Abstract Amyloid β protein deposition in cortical andleptomeningeal vessels, causing the most common type ofcerebral amyloid angiopathy, is found in sporadic and fa-milial Alzheimer’s disease (AD) and is the principal fea-ture in the hereditary cerebral hemorrhage with amyloido-sis, Dutch type. The presence of the Apolipoprotein E(APOE)-ε4 allele has been implicated as a risk factor forAD and the development of cerebral amyloid angiopathyin AD. We report clinical, pathological and biochemicalstudies on two APOE-ε4 homozygous subjects, who hadsenile dementia and whose main neuropathological fea-ture was a severe and diffuse amyloid angiopathy asso-

ciated with perivascular tau neurofibrillary pathology.Amyloid β protein and ApoE immunoreactivity were ob-served in leptomeningeal vessels as well as in medium-sized and small vessels and capillaries in the parenchymaof the neocortex, hippocampus, thalamus, cerebellum, mid-brain, pons, and medulla. The predominant peptide formof amyloid β protein was that terminating at residue Val40,as determined by immunohistochemistry, amino acid se-quence and mass spectrometry analysis. A crown of tau-immunopositive cell processes was consistently presentaround blood vessels. DNA sequence analysis of the Amy-loid Precursor Protein gene and Presenilin-1 (PS-1) generevealed no mutations. In these APOE-ε4 homozygouspatients, the pathological process differed from that typi-cally seen in AD in that they showed a heavy burden ofperivascular tau-immunopositive cell processes associatedwith severe amyloid β protein angiopathy, neurofibrillarytangles, some cortical Lewy bodies and an absence ofneuritic plaques. These cases emphasize the concept thattau deposits may be pathogenetically related to amyloid βprotein deposition.

Key words Alzheimer’s disease · Cerebral amyloid angiopathy · Amyloid β protein · Neurofibrillary tangles ·Senile plaques

Introduction

The association of amyloid β protein (Aβ) deposits in senile(neuritic) plaques and in some cerebral vessels as well asfibrillary tau pathology in nerve cells constitutes the neu-ropathological hallmark of Alzheimer’s disease (AD) [6].In the brain, Aβ deposition may occur in both the paren-chyma and the vascular compartment. In the parenchyma,the deposition is in the form of diffuse deposits (preamy-loid lesions) and neuritic plaques (NP). Aβ, a peptide con-taining 39- to 44-residues with heterogeneous N and Ctermini, is a fragment of a larger precursor protein (AβPP)that is encoded by a single gene, the Amyloid Precursor

Rubén Vidal · Miguel Calero · Pedro Piccardo ·Martin R. Farlow · Frederick W. Unverzagt ·Enrique Méndez · Adolfo Jiménez-Huete ·Ronald Beavis · Gloria Gallo · Estrella Gomez-Tortosa ·Jorge Ghiso · Bradley T. Hyman · Blas Frangione ·Bernardino Ghetti

Senile dementia associated with amyloid â protein angiopathy and tau perivascular pathology but not neuritic plaques in patients homozygous for the APOE-ε4 allele

Acta Neuropathol (2000) 100 :1–12 © Springer-Verlag 2000

Received: 18 August 1999 / Revised, accepted: 15 October 1999

REGULAR PAPER

R. Vidal · M. Calero · A. Jiménez-Huete · G. Gallo · J. Ghiso ·B. FrangioneDepartment of Pathology, New York University School of Medicine, New York, NY 10016, USA

P. Piccardo · B. Ghetti (Y) Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USAe-mail: bghetti@iupui.edu

R. BeavisDepartment of Pharmacology, New York University School of Medicine, New York, NY 10016, USA

M. R. FarlowDepartment of Neurology, Indiana University School of Medicine, Indiananpolis, IN 46202, USA

F. W. UnverzagtDepartment of Psychiatry, Indiana University School of Medicine, Indianapolis, IN 46202, USA

E. MéndezUnidad de Análisis Estructural de Proteínas, Centro Nacional de Biotecnología, Madrid, Spain

E. Gomez-Tortosa · B. T. HymanAlzheimer Reasearch Unit, Massachusetts General Hospital,Charleston, MA 02129, USA

Protein (APP) gene on chromosome 21 [14]. The depositionof Aβ in the extracellular compartment has been linked ina causal relationship with the development of cytoskeletaltau pathology [32]. The consequences of the vascular Aβdeposits are less clear [42].

Aβ deposition in the vascular compartment is limitedto the vessels of the brain and involves large and smallleptomeningeal vessels as well as intraparenchymal me-dium-sized and small vessels [42]. Cerebral amyloid an-giopathy (CAA) related to Aβ may often be severe in spo-radic and familial AD, may occur in non-demented el-derly and is the principal feature in the hereditary cerebralhemorrhage with amyloidosis, Dutch type (HCHWA-D)[38]. This condition is associated with a glutamine substi-tution for glutamic acid at codon 693 of the APP gene(ΑPP770) corresponding to residue 22 of Aβ [15]; it ischaracterized clinically by recurrent strokes and fatal cere-bral bleeding in the fifth or sixth decade of life and neu-ropathologically by parenchymal deposition of preamy-loid lesions or diffuse plaque structures that are Congo rednegative, without the presence of NP and neurofibrillarytangles (NFT) [38]. In HCHWA-D, Aβ1–40 is the pre-dominant form present in the amyloid deposits, with C-terminal truncated derivatives found in both leptomenin-geal and cortical vessels [7]. In addition to mutation(s) atcodon 693, a mutation at codon 692 of the APP genecausing a glycine for alanine substitution co-segregates inone family with presenile dementia and cerebral hemor-rhage due to CAA [12], causing a dual phenotype of con-gophilic angiopathy and AD.

Since the discovery of the association between Aβ depo-sition in AD and apolipoprotein E (ApoE), the role of ApoEin the central nervous system is being intensively studied[29]. ApoE, a sialoglycoprotein composed of 299 aminoacids, is secreted as a protein with a molecular mass of34.2 kDa [44] and plays a central role in the metabolismof cholesterol and triglycerides. ApoE, which is encodedby a single gene locus on the long arm of chromosome 19(19q13.2), determines three co-dominant alleles, ε2, ε3and ε4. APOE alleles have been implicated in dietary hy-percholesterolemia, cardiovascular disease and more re-cently in the pathogenesis of AD [29, 35]. Of the threealleles, APOE-ε4 has been highly associated with late-onset familial and sporadic AD [35]. A high frequency ofthe APOE-ε4 allele has been found in patients with ADand prominent CAA [28], dementia and stroke [34], Aβdeposits subsequent to head injury [24], and finally, stillneeding further confirmation, in patients with Lewy bodydisease [1, 16]. In contrast, one report emphasizes the lowprevalence of the APOE-ε4 allele in the NFT predominantform of senile dementia [4]. Although ApoE has been re-ported to have multiple roles in the pathogenesis of AD,the observation that AD can occur in persons without theAPOE-ε4 allele is consistent with the notion that AD is aheterogeneous disorder in which multiple factors may in-fluence its development.

We now report two cases of individuals homozygousfor the APOE-ε4 allele suffering from dementia with se-vere Aβ-CAA. The Aβ deposition in the vessel walls was

not associated with a mutation in the ΑPP or PS-1 gene.In both cases, the most extensive deposition of Aβ amy-loid was associated with the vascular compartment, whilethere was a virtual absence of neuritic plaques. Aβ vasculardeposition coexisted with severe perivascular tau pathol-ogy, while NFT and neuropil threads (NT) were also seenthroughout the neocortex.

Materials and methods

Clinical studies

Both patients underwent a standard neurological examination. Pa-tient 1 was assessed cognitively, using the neuropsychological bat-tery of the Consortium to Establish a Registry for Alzheimer Dis-ease (CERAD) [23]. Patient 2 was evaluated in the context of aclinical drug trial using the Alzheimer Disease Assessment Scale(ADAS) [31].

Neuropathology

Neurohistology

Postmortem neuropathological studies were done on both patients.Tissue was obtained from superior frontal and cingulate gyri, su-perior parietal lobule, calcarine cortex, superior and middle tempo-ral gyri, entorhinal cortex, hippocampus, amydgala, caudate nucleus,putamen, globus pallidus, thalamus, cerebellum, midbrain, ponsand medulla. Following fixation in 4% formaldehyde, the tissuewas dehydrated and embedded in paraffin. Sections, 10 µm thick,were stained using the following methods: hematoxylin and eosin,Congo red, thioflavin S, Woelcke-Heidenhan and Bodian.

Immunohistochemistry

Sections were incubated with antibodies (Ab) raised against micro-tubule-associated protein tau (AT8, Innogenetics and Alz50, do-nated by Dr. P. Davis), Aβ protein (10D5, Athena Neurosciences),glial fibrillary acidic protein (GFAP; Dako), ubiquitin (Carpinte-ria), and with an antiserum raised against a synthetic peptide cor-responding to residues 119–137 of α-synuclein [26]. All Ab wereused at a dilution of 1:500, except for Ab to α-synuclein that wasused at 1:300. Aβ species containing 40 or 42 residues were de-tected using antisera raised against Aβ ending at Val40 or at Ala42(1:100) [13]. When Ab against Aβ were used, tissue sections werepreincubated for 5 min in 90% formic acid before incubation withthe primary Ab. Sections from case 1 were also incubated with poly-clonal Ab against ApoJ α and β chains [18] (1:100), P-component(Dako; 1:100), and cystatin C (1:500) [9], as well as monoclonal Abagainst ApoE (3D12; Biodesign; 1:100), Aβ17–24 (4G8; Senetek;1:200), Aβ1–17 (6E10; Senetek; 1:200), vitronectin (Biosource;1:1,000), and prion protein (3F4; Senetex; 1:500). Tissue sectionswere incubated overnight at 4 °C with the primary Ab and thenprocessed for staining. Polyclonal Ab were visualized by the per-oxidase-antiperoxidase (PAP) method with goat anti-rabbit immu-noglobulin (Ig) and anti-rabbit PAP; monoclonal Ab were visual-ized using goat anti-mouse Ig and anti-mouse PAP; 3,3′-di-aminobenzidine (DAB) was used as a chromogen.

Frozen sections of unfixed purified leptomeningeal vesselsfrom patient 1 were mounted on slides, fixed in acetone for 2 min,washed in 20 mM TRIS, 150 mM NaCl, pH 7.5 (TBS) for 10 min,and then incubated in a moist chamber with each of the correspond-ing Ab. As primary Ab we used 3D12, anti-ApoJ α and β chains,anti-P-component, 4G8, 6E10, anti-vitronectin, anti-cystatin C and3F4. All Ab were used at 1:100. Slides were then incubated withthe corresponding fluorescein-conjugated goat antisera diluted 1:10(Dako). Adjacent sections were stained with Congo red.

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Confocal microscopy

Paraffin embedded tissue blocks from the temporal cortex of thetwo patients were cut at 7 µm and processed for triple immunoflu-orescence immunohistochemistry using Ab R1282 (polyclonal Abagainst Aβ, courtesy of Dr. D. Selkoe), Ab 6F2 against GFAP (mono-clonal IgG, Dako), and either Tg3 or Alz50 (IgM monoclonal Abdirected against paired helical filament-associated epitopes on tau,courtesy of Dr. P. Davies). Appropriate secondary Ab labeled withbodipy-fluorescein (Molecular Probes), cy-3, or cy-5 (Jackson Im-munoResearch) were used for detection. Imaging was carried outon a Bio-Rad 1024 confocal microscope using a Krypon/Argonlaser.

Biochemistry

Isolation of Aβ from leptomeningeal vessels

Leptomeninges from patient 1 were carefully dissected from coro-nal sections of the unfixed brain, cut with scissors into 1- to 3-mmpieces, placed in buffer A (0.1 M TRIS-HCl, pH 7.4, 2 mM EDTA),and a cocktail of protease inhibitors (PI; Complete, 1 µM pepstatin,100 µM TLCK-HCl, 200 µM TPCK, and 1 µM leupeptin, all fromBoehringer Mannheim) on ice. Tissue was then washed by resus-pension in buffer A with PI, and centrifuged at 5,000 g for 10 minat 4 °C; the procedure was repeated five times. The material wascollected by filtration through a 45-µm nylon mesh (Spectrum),washed three times with 0.1 M TRIS-HCl, pH 7.4, 1% SDS, with-out PI, centrifuged at 5,000 g for 10 min at 4 °C. The pellet was re-suspended in 20 vol 2 mM CaCl2 in 0.1 M TRIS-HCl, pH 7.5, 3 mMNaN3 and 2.0 mg/ml collagenase CLS-3 (Worthington) and 10 µg/mlDNase I (Worthington) were added, and the mixture was incubatedfor 18 h at 37°C. After digestion, the suspension was centrifugedat 5,000 g for 10 min at 4 °C, washed three times with 0.1 M TRIS-HCl, pH 7.4 and the pellet was incubated for 1 h at room tempera-ture with 99% formic acid (Sigma). After centrifugation at 10,000 gfor 5 min, the supernatant was dried under N2. Amyloid was sepa-rated by reverse-phase (RP)-HPLC using a Vydac C4 narrow borecolumn (The separations Group; 214TP52; 0.21 × 25 cm) and a60-min 20–80% linear gradient of acetonitrile in water with 0.1%trifluoroacetic acid at a flow rate of 0.2 ml/min. As a control, lep-tomeninges from a patient without brain amyloidosis were treatedidentically.

Isolation of cortical vascular amyloid

Brain (case 1) stored at –70°C was incubated overnight at –20°Cin Hanks’ balanced salt solution (HBSS) and for 1 h at 4 °C beforestart. Cerebral cortex was dissected from white matter and sus-pended in HBSS. The material was homogenized and then cen-trifuged at 3,000 g for 15 min at 4 °C. The pellet was resuspendedin 15% dextran, 5% horse serum, shaked well and then centrifugedat 5,500 g for 20 min at 4 °C. The pellet was rinsed with HBSS andresuspended in HBSS and filtered through 210-µm mesh collect-ing the filtrate for microvessel isolation through a mesh of 53 µm.The retained material (fraction 53) was centrifuged at 10,000 g for30 min at 4 °C, and washed with 2 mM CaCl2 in 0.1 M TRIS-HClpH 7.5 and digested with 0.3 mg/ml of collagenase CLS-3 and 10 µg/ml of DNase I in the same buffer. After 18 h at 37°C, the suspen-sion was centrifuged at 10,000 g for 12 min, and the pellet was in-cubated for 2 h at room temperature in 100 vol 2% SDS in 0.1 MTRIS-HCl pH 8.0. After centrifugation at 10,000 g for 12 min at 4 °Cthe insoluble pellet was washed with 0.1 M TRIS pH 7.5. Amyloidwas solubilized in 99% formic acid and separated by RP-HPLC us-ing a Vydac C18 narrow bore column (218TP52; 0.21 × 25 cm) anda 70-min 0–70% linear gradient of acetonitrile-water (pH 2.1) at aflow rate of 0.2 ml/min.

Western blot analysis of cerebrovascular amyloid

Amyloid extracted from leptomeningeal vessels and fraction 53 wassubjected to 16% acrylamide, tris-tricine-SDS PAGE. Proteinswere electrophoretically transferred for 1 h at 400 mA at 4 °C topolyvinylidene fluoride membranes (Immobilon-P, Millipore) us-ing 10 mM CAPS buffer, pH 11, containing 10% methanol. Themembranes were blocked with 5% non-fat dried milk in 10 mMphosphate buffer, 137 mM NaCl, 2.7 mM KCl (PBS) pH 7.4 with0.1% Tween-20 (PBS-T) overnight and then incubated for 2 h atroom temperature with the corresponding primary Ab. As primaryAb, monoclonal 4G8 was used diluted 1:500 in PBS-T. Alterna-tively, monoclonal 6E10 was used diluted 1:500 in PBS-T. To de-tect Aβ species containing 40 or 42 residues, polyclonal anti-Aβending at Val40 or Ala42 Ab were used (1:500 dilution). Horserad-ish peroxidase-conjugated goat anti-mouse (rabbit) (Amersham) wasused as the second Ab at a dilution of 1:5,000 in PBS-T. Im-munoblots were visualized by chemiluminescence (Amersham)according to the manufacturer’s specifications.

Protein sequence analysis

Automated Edman degradation sequence analysis was carried outon a 477A protein-peptide sequencer, and the resulting phenylthio-hydantoin amino acid derivatives were identified using the on-line120A PTH analyzer (Applied Biosystems).

Mass spectrometry

Aβ from formic acid extracts, RP-HPLC purified and immunopre-cipitated samples were prepared as described [10] for matrix-as-sisted laser desorption-ionization mass spectrometry (MALDI-MS)using the dried droplet method. The matrix used was α-cyano-4-hydroxy-cinnamic acid (Sigma), which was purified by recrystal-lization. The linear, time-of-flight mass spectrometer used wascustom built at the Skirball Institute of the New York UniversitySchool of Medicine.

Genetic analysis

APOE genotyping

Genomic DNA was extracted from frozen brain tissue by standardmethods and the ApoE genotyping was performed essentially asdescribed [43]. Polymerase chain reaction (PCR) products (227 bp)were separated on 5% polyacrylamide gels and visualized by ethid-ium bromide staining. PCR products were digested for 2 h at 37°Cwith 10 units of HhaI (Gibco, BRL), subjected to electrophoresis on4% Methaphor (FMC) and photographed under ultraviolet light.

Sequencing of exons 16 and 17 of the ΑPP gene

Amplification of exons 16 and 17 of the ΑPP770 gene was per-formed as described [15]. PCR products (242 bp for exon 16 and319 bp for exon 17) were separated on 5% polyacrylamide gels, vi-sualized by ethidium bromide staining and subcloned into thepCR2.1 vector (TA cloning kit, Invitrogen). Recombinant plasmidDNA was isolated from eight to ten clones of different PCR reac-tions and sequenced in both directions.

Sequencing of PS-1

Total cellular RNA was extracted from brain tissue by the guani-dine isothiocyanate method using Trizol LS reagent (Gibco BRL).Reverse transcription of RNA (1 µg) was performed with the firststrand cDNA synthesis kit (Boehringer Mannheim) as described[39]. Of the PCR products, 10 µl were reamplified using oligonu-

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cleotide primers forward (F1:5′-GAG CAA GAT GAG GAA GAAGAT-3′, exon 4) and reverse (R1:5′-TGA AAT CAC AGC CAAGAT GAG-3′, exon 7) for 30 cycles of 94°C 1 min, 45°C 1 minand, 72°C 2 min. Of the PS1 cDNA, 10 µl were also reamplifiedusing oligonucleotides forward (F2:5′-GAT TTA GTG GCT GTTTTG TG-3′, exon 8) and reverse (R2:5′-ATC AGC GGC CGC TAACCG CAA ATA TGC, exon 12) under the same conditions. Afteramplification, the resulting PCR products were separated on 5%polyacrylamide gels, visualized by ethidium bromide staining andsubcloned into the pCR2.1 vector. Recombinant plasmid DNA wasisolated from ten clones in each case and sequenced in both direc-tions.

Results

Clinical studies

Patient 1

The subject, a 75-year-old male physician, presented witha gradual decline in memory and difficulty in completingactivities of daily living. At age 77, a computerized tomog-raphy of the head revealed no structural abnormalities, butelectroencephalography showed diffuse slowing, disorga-nization, and reduced amplitudes in the left mid-temporalregion. At age 78, he presented at the Indiana AlzheimerDisease Center (IADC). Clinical examination found the pa-tient to have poor short-term memory, poor verbal com-prehension, word finding difficulty and tangential speech.A neuropsychological evaluation at that time revealed aMini-mental State Examination (MMSE) score of 8/30,indicating severe generalized impairment. He was quiterepetitive in his verbal responses. After responding to theMMSE question asking for the current month and provid-ing the (wrong) answer of April, he proceeded to answerthe next 5 questions, pertaining to orientation to place, byalternately saying February and January. Verbal memorywas severely impaired (he never recalled more than 3 of10 words at any of the three learning trials) and he pro-duced an enormous number of non-list word intrusions onthis test (numbering 25 in total). Graphic reproduction ofgeometric figures showed mild tremor and dyspraxia(Constructional Praxis = 7/11); however, he did demon-strate a surprising degree of retention in recalling two ofthe five figures after a brief filled delay interval. Over theensuing 6 months, the patient’s behavior deteriorated be-ing marked by significant agitation requiring treatment withneuroleptics. The patient continued to decline and wasplaced in a nursing home where he died at age 81. The clin-ical diagnosis was “probable Alzheimer disease” [20]. Therewas a family history of dementia; the patient’s mother wasreported to have had senile dementia.

Patient 2

The subject, a 69-year-old salesman, had become depressedover the death of three siblings. At age 71, he became for-getful, his driving skills deteriorated, he developed ex-pressive and receptive aphasia and he became irritable and

stubborn. At age 72, he presented at the IADC, where hisneurological examination showed him to be disoriented totime and place and unable to recall any of 3 items at 3 min.His modified Hachinski-scale score was 0. On neuropsy-chological testing, the patient’s MMSE score was 22/30with errors reflecting memory loss primarily (problemswith orientation and delayed recall of objects). Word ListLearning was severely impaired (only 6/30 words recalledacross the three learning trials). Confrontation naming toactual objects was mildly impaired (ADAS Object Nam-ing 7/12) as was ability to follow commands (ADAS Fol-lowing Commands 3/5). Spatial skills were intact (CERADConstructional Praxis 10/11, ADAS Ideomotor Praxis 5/5).

Over the next year, there was rapid deterioration (Clin-ical Dementia Rating (CDR) score = 2). He developed mo-tor agitation, mild cogwheel rigidity, bradykinesia, shuf-fling gate, and severe delusions. His symptoms did notfluctuate. Behavioral symptoms responded only modestlyto trials of risperidone, trazadone, and lorazepam. Cogni-tive testing at this time paralleled the general clinical de-terioration with an eight point drop in MMSE score (15/30 to 7/30) over a period of 5 months. Verbal short-termmemory was essentially absent at that point (ADAS WordRecognition = 34/72 which is chance level performance).Tremor was apparent in graphic reproduction, although thepatient was still able to accurately copy the four CERADdesigns reasonably well (Constructional Praxis 9/11). Thepatient’s ability to follow commands was severely impaired.He died at age 74, 3 years after symptom onset. The clin-ical diagnosis was “Alzheimer disease with prominent ex-trapyramidal signs.” Reviewing the family history, the pa-tient’s mother and three siblings were reported to havehad dementia.

Neuropathological studies

For patient 1, the fresh brain weighed 1,390 g and showedmild atrophy of the frontal and temporal regions as well asmoderate atheromatous changes of the cerebral vessels.The fresh brain of patient 2 weighed 1,310 g and showedmoderate atrophy of the frontal and temporal regions.

Neurohistology and immunohistochemistry

On microscopic examination the neuropathological fea-tures observed in patients 1 and 2 were similar and aretherefore reported together. Congo red and thioflavin Sstaining of brain sections showed apple-green birefringenceunder polarized light and fluorescence, respectively, at thelevel of the vessel wall and around vessels (Fig.1A). Im-munohistochemistry using 10D5 showed intense stainingof the vessel wall and paravascular parenchyma (Fig.1B).Severe Aβ angiopathy was observed in leptomeningeal andcerebral medium-sized and small vessels as well as capil-laries of the neocortex, basal ganglia, hippocampus, thal-amus, cerebellum, midbrain, pons, and medulla. Using anantiserum against Aβ Val40 vessel walls and perivascular pa-

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renchyma were strongly immunolabeled (Fig. 2A), whereasantiserum against AβAla42 only stained a few vessels weak-ly (Fig. 2B). Neuritic plaques were absent. In the neocortex,dystrophic neurites were prominent in the parenchymasourrounding medium-sized and small vessels and capil-laries (Fig.3A). In addition, Alz50-positive NFT and NTwere seen in the neocortex (Fig.3B), amygdala, hippocam-pus, thalamus and pons. The parechymal vessels were im-munostained with Ab against P-component, vitronectinand ApoJ. ApoE immunopositivity was observed in lep-tomeningeal and parenchymal vessel walls as well as inthe neuropil in perivascular locations. No immunolabelling

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Fig.1A, B Cerebral vessels show amyloid angiopathy. A The flu-orescence is seen at the level of the vessel walls and in the sur-rounding parenchyma, in a thioflavin S preparation; B the vesselwalls and surrounding parenchyma are immunolabeled with mon-oclonal antibody 10D5. A × 66, B × 54

Fig.2 Blood vessels immunolabeled with antibody recognizingAβ1–40 (A), and Aβ1–42 (B). Strong labeling is observed in A. A,B × 166

Fig.3 Alz50-immunopositive neurites around blood vessels (A);Alz50-immunopositive NFT and NT in the cortical parenchyma(B) (NFT neurofibrillary tangles, NT neuropil threads). A, B × 166

was seen in leptomeningeal or parenchymal vessels us-ing Ab against cystatin C and prion protein (3F4). In pa-tient 1, ubiquitin- and α-synuclein-immunopositive Lewybodies (Fig.4A, B) were observed in neurons of the cin-gulate, frontal, temporal cortices, amydgala, thalamus andsubstantia nigra. In patient 2, rare α-synuclein-immunopos-itive inclusions were seen in the neocortex; in the substan-tia nigra, mild neuronal loss and several NFT were seen.

Samples from isolated leptomeningeal vessels of pa-tient 1 showed apple-green birefringence under polarizedlight after Congo red staining. Immunofluorescence mi-croscopy with monoclonal Ab 4G8 and 6E10 showed verystrong labeling of the vessel walls (Fig.5). Strong im-munoreactivity was also observed with Ab against amy-loid-associated proteins ApoJ (α- and β-chains), P-com-ponent, and vitronectin.

Confocal microscopy

This immunostaining revealed marked amyloid angiopa-thy, occasional diffuse amyloid deposits in the neuropil,and numerous Alz50- and Tg3-immunoreactive NFT, NT,

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Fig.4 A Neuron with a Lewy body, H&E preparation. B α-Synu-clein-immunopositive Lewy bodies. A × 416, B × 166

Fig.5 Isolated leptomeningeal vessels, immunofluorescence withmonoclonal antibody 6E10. × 150

Fig.6A–C Confocal microscopic images of temporal cortex withtriple immunostaining for tau (red), amyloid (blue) and astrocytes(green). A Tau immunoreactivity in a “crown” of small, punctatedeposits surrounding amyloid-encased vessels. B Marked astro-cytic response surrounding the vessels. C Occasionally, tau-posi-tive immunoreactivity showed close association with astrocyticprocesses. Bars A, B 30 µm; C 10 µm

and crowns of small, punctate immunoreactivity sur-rounding amyloid-encased vessels (Fig.6A). There wasalso marked astrocytic response surrounding the vessels(Fig.6B). At × 100 magnification, examination of a fewof these punctate and linear tau-positive features showedclose association with GFAP-positive processes (Fig.6C).Although the majority of tau-positive processes in thecrown of deposits around vessels did not colocalize withGFAP, instances in which the tau immunoreactivity ap-peared to be within the fine processes of astrocytes, even“pushing aside” the GFAP filament, were evident.

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Fig.7 A Western blot of formic acid-soluble extracts from lep-tomeningeal vessels (lane 2) and from cortical vessels (fraction 53,lane 3) with 6E10 antibody as the primary antibody as describedunder Material and methods. Lane 1 is synthetic Aβ1–40 peptide.Left Molecular weight markers from top to bottom: 14.3, 6.5, and3.4 kDa. Arrowhead indicates monomer Aβ. B Mass/charge ratio(molecular weight of protonated parent ion, m/z) in MALDI-MSof the amyloid isolated from formic acid extract from lep-tomeningeal vessels. Insets Calculated and observed masses of Aβspecies with their predicted sequences. The presence of formylatedAβ species is indicated (asterisks). C MALDI-MS of immunopre-cipitated Aβ from leptomeningeal vessels using a mix of 4G8 and6E10 antibodies. Note that the immunoprecipitated material is lessformylated than in A due to a shorter treatment with formic acid.D MALDI-MS of immunoprecipitated Aβ from fraction 53 fromcortical vessels using a mix of 4G8 and 6E10 antibodies. The presence of formylation is indicated by asterisks

Biochemical studies

Amyloid extracted from leptomeningeal vessels and corti-cal vessels (fraction 53) consisted largely of 4-kDa mono-meric Aβ together with dimeric and polymeric species(Fig.7A). No immunoreactivity was observed using spe-cific Ab for Aβ ending at position Ala42 (not shown). Massspectrometry analysis of formic acid extracts from lep-tomeningeal vessels (Fig.7B) showed a major componentwith a mass of 4,328.5 Da (peak 7) corresponding to Aβ1–40 (calculated mass of 4,327.17). In addition, we observedthree minor peaks at 4,018.0 (peak 1), 4,125.7 (peak 3) and

4,215.4 mass units (peak 5) corresponding to Aβ1–36,1–38 and 2–40 species, respectively. A small peak at4,508.9 mass units was indicative of the presence of Aβ1–42. Peaks 2, 4, 6, and 8 corresponded to the formylatedAβ species. When synthetic peptide Aβ1–40 was subjectedto MALDI-MS for comparison, a peak was observed at4,327.9 mass units. The major Aβ species found in theimmunoprecipitate from leptomeningeal vessels using amixture of monoclonal 4G8 and 6E10 Ab (Fig.7C) wasAβ1–40 (peak 4, 4,328.9 mass units), with minor Aβ1–38(peak 2, 4,125.5 mass units), Aβ1–36 (peak 1, 4,013.7 massunits), 2–40 (peak 3, 4,215.0 mass units), and Aβ1–42

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Fig.8 A Elution profile of theextracted Aβ from lep-tomeninges, run on a C4 RP-HPLC column. The formicacid-soluble material obtainedfrom leptomeningeal vesselswas run on a C4 narrow boreRP-HPLC. Monomer Aβ pep-tides eluted at 46% acetoni-trile. The arrowhead indicatesthe position of elution of syn-thetic peptide Aβ1–40 at 47%acetonitrile. Inset is a fluoro-gram developed using mono-clonal 4G8 (1: 500) as a pri-mary antibody of a Westernblot of the HPLC fractions[2–9] after 16% SDS-Tris-tricine-PAGE. Aβ amyloidmonomers, dimers and highermolecular mass polymers dis-play a strong immunoreactivitywith 4G8. Synthetic peptideAβ1–40 (400 ng) was run onlane 1 as a control. B Massspectrometry of Aβ species pu-rified by RP-HPLC (peak 4) asdepicted in the chromato-graphic profile in A. InsetsPredicted and observed molec-ular masses of Aβ species. Thepresence of one formic acidmolecule is indicated by anasterisk (RP reverse phase)

(peak 5, 4,510.4 mass units). A similar pattern of Aβ pep-tides was obtained with Aβ immunoprecipitated from frac-tion 53 (Fig. 7D). We found Aβ1–40 (peak 6, 4,328.8 massunits) as the major component, with Aβ1–36 (peak 1,4,013.9 mass units), Aβ1–38 (peak 3, 4,124.9 mass units),Aβ2–40 (peak 5, 4,215.7 mass units), and Aβ1–42 (peak8, 4,516.7 mass units) as minor components. Peaks 2, 4,and 7 correspond to the formylated isoforms. Mass spec-trometry of Aβ immunoprecipitated from leptomeningesprocessed in the same way from a normal control wasnegative (not shown).

The leptomeningeal amyloid was further purified byRP-HPLC (Fig.8A). Western blot analysis using anti-Aβ4G8 Ab showed a band of a molecular weight of 4 kDa and

the presence of polymers (Fig.8A, inset). Mass spectrom-etry analysis of the fraction eluting at 47% acetonitrile(fraction 4) showed a similar pattern (Fig. 8B) of Aβ speciesas that obtained from formic acid extracts from crude lep-tomeningeal vessels preparations (Fig.7B) with Aβ1–40 asthe major form (peak 7) and Aβ1–38 (peak 3), Aβ1–36(peak 1), 2–40 (peak 5), and Aβ1–42 (peak 9) as minorforms. Application of the partially purified amyloid directlyonto the HPLC gave a higher yield but with slightly morecontaminants than when the material was first purifiedthrough Sephadex G-100 in 3 M formic acid (not shown).

Fraction 53 from microvessels was also purified by RP-HPLC (Fig. 9A). Aβ was detected by Western blot (Fig.9A,inset) using 4G8 Ab. The fraction eluting at 40–45% ace-

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Fig.9 A RP-HPLC of Aβ ex-tracted from cortical microves-sels, run on a C18 column.Formic acid-soluble materialextracted from fraction 53 wasrun on a C18 narrow bore col-umn. The arrowhead indicatesthe position in which syntheticAβ peptide elutes in this col-umn. In the inset, an aliquot ofeach fraction [2–9] was ana-lyzed by Western blotting with4G8 antibody as the primaryantibody after 16% SDS-Tris-tricine-PAGE. In lane 1 400 ngof synthetic Aβ was loaded forcomparison. B MALDI-MS ofpeak 4 purified as indicated inA. Inset Calculated and ob-served molecular masses of Aβspecies with their positions inthe amino acid sequence. Theasterisk indicates a formylatedAβ peptide and double asteriskindicates the presence of twoformyl groups

tonitrile (fraