulation of solubleforms of Aβ in cerebral extracellular fluid has been implicated in early synaptic dysfunction(10, 14). Furthermore, pathology involving cerebral microvasculature often parallels or evenprecedes AD pathology (15). In light of these findings, impaired clearance of Aβ across theBBB is increasingly a focus of possible early AD pathology (8, 13).

A primary HSPG core protein associated with both AD plaques and basement membranes ofthe BBB is AGRIN (16–18). AGRIN may contribute to Aβ deposition and AD pathologythrough multiple mechanisms. Like other HSPGs, the in vitro binding of Aβ and the heparansulfate moieties of AGRIN promotes fibril formation (17). The direct interaction of AGRINand Aβ may lead to a positive-feedback cycle of Aβ fibril formation causing amyloid toaccumulate along with additional HS, thereby further promoting fibrillogenesis (19).Alternatively, the presence of AGRIN in the cerebral vasculature coincides with theformation of the BBB (20). AGRIN may influence AD pathology through its role in BBBintegrity and/or function (21).

Testing the in vivo functional consequences of AGRIN/Aβ association has been hampered(even in animal models) because the disruption of the Agrin gene (mouse gene symbolAgrn) causes perinatal lethality due to its critical function in neuromuscular junction (NMJ)formation (22). We have generated mouse models that allow us to address roles of AGRINin Aβ deposition by reducing AGRIN selectively in the vasculature or on neurons or byoverexpressing Agrn throughout the brain. We then asked whether altered Agrn expressionaffected accumulation of insoluble Aβ in a transgenic mouse model of AD expressingmutant forms of amyloid precursor protein (APP) and presenilin1 (PS1) [APP(Swe)/PS1(Dex9)line85] (23).

MATERIALS AND METHODSStudy Animals

All mice used in these experiments were housed under standard conditions in the ResearchAnimal Facility at The Jackson Laboratory and provided food and water ad libitum. TheInstitutional Animal Care and Use Committee of The Jackson Laboratory approved allanimal procedures.

The AGRIN-cyan fluorescent protein (CFP) bacterial artificial chromosome (BAC)transgenic mice (C57BL/6J-Tg[MGS1-19375/CFP]2R9Rwb, abbreviated AGRIN-CFP)express all AGRIN isoforms under the control of the endogenous regulatory elements andsplicing (24). The Agrn-loxP conditional allele allows the deletion of exons 7 through 33

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(B6;129-Agrntm1Rwb abbreviated Agrnfl) in response to Cre expression and has beenpreviously described (25). The APP (Swe)/PS1(Dex9)line85 co-integrated transgenic mice(abbreviated APP/PS1) were generously provided by Dr. David Borchelt (23). Transgenicmice expressing Cre in endothelial cells driven by the Tie2-promoter (B6.Cg-Tg[Tek-Cre]1Ywa/J, abbreviated Tie2-Cre) were obtained from The Jackson Laboratory (stock 8863)(26).

Deletion of Short Isoform-AgrnThe short isoform (SN)-specific exon of Agrn and approximately 2 kb of upstream sequencewere deleted from the mouse genome by homologous recombination. A targeting vectorconsisting of an upstream EcoRI/BglII genomic fragment and a second downstream BglIIfragment was used to replace the SN exon with a loxP-flanked Neor cassette that is in theopposite orientation relative to the Agrn gene. A thymidine kinase-negative selectioncassette was also included in the vector outside of the regions of homology. Mouse RIembryonic stem cells were targeted by electroporation and G418 and FIAU selection usingstandard techniques. Homologous recombinant embryonic stem cell clones were identifiedby polymerase chain reaction (PCR) and confirmed by Southern blotting. Proper targetingresulted in the introduction of an XmaI site and the loss of an SphI site, causing a decreasefrom 14 kb to 6kb in the XmaI fragment detected by a downstream probe, and an increasefrom 8.2kb to 16.3 kb in the SphI fragment. The second, smaller SphI fragment detected bythe probe was unaffected, as anticipated. Chimeric mice were generated from recombinantembryonic stem cell clones by standard blastocyst injection and were bred for germlinetransmission of the mutation. To insure that expression of LN forms of AGRIN wereunaffected, the floxed Neo cassette was deleted by breeding to b-Actn-Cre transgenic mice(27). These mice were subsequently bred to pass the allele, which lacks the SN exon andcarries a LoxP site in its place, with no deleterious effects on long isoform (LN)-AGRINexpression or any common exons.

Acetylcholine Receptor Clustering Assay of AGRIN ActivityThe acetylcholine receptor clustering (AChR) clustering activity of brain homogenates fromcontrol and SN-Agrn knockout animals (n = 3 for each genotype) was tested as previouslydescribed(28). In brief, cultured C2C12 myotubes were treated with homogenates preparedin DMEM media and standardized for tissue weight/volume. Extracts were then diluted andapplied to the cultures and clusters/myotubes were counted after 24 hours. Treatment withan inactive recombinant AGRIN construct was used to establish background levels of AChRclustering, and a saturating dose of recombinant Z8 AGRIN was used to define 100%activity.

Filter Trap Assay for Insoluble AβStudy mice were killed by CO2 inhalation at 186 ± 7 days. The brain was immediatelyremoved and divided sagittally with half placed in 4% paraformaldehyde (PFA) forsectioning and immunocytochemistry and the other half processed for use in an enzyme-linked immunosorbent assay (ELISA) or filter trap assay (29). Half brains were eitherimmediately homogenized in TBS with 1% protease inhibitors (Complete, Roche), TBS-PI,using a mechanical homogenizer and placed at −80°C, or flash frozen in liquid N2, stored at−80°C and homogenized by a Dounce homogenizer in TBS-PI at the time of performing theassays. Samples were initially centrifuged at slow speed (3000 RPM, 5 min in a microfuge)to remove incompletely disrupted tissue, which interferes with the filter trap assay (29). Thisinitial low speed spin changed absolute values by reducing total protein but did not affectrelative levels of soluble or insoluble total Aβ, Aβ-40, or Aβ-42, as determined by ELISAperformed on the same samples prepared with and without the initial spin (data not shown).

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For the filter trap assay, total protein was measured with a BCA Assay kit (Pierce, Catalog #23227, Rockford, IL) and equal amounts of protein from each study brain were furthersolubilized by addition of SDS (1% final concentration), loaded in a dilution series on a 96-well dot-blot apparatus, and vacuum-filtered through a 0.2-μm cellulose acetate membrane(OE 66, Schleicher and Schuell, Keene, NH). For detection of insoluble Aβ, a rabbitpolyclonal anti-Aβ antibody was used to probe the membrane after filtration (1:500 dilutionin block of 5% nonfat dry milk and 0.1% Tween in phosphate-buffered saline (PBS; Zymed,South San Francisco, CA), followed by an HRP-conjugated goat anti-rabbit IgG secondaryantibody (1:10,000 Perkin Elmer, Boston, MA). Signals were developed using enhancedchemiluminescence (Western Lighting, Perkin Elmer) to expose the film, which was thenscanned. The densities of the resulting dots were measured using Image J software(www.nih.gov). Background was determined by scanning an equivalent area adjacent to thespots and background values were subtracted from signal intensities.

ELISA Assay for AβELISAs measuring Aβ-40 and Aβ-42 were performed using commercial kits according tothe manufacturer's instructions (KHB3442 and KHB3482, Invitrogen, Camarillo, CA), withthe following modifications: Homogenates were further separated into soluble and insolublefractions by treatment with diethylamine (DEA) to a final concentration of 2%, sonication(30 seconds at 10 watts in a 500-μl sample volume), and then centrifugation at 100,000 g for30 minutes. The supernatant was used to determine soluble Aβ. The pellet was resuspendedin 20 μl 5M guanidine in TBS-PI and used to measure insoluble Aβ.

ELISA Assay for Aquaporin 4Aquaporin 4 (AQP4) protein concentrations in mouse hemi-brain samples were measuredusing a solid-phase sandwich ELISA method (wild type, n = 6; Agrnfl/fl;+/+ [no Cre], n = 3;and Agrnfl/fl;Tie2-Cre/+, n = 6). Samples of brain tissue were snap-frozen in liquid N2 andthen ground over a bed of dry ice. 100 mg of each sample were homogenized thoroughly in8 volumes of a cold 5 M guanidine HCl/50 mM Tris-HCl, pH 8.0, buffer. The resultanthomogenates were mixed for 3.5 hours at room temperature and then diluted 10-fold with 1×protease inhibitor cocktail (Sigma-Aldrich, Catalog # P-2714, St. Louis, MO) in coldDulbecco's PBS. Samples were centrifuged at 16,000 × g for 20 minutes at 4°C, and thesupernatant was stored on ice until use with the assays.

Following protein extraction, AQP4 concentrations were measured using an ELISA kit(USCN Life, Catalog # E0582m, Wuhan, China), in accordance with the manufacturer'sinstructions. Samples and standards were added in duplicate to microtiter plate wells pre-coated with an antibody directed against AQP4. After a 2-hour incubation at 37°C, wellswere aspirated and then incubated for 1 hour at 37°C with a biotin-conjugated polyclonalantibody preparation specific for AQP4. Wells were washed 4 times prior to the addition ofavidin-conjugated horseradish peroxidase for 1 hour at 37°C. After a final series of 4washes, the color was developed by adding a 3,3',5,5'-tetramethylbenzidine (TMB) solutionto each well. The reaction was terminated 30 minutes later using the stop solution from thekit. The plate was read on a Multiskan EX spectrophotometer (Thermo ElectronCorporation, Catalog # 1507300, Vantaa, Finland) at 450 nm; data were analyzed usingAscent Software (Thermo Electron Corporation). Immunoassay results were corrected fortissue weight and the total protein content of each sample was determined using a BCAProtein Assay kit, with absorbance read at 562 nm.

ImmunocytochemistryThe half brains were immersed in PFA for 24 hours, placed in 30% sucrose for 24 hours,embedded in OCT media, and frozen using liquid N2for sectioning in a cryostat. Sections

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(14 mm) were cut coronally through hippocampus and cortex and placed on slides. Foramyloid plaque identification, sections were immersed in 70% formic acid for 30 minutesthen rinsed in TBS, blocked using 5% normal goat serum or 1% bovine serum albumen and0.5% Tween-20 in TBS, and stained with antibody in the same blocking solution. Primaryantibody was mouse monoclonal anti-Aβ (4G8, Covance Research, Dedham, MA), andsecondary antibody was fluorescent-tagged goat anti-mouse Ig2b (AlexaFluor 568,Molecular Probes, Eugene, OR). A Nikon E600 fluorescent microscope equipped withSPOT camera and software was used for individual plaque area measurement andmorphology examination.

For evaluation of BBB immunocytochemistry, sections from older mice (includinghomozygous Agrnfl/fl with and without the Tie2-Cre transgene) were prepared as above butwithout formic acid treatment. The sections were evaluated using polyclonal rabbit primaryantibodies to mouse occludin (1:100) and claudin-5 (1:100) (both from Zymed). For LRP -1detection, primary antibody was goat anti-LRP-1 (N20 1:100, Santa Cruz Biotechnology,Santa Cruz, CA). Secondary antibody was a fluorescently tagged goat anti-rabbit IgG(1:200, AlexaFluor 500, Molecular Probes), except for LRP-1, which was a fluorescentlytagged chicken anti-goat IgG (AlexaFluor 594, Molecular Probes).

AQP4 and AGRIN protein in the brain vasculature was also evaluated. After quick rinses in95% ethanol, 70% ethanol and distilled water, sections were treated for antigen retrievalwith hot (85°C) 10 mM citrate buffer (pH 6.0) for 15 minutes. Sections were then washedwith distilled water and then quenched with a peroxidase-blocking reagent (DAKO,Carpinteria, CA; S2001) for 10 minutes at room temperature to eliminate endogenousperoxidase activity. Non-specific binding sites were blocked by incubation with 5% normalgoat or rabbit serum for 24 hours at 4°C prior to incubation with one of the followingprimary antibodies: polyclonal rabbit anti-rat AQP4 antibody (Alpha DiagnosticInternational, San Antonio, TX; AQP41-A; diluted 1:250), polyclonal rabbit anti-AGRINC95-Rb-204 antibody (courtesy of Dr. M. Ruegg, University of Basel, Basel, Switzerland;diluted 1:2000), or polyclonal sheep anti-rat AGRIN GR-14 antibody (courtesy of Dr. J.Berden, University Medical Center Nijmegen, Nijmegen, The Netherlands; diluted 1:800).Following an overnight incubation at 4°C, the tissue sections were subjected to a modifiedABC technique using the Vectastain Elite ABC rabbit or sheep peroxidase system (VectorLaboratories, Burlingame, CA). 3,3-diaminobenzidine (DAKO; K3468) was used as thechromogen, and the slides were coverslipped and sealed using Cytoseal, a xylene-basedmounting medium (Stevens Scientific, Riverdale, NJ). Antibody specificities have beenpreviously demonstrated (30, 31). Primary antibody omission controls were run alongsidethe other samples to check for nonspecific binding caused by the secondary antibody, alongwith positive control tissue (kidney, thymus gland and lung). Images of mouse brain sectionswere obtained using an Olympus BH2-RFCA microscope (Olympus America, Inc.,Melville, NY), and acquired with a CoolSNAP cf camera (Roper Scientific, Tucson, AZ).

Electron Microscopy and Elemental AnalysisBrain samples of 1-year-old wild type and of Agrnfl/fl;Tie2-Cre mice were prepared forelectron microscopy using standard techniques. Tissue was fixed in 2% PFA/2%glutaraldehyde in 0.1M cacodylate buffer. A JEOL JEM-1230 electron microscope withAMT digital camera and an attached Genesis 2000 EDAX unit were used, which alsoallowed spectroscopic analysis of elemental content within a given EM field. Measurementsof the brain vascular basement membrane sulfur content relative to phosphate were made at200,000× magnification to maximize the area represented by the basement membrane. Eightindependent measurements were made from 2 mice of each genotype.

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Quantitative PCRAdult mice were euthanized under CO2 and the brain immediately removed and processedfor RNA by Trizol extraction and DNAse treatment to remove contaminating genomicDNA. Equal amounts (5 μg) of total RNA were reverse transcribed following manufacturerdirections using Superscript III reverse transcriptase and a mix of random and oligo dTpriming (Invitrogen). Equal volumes of the resulting cDNA were used for quantitative PCRusing an ABI Prism 7000 unit (Applied Biosystems, Foster City, CA). Samples were runminimally in triplicate and SYBR green (SYBR green PCR Master Mix, AppliedBiosystems) was used for quantification of amplification products. β-actin mRNA was usedas the standard for comparison by the DDCT method. β-actin primers were F: CAT TGCTGA CAG GAT GCA GAA and R: GCC ACC GAT CCA CAC AGA GT. Pan-Agrnprimers were F: GGT GCT GTG GAT TGG AAA GGT and R: TCA CAG TGG AGC GCAGCA. Z+ isoform specific primers were F: TCC CAG CCC CCG AAA CT and R: GTAGTC TGC ACG TTC TCC AAC CTT. Primers used for APP were F: CGA CAT GAC TCAGGA TAT GAA GTT CA and R: ACC ATG ATG AAT GGA TGT GTA CTG TT.

Sulforhodamine B and Fluorescently Conjugated DextranSulforhodamine B (approximately 1 kD) and 4kD fluorescein isothiocyanate (FITC)-Dextran (both from Sigma-Aldrich) were injected i.p. The mice were killed 3 hours later andblood was drained via right atrial cut and replaced with PBS then 4% PFA by transcardiacperfusion. The brains and livers were removed and processed as above for fluorescentmicroscopic examination without antibody treatment and using appropriate excitation andemission wavelengths.

Data Analysis and StatisticsValues are presented as means ± SD or percent of control values ± SE. Statisticalsignificance was determined using a t test pair-wise comparison of mutant vs. control values.Significance was not different when comparing absolute empirical quantities or percent ofcontrol values. A threshold of significance of 0.05 was used.

RESULTSDeletion of Agrn from Endothelial Cells

To examine the role of AGRIN in the BBB, and to determine if AGRIN-related changes inBBB integrity or composition may result in altered amyloid deposition in mouse models ofAD, we selectively deleted Agrn from endothelial cells using a loxP-flanked conditionalallele of the mouse gene (Agrnfl) (25). The Agrnfl allele was combined with transgenic Credriven in endothelial cells by the Tie2 promoter (26).

Deletion of Agrn from endothelial cells resulted in a reduction in vessel-associated AGRIN.Studies sing a primary antibody against the C-terminus of AGRIN (31) and indirectimmunofluorescence showed a reduction of AGRIN staining intensity in cerebral bloodvessels, but not in the pia, in Agrnfl/fl;Tie2-Cre mice compared to controls (Fig. 1A, B). Inadditional tests using a polyclonal antibody specifically recognizing the LN isoform ofAGRIN (30), immunoreactivity was again observed in the cerebral microvasculature, withmore robust LN-AGRIN staining in control mice (Fig. 1C, E) than in Agrnfl/fl;Tie2-Cre mice(Fig. 1D, F). Extracellular matrix-associated LN-AGRIN is the major AGRIN isoform in thecerebral microvasculature, consistent with the reduced staining with the LN-specificantibody. Deletion of the Agrnfl allele results in an N-terminal protein product that can bedetected by the antibody used in Figure 1C–F, but not the C-terminal antibody used inFigure 1A, B (25). Because the staining intensity of AGRIN immunocytochemistrydepended on both the primary and secondary antibodies used, and because we were unable

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to specifically determine the levels in blood vessels apart from the remainder of the brain byWestern blotting or ELISAs, we did not quantify the loss of AGRIN in cerebral vessels.

Effects on the BBB and AQP4 Mediated by Loss of Endothelial cell AGRINTo assess BBB integrity, tracers were used to examine permeability; no leakage ofsulforhodamine B (<1 kD) or FITC-labeled dextran (4 kD) from the circulation into thebrain was observed following the loss of endothelial cell AGRIN (Fig. 2A, B). In somecases, the vessels retained fluorescent tracers following perfusion, but this was not aconsistent result and entry of the tracers into the brain parenchyma causing diffuse halosaround vessels was not observed in either genotype. Clarified tissue homogenates were alsoanalyzed by fluorescence photospectroscopy for the presence of either rhodamine or FITC,but these were below detection in both wild type and Agrnfl/fl;Tie2-Cre mice (not shown).The basement membranes surrounding cerebral vessels were examined by transmissionelectron microscopy (Fig. 2C, D). No thinning or obvious gaps were observed, suggestingthat the loss of AGRIN does not result in a dramatic change in integrity, or that there iscompensation by other matrix molecules such as perlecan or laminin. However, elementalanalysis spectroscopy performed using transmission electron microscopy did detect adecrease in the sulfur content (expressed as a ratio relative to phosphorous) in the basementmembranes of mice lacking endothelial cell AGRIN (Fig. 2E). The majority of the sulfur inthe basement membrane is presumably contributed by sulfated glycans, and the decreasesuggests that the reduction of AGRIN is not compensated for by another HSPG such asperlecan. The reduction also suggests that there is probably a reduction in the negativecharge associated with the basement membrane, which might influence the permeability ofthe barrier to some solutes.

Despite retaining sufficient integrity to be impermeable to the tracers tested, samples stainedwith the C-terminal anti-AGRIN antibody using the enhanced peroxidase detection methoddemonstrated that immunopositive microvessels in Agrnfl/fl;Tie2-Cre mice have attenuateddiameters and ragged profiles when compared to controls (Fig. 2F, G), consistent with thecontribution of AGRIN to the structure of the microvessels.

Other protein components of the BBB that may depend on basement membrane anchoringfor their localization and stabilization were also examined. No changes in occludin,claudin-5, or LRP-1 staining were seen following the loss of endothelial cell AGRIN (notshown). However, AQP4, which is arranged in orthogonal arrays that are reported to interactwith AGRIN and dystroglycan (7, 32–35), was reduced in staining intensity in mice lackingendothelial cell AGRIN, although its distribution was not changed (Fig. 2H–K). Using anELISA, levels of AQP4 were reduced to 41% (p < 0.001) relative to strain and age-matchedcontrol samples (Fig. 2L). Therefore, the cerebral vessels have reduced but not eliminatedAGRIN. This reduction does not result in obvious leakiness or failure of integrity of theBBB, but does impact AQP4, and presumably other proteins that interact with the vascularbasement membrane.

Deletion of SN-AgrnAgrn has 2 alternative transcriptional and translational start sites, generating a secreted,matrix-associated LN-AGRIN, which is found extensively around cerebral blood vessels andat the neuromuscular junction, and a type II transmembrane SN-AGRIN that is expressedprimarily by CNS neurons (28, 36–38). We took advantage of this differential distribution todecrease levels of AGRIN in neurons without affecting the BBB. To this end, we generatedan isoform-specific knockout of Agrn that lacks expression of the SN form of the protein.This was accomplished by targeting the SN-specific first exon and approximately 2 kb ofupstream sequence, while leaving the upstream LN-specific exons, as well as all common

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downstream exons, intact (Fig. 3A). In situ hybridization with a probe that recognizes bothSN- and LN-Agrn isoforms revealed that expression in brain regions such as the adulthippocampus was effectively eliminated in the SN-Agrn knockout samples (Fig. 3B–E). Insitu hybridization with probes recognizing just the SN-isoform gave hybridization patternssimilar to those of the pan-Agrn probe in wild-type mice, but no signal in the SN-Agrnmutant. In younger mice (postnatal day 7), the reduction in expression by in situhybridization using pan-Agrn probes was less dramatic, suggesting that at early postnatalages, some neurons may express LN-Agrn as well as SN (not shown). Sites of LN-Agrnexpression, such as the NMJ and basement membranes in the kidney glomeruli, wereunaffected by the loss of SN-AGRIN, and were still immunoreactive for anti-AGRINantibodies (Fig. 3F–I). QPCR demonstrated that Agrn mRNA in the brain was reduced byapproximately 70% in the SN-Agrn mutant (data not shown).

As a second test of the efficacy of the SN-Agrn allele, we used an AGRIN activity assay inwhich cultured C2C12 myotubes were treated with brain homogenates and the acetylcholineesterase (AChE) clustering activity was quantified (Fig. 3J). This activity depends on theinclusion of “Z” exons at a C-terminal alternative splice site, and these Z+ isoforms ofAGRIN are only made by neurons. An 80% reduction in Z+ AGRIN in the brain wasdemonstrated by a 1:50 dilution of a wild type brain extract containing roughly the sameAChR clustering activity as a 1:10 dilution of an SN-AGRIN mutant extract.

Surprisingly, mice lacking SN-AGRIN expression appear normal and have no overtneurological phenotypes such as ataxia, seizures, or spasticity. These mice breed ashomozygotes and have no reduction in lifespan to the extent it has been tested (>18 months).Based on these results, we used SN-Agrn knockout mice to eliminate the predominantneuron-associated form of AGRIN in the brain, while leaving intact blood vessels andNMJs, which rely on LN-AGRIN (28).

Amyloid Plaque-Forming Transgenic MiceTo examine the in vivo relationship between AGRIN and Aβ deposition, we used the animalmodels above that allowed us to manipulate Agrn expression in the presence of transgenesthat promote the formation of amyloid deposits in the mouse brain. Experiments below wereperformed in the background of APP(Swe)/PS1(Dex9)line85, a transgenic model (hereafterreferred to as APP/PS1) (23). These mice express the Swedish (K594N, M595L) form ofAPP, as well as the Presenilin1 gene with a deletion of exon 9. Both of these alleles areassociated with human early-onset familial AD (39, 40). In the mice, both transgenes aredriven by the prion promoter and are co-integrated in the genome so that they segregate as asingle locus. These mice begin to show amyloid deposits detectable byimmunocytochemistry at 5.5 to 6 months of age (Fig. 4A, B). The composition of plaquesclosely resembles human amyloid plaques with associated-AChE and astrocytosis in thecortex and hippocampus of transgenic animals (Fig. 4C, D). Immunoreactivity for HSPGswas detected in the Aβ deposits in the transgenic mouse brain (Fig. 4E), but (consistent withother studies) not all deposits labeled with HSPG (41). In these mice, Aβ levels werequantified using a filter trap assay (Fig. 4F) and by quantitative ELISA to examine 40 vs. 42amino acid forms of Aβ. As reported for other similar models, female mice accumulate 2- to3-fold higher levels of insoluble Aβ than age-matched male mice (42–45).

Endothelial Cell Deletion of Agrn, but not Deletion of SN-Agrn, Increases Aβ AccumulationTo assess the functional relationship between AGRIN and Aβ, we first combined the plaque-forming APP/PS1 mice with the Agrnfl allele and the Tie2-Cre transgene to delete Agrnfrom endothelial cells. Mice were examined at 6 months of age, when amyloid accumulationis underway and easily documented but not yet highly variable between animals. In this

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way, we sought to determine if the age of onset of amyloid deposition was markedlychanged (hastened or delayed) as an indicator of the severity of the eventual pathology andof changes in Aβ biochemistry.

Reduction in vessel-associated AGRIN resulted in a significant increase in the levels ofAβ-40 in female mice (Fig. 5A, p = 0.02). In all mice, we found levels of Aβ-42 to beconsistently 3 to 5 times higher than levels of Aβ-40, consistent with previously publishedstudies using these mice (23), and reflecting the aggressive plaque-forming nature of themodel. Results in Figure 5 are presented as percentage of control values. Because there waslittle change in Aβ-42, the predominant form of Aβ found in this transgenic model, therewas no significant decrease in total Aβ levels. No significant changes were seen in malemice (not shown). The increase in Aβ-40 following endothelial cell deletion of Agrnindicates that Aβ deposition is influenced by AGRIN associated with the BBB, possiblythrough direct interactions or through indirect mechanisms such as altered localization ofBBB components such as transporters of Aβ.

We next examined the impact of deleting neuron-associated SN-Agrn on Aβ levels in thebrain. Despite the large reduction in AGRIN levels in the brain, and the almost completeelimination of Agrn expression from regions such as the adult hippocampus, no significanteffect was seen on Aβ levels in mice lacking SN-AGRIN (Fig. 5B). Neither males norfemales showed statistically significant differences in soluble or insoluble total Aβ, Aβ-40,or Aβ-42 at 6 months of age. Male mice (shown) trended towards an increase in Aβ levels,whereas female mice had no differences in Aβ levels between wild type and SN-deletedanimals or trended towards a decrease in Aβ. Other studies have suggested that AGRIN isonly associated with 10% to 30% of plaques in APP/PS1 mice, and this may decrease theimpact of the loss of SN-AGRIN (41). However, the finding that Aβ levels were changed byendothelial cell deletion of Agrn indicates that AGRIN levels do have a functionalconsequence on Aβ homeostasis and that this effect appears to be primarily mediated byvessel-associated AGRIN isoforms.

Transgenic Overexpression of Agrn Reduces Aβ AccumulationHaving determined that Aβ increases as a consequence of Agrn loss-of-function inendothelial cells, we also wanted to address the converse question of whether increasingAgrn expression could have a protective effect and reduce Aβ deposition in the brain. Tothis end, we used Agrn transgenic mice that carry extra copies of the entire Agrn gene on aBAC incorporated into chromosome 8. CFP cDNA was fused to the 3' end of the Agrncoding sequence in the BAC to mark AGRIN. This strain (2R9) overexpresses Agrn 2- to 4-fold depending on the age and tissue examined, and the transgene is fully functional basedupon its ability to rescue the NMJ and perinatal lethality phenotypes of Agrn knockout mice(24). When bred to APP/PS1 mice, transgenic AGRIN protein was detected strongly inblood vessels and also in plaques using anti-green fluorescence protein antibodies (whichcross-react with CFP) (Fig. 6A).

The effect of Agrn overexpression on the accumulation of insoluble Aβ was tested usingboth the filter trap assay and ELISA. A decrease in SDS-insoluble Aβ was observed by filtertrap assay in the Agrn transgenic samples compared to mice that were wild type for Agrn at6 months of age (Fig. 6B, p = 0.02). The antibody used for detection in this assay recognizeshuman amyloid, but does not distinguish between Aβ-40 and Aβ-42; only SDS-insolubleaggregates are bound in the filter. To examine these effects in more detail, brain tissue wassolubilized in DEA and used in a quantitative ELISA. Extracts were fractionated intosoluble and insoluble fractions by centrifugation and independent assays were performed toquantify both 40 and 42 amino acid forms of Aβ. Agrn transgenic mice had significantlylower levels of both Aβ-40 and Aβ-42, contributing to decreased total Aβ (Fig. 6C),

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consistent with the filter trap results. The bulk of this decrease came from a decrease in theAβ insoluble forms,, suggesting that less Aβ is deposited into insoluble plaques in thesemice (Fig. 6D). The differences related to Agrn overexpression were significant in malemice (shown), and while female mice continued to show higher Aβ levels than age-matchedmales, no significant changes in Aβ levels were seen in response to Agrn overexpression infemales.

Plaques were also qualitatively examined by immunofluorescence using the 4G8 antibodyagainst human Aβ (not shown). This analysis did not reveal a change in the number, size, ordistribution (hippocampal and cortical) of the plaques. This may reflect lack of sensitivity inthe technique.

DISCUSSIONWe have demonstrated that genetically altering Agrn expression results in differences in Aβaccumulation in the brains of transgenic and knockout mice. Deletion of Agrn fromendothelial cells resulted in an increase in Aβ-40 in female mice, whereas increasing Agrnexpression resulted in decreased insoluble Aβ in male mice. In contrast, deletion of thepredominant neuronal isoform of Agrn (SN-AGRIN) did not result in a change in Aβ levelsin either sex. Whether the sex-specific differences observed in the response to altered Agrnexpression reflect real differences in physiology and biochemistry between males andfemales or are secondary to differences in Aβ deposition resulting from differences in prionpromoter expression between the sexes remains unclear. Nevertheless, these results indicatethat AGRIN levels do indeed influence amyloid accumulation in vivo, and that AGRINassociated with the cerebral vasculature is largely responsible for these effects.

The inverse correlation between Agrn expression and Aβ accumulation does not support amodel in which the interaction of Aβ with the heparan sulfate side chains of AGRINpromotes fibril formation and plaque accumulation in vivo. In that scenario, increasedAGRIN would be expected to result in increased Aβ interaction and therefore increasedfibrillogenesis and insoluble Aβ accumulation, whereas the opposite was seen. Instead, thedata support an interpretation in which the presence of AGRIN presence around thevasculature influences Aβ levels. This is indicated most directly by the changes in Aβ seenwith endothelial cell-specific deletion of Agrn, but not with deletion of the neuronal SN-isoform.

The effects of altered Agrn expression on Aβ levels may in part be indirect and result fromchanges in the BBB and associated cellular proteins or changes in the microvasculature itselfin response to reduced AGRIN levels. AD-associated changes in the cerebralmicrovasculature include a hypercontractile state of vessel smooth muscle, altered cerebralblood flow, and plaque-associated diseased blood vessels (46, 47). Furthermore, co-morbidities such as cerebral amyloid angiopathy (CAA), which is found in 40% of ADpatients (48), suggest a common link between vascular/BBB dysfunction and AD. However,we did not find evidence that BBB integrity is severely compromised by the removal ofendothelial cell AGRIN. AGRIN levels were reduced but not eliminated in the vasculature.This may be explained by a combination of several factors, including some AGRIN beingcontributed by astrocytes, inefficient deletion of the Agrn gene in endothelial cells by theTie2-Cre, and by the stability of AGRIN protein in the ECM even after the gene is deleted.The inability to completely eliminate vessel-associated AGRIN may have reduced themagnitude of the effects observed, we were nonetheless able to document a significantreduction in sulfur content in the basement membrane of the cerebral vasculature byelemental analysis spectroscopy that was presumably from a decrease in heparan sulfatecontent after Agrn deletion. This change should also result in a decrease in negative charge

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in the ECM. Despite these effects, the BBB remained impermeable to tracers such assulforhodamine B and fluorescein-conjugated dextrans, which did not leak from thecirculation into the brain in mice lacking endothelial cell Agrn. These results are consistentwith a lack of apparent effect on the tight junction proteins claudin 5 and occludin with thereduction of endothelial cell AGRIN.

Instead of overt compromise of BBB integrity, our results indicate that reduced AGRINlevels affect other cell surface proteins that depend on ECM interactions for theirlocalization or stability. We noted a decrease in AQP4 levels in mice lacking endothelial cellAgrn expression. The exact mechanisms governing the complex relationship betweenAGRIN and AQP4 remain to be determined, but basement membrane AGRIN depletionmay lead to the loss of glial AQP4 along the basal lamina and a breakdown of the BBB inhuman patients with high-grade gliomas (49). Our data in mice indicate that the reduction ofbasement membrane AGRIN in cerebral capillaries leads to a decrease in AQP4immunoreactivity along the basal lamina. This is the first demonstration of a functionalconsequence of the interaction between AGRIN and AQP4 in situ. A decrease in AQP4 hasalso been described in other mouse models of AD, as well as in AD patients (50). It will beof great interest to determine if the loss of perivascular AQP4 immunoreactivity inAgrnfl/fl;Tie2-Cre mice leads to a physiological derangement of the BBB that may contributeto AD-like pathology. Thus far, we have only tested for leakage of the tracerssulforhodamine B and fluorescein-conjugated dextrans in the Agrnfl/fl;Tie2-Cre mice;whether BBB permeability to small peptides like Aβ is altered will require furtherinvestigation.

The impact of changes in AGRIN levels on amyloid deposition in the cerebral vesselsthemselves remains unclear. Although Aβ deposition in capillaries (dyshoric angiopathy)has been recognized for many years, the effects of Aβ on capillary structure and functionremain poorly understood. We previously reported a decrease in basal lamina AGRINcontent in AD patients that was most pronounced in patients with the APOE 4,4 genotype(51). Capillary Aβ correlates significantly with recognized CERAD, Braak, and NIA-Reagan-Institute criteria for diagnosing AD (52). Two distinct types of sporadic CAA havebeen reported: in type I CAA, Aβ deposits are detectable in cortical capillaries, whereas intype II CAA, they are not. (53). The APOE4 allele is over 4 times more common in type ICAA than in type II CAA (53) and we previously found that reduced AGRIN within thecapillary basement membrane correlated strongly with the presence of 1 or more APOE4alleles (51). Accordingly, APOE4, which directly interacts with HSPGs, may cause areduction in basement membrane AGRIN content that may in turn be a predisposing factorfor Aβ deposition. However, our ability to address the role of AGRIN in CAA may belimited by differences in expression between mice and humans, and even more severely bylimitations of the APP/PS1 plaque-forming mice. The vast majority of these models,including the strain studied here, use the prion protein promoter to drive transgeneexpression primarily in neurons. A different model with more targeted amyloidaccumulation in the vasculature (54) may be required to address the relationship of AGRINin the microvascular basement membrane and changes in amyloid deposition in the brainparenchyma vs. in the vasculature itself. Such differences in expression of both Agrn andAPP between mice and humans may also explain the decreased association of AGRIN intransgenic mouse plaques compared to human samples (41).

Changes in Aβ receptor protein expression may have a more direct impact on Aβaccumulation. The decrease in AQP4 suggests that such changes may be downstreamconsequences of the decrease in vessel-associated AGRIN. For example, the proteins LRP-1and RAGE are involved in Aβ transport, with LRP-1 removing Aβ from the brain into thecirculation, and RAGE functioning in the opposite direction (55). Imbalance in the activity



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of these proteins could, therefore, directly impact cerebral Aβ levels. The distribution ofLRP-1 did not appear to be altered by the removal of endothelial cell Agrn, but subtlechanges or changes in other proteins with similar properties could have been missed.Furthermore, the treatment of pericytes in culture with Aβ results in an upregulation ofAGRIN, LRP-1, glypican (another HSPG), and the low-density lipoprotein receptor,suggesting a complex co-regulated relationship among these proteins (56). In such a system,changes in the composition of the BBB, such as reduced AGRIN, could result in alterationsin other proteins that are directly involved in Aβ homeostasis. Our results using geneticmanipulation of Agrn in mice suggest that such changes do indeed influence Aβ levels. Itwill be important to determine whether similar changes occur with age or following injury inhumans, and if such changes correlate with increased risk or severity of AD. Additionalstudies in mice will also be required to determine if the changes in Aβ observed in responseto altered Agrn expression also correlate with changes in other cellular pathologicalresponses such as reactive astrogliosis, or with changes in cognitive performance.

Previous studies have confirmed that AGRIN is a major component of amyloid plaques inAD brains and is an important basement membrane-associated HSPG in cerebral capillaries,as well as in other organs with blood-tissue barriers (e.g. thymus, testis etc.). Our studies inhuman brain have indicated that fragmentation of the basal lamina is an early event in thepathogenesis of AD, as well as in aging-related microvascular injury (16, 21). BecauseAGRIN is an integral component of the basement membrane, and its reduction has asignificant effect on proteins such as AQP4, the complex interactions of AGRIN with Aβand other BBB-associated proteins may provide important new insights into thepathogenesis of AD and lead to the development of novel treatment regimens.

AcknowledgmentsWe would like to thank the scientific services at The Jackson Laboratory for their assistance, particularly theHistology and Electron Microscopy service and the Cell Biology and Microinjection service. We would also like tothank Dr. Kevin Seburn for his comments on the manuscript, and Renee and Scott Relf for their generous supportof this work.

This work was supported by The Alzheimer's Association (R.W.B and E.G.S.), the NIH (5R01AG027910-04 toCEJ and 5R37NS019195-29 and 5R01AG032322-03 to JRS), and the philanthropic generosity of Renee and ScottRelf (R.W.B.).

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Figure 1.AGRIN deletion from endothelial cells. Combining the Agrinfl/fl allele with transgenic Tie2-Cre expression results in a reduction in blood vessel-associated AGRIN in the brain, asdetected by immunostaining in adult mice (6 months of age and greater). (A) By indirectimmunofluorescence, cerebral blood vessels and the pia separating the cortex and midbrainin a control mouse are strongly immunoreactive for carboxy-terminal anti-AGRIN. (B) Theanti-AGRIN staining intensity is reduced to near the threshold of detection in Agrnfl/fl; Tie2-Cre mice, although the pia remains intensely labeled. (C, D) Using more sensitive enhancedperoxidase immunohistochemistry and a long isoform (LN-AGRIN)-specific amino-terminalantibody, anti-AGRIN labeling is reduced but not eliminated from the cerebral vasculaturein Agrnfl/fl; Tie2-Cre samples. (E, F) Vessel morphology and the reduction in stainingintensity at higher magnification.



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Figure 2.Effects of endothelial cell Agrn deletion on blood-brain barrier composition. (A, B) FITCdextran and sulforhodamine injected intravenously did not enter the brain parenchyma ineither control (A) or Agrnfl/fl; Tie2-Cre (B) samples from mice ages 6 months and greater.Arrows denote blood vessels. (C, D) Transmission electron microscopy of cerebralbasement membrane on the abluminal surface of endothelial cells (between arrows) incontrol (C) and Agrnfl/fl; Tie2-Cre (D) mice does not reveal any thinning or breaches in thestructural integrity of the matrix. (E) Elemental spectroscopy analysis of the basementmembrane revealed a decrease in the ratio of sulfur to phosphorus following endothelial cellAgrn deletion, suggesting that the decrease in AGRIN is not compensated for by an increasein other sulfated proteoglycans. Results are from 8 independent fields examined in each of 2mice of each genotype. (F, G) Cerebral blood vessels labeled with anti-AGRIN in control(F) and Agrnfl/fl; Tie2-Cre (G) mice demonstrate a reduction in labeling surrounding thevessels, smaller vessel diameters, and more ragged profiles in the Agrnfl/fl; Tie2-Cresamples, consistent with changes in the neurovascular unit in response to endothelial cellAgrn deletion. (H–K) Immunostaining for Aquaporin4 (AQP4) in wild type (H, J) andAgrnfl/fl; Tie2-Cre (I, K) mice demonstrates a marked decrease in immunoreactivityfollowing endothelial cell Agrn deletion. Higher magnification panels (J, K) indicate thatthe localization of the AQP4 was not changed. (L) An enzyme-linked immunosorbent assay(ELISA) was used to quantify AQP4 in brain homogenates from age and strain matchedcontrol mice (n = 6), Agrnfl/fl mice with no transgenic Cre expression (n = 3), and Agrnfl/fl;Tie2-Cre mice lacking endothelial cell Agrin expression (n = 6). Consistent with the reducedstaining intensity in immunocytochemistry, AQP4 levels were reduced from 0.68 ± 0.15 ngAQP4/mg of tissue in wild type controls vs. 0.28 ± 0.07 in Agrnfl/fl; Tie2-Cre mice (mean ±SD, t test. p = 0.0002).



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Figure 3.Deletion of short isoform (SN)-Agrn. (A) The single exon encoding the SN amino terminusof AGRIN and approximately 2 kb of upstream sequence were targeted by homologousrecombination; upstream exons encoding the long isoform (LN)-N-terminus anddownstream exons encoding common sequences were left intact. The loxP-flankedselectable marker, Neo, for embryonic stem cells was removed from the genome by matingto Cre transgenic mice and a single loxP site and flanking restriction sites were left in theAgrn gene. Southern blotting DNA samples from control and homozygous SN-deletion miceconfirmed the homologous recombination event with the anticipated shifts in restrictionfragment sizes. (B) In situ hybridization with a probe specific to the SN-isoform of Agrnrevealed strong expression in the adult hippocampus. (C) Hybridization with a proberecognizing all Agrn isoforms showed a similar pattern of expression. D, E: Hybridizationwith SN-specific (D) or pan-Agrn (E) probes indicated a lack of Agrn expression in thehippocampus of adult SN-Agrn knockout mice, indicating that SN-Agrn is the predominantisoform expressed in the hippocampus. (F, G) Adult neuromuscular junctions stained withα-bungarotoxin to label AChRs (red) and an antibody against the C-terminus of AGRIN(green) revealed no changed in AGRIN localization or NMJ morphology in the SN-Agrnknockout mice. (H, I) Kidney glomeruli stained with an antibody against the C-terminus ofAGRIN revealed abundant basement membrane staining in both control (H) and SN-Agrnknockout (I) kidneys. Data in panels F–I are consistent with preserved expression of LN-AGRIN. (J) SN-AGRIN is the predominant isoform in brain. In AChR clustering activityassays, homogenates from SN-Agrn knockout brains were reduced in clustering activity by4- to 5-fold compared to homogenates from control brains (n = 3 mice of each genotype),indicating SN-AGRIN is the predominant Z+ isoform of AGRIN expressed by CNSneurons. Background AChR clustering activity was determined by treating myotube cultureswith recombinant inactive AGRIN and 100% activity was determined using saturating dosesof recombinant Z8 AGRIN.



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Figure 4.Composition of APP/PS1 transgenic mouse plaques. Plaques are detectable in the brains ofAPP/PS1 mice beginning between 5 and 6 months of age. (A) Low magnification of plaquesstained with 4G8 (red), a monoclonal antibody specific for human β-Amyloid (Aβ); nucleiare counterstained in blue (DAPI). (B) High magnification of a plaque stained with 4G8shows a dense core of Aβ surrounded by fibrillar protein. (C) Plaques are also reactive foracetylcholine esterase (AChE, brown). (D) Activated astrocytes detected with anti-glialfibrillary acidic protein (GFAP, green) surround plaques labeled with 4G8 (red). (E) Plaquesin the transgenic mice are also positive for heparan sulfate proteoglycans (HSPG, green). (F)Filter trap analysis of SDS-insoluble Aβ. Nine mice were examined at 6 most of age; theyconsistent of 3 male APP/PS1 transgenics (columns 1–3), a non-transgenic wild type mouse(column 4), and 5 female APP/PS1 transgenics (columns 5–9). Each column represents adilution series for each sample, amounts of total protein loaded per well are 100 μg in the



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top row, 80, 60, 40 and 20 μg in the bottom row. Note the consistency of the technique andthe relative quantification (20–40 μg of protein in females is equivalent in intensity to 80 to100 μg of protein in males, consistent with other results indicating that females expressapproximately 3-fold higher levels of Aβ). The male/female difference has been reported byothers and provided an internal control for subsequent assays. Note the total lack of signal incolumn 4, the non-transgenic mouse.



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Figure 5.β-Amyloid (Aβ) levels in mice lacking endothelial cell or short isoform (SN)-Agrn. (A)Female mice 6 months of age and lacking endothelial cell AGRIN (Agrnfl/fl; Tie2-Cre)showed significantly higher levels of Aβ-40 vs. control mice (t test p value = 0.018); levelsof Aβ-42 and total Aβ were not different. Resul ts are reported as mean percent of controlvalues ± SEM; animal numbers are indicated. (B) Aβ in SN-Agrn KO mice. Deletion of SN-Agrn did not result in significant differences from controls in Aβ levels at 6 months of age,as determined by ELISA analysis. Data shown are for male mice; values are mean percent ofcontrol ± SEM; animal numbers are indicated.



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Figure 6.Effect of transgenic Agrn overexpression on β-Amyloid (Aβ) levels. (A) Staining with ananti-GFP antibody detects transgenic AGRIN-CFP in both plaques and blood vessels,indicating that the transgenic protein localizes to plaques and the blood-brain barrier. Notethe staining intensity is much higher in the blood vessel (arrow) than in the plaque. (B) Infilter trap assays, 6-month-old Agrn-CFP transgenic mice had significantly reduced SDS-insoluble Aβ levels vs. age-matched control littermates. Data shown are for male mice only;mean relative signal intensity based on densitometry ± SD, t test p = 0.02; animal numbersare shown. (C) Levels of Aβ were further examined by ELISA in 6-month-old mice.Transgenic expression of Agrn-CFP significantly reduced total amyloid, Aβ-42, and Aβ-40levels compared to littermate controls. Asterisks indicate t test p values <0.05, animalnumbers are given. Results shown are for male mice, values are mean percent of controlvalues ± SEM. (D) Further subdividing Aβ species into DEA-soluble and insolublefractions, indicated that the greatest contribution of the reduction seen with Agrn-CFPoverexpression came from reduced Aβ-42 insoluble amyloid, although differences did notreach statistical significance (p values 0.06–0.08).



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Changes in Brain β-Amyloid Deposition and Aquaporin 4 Levels in Response to Altered Agrin Expression in Mice

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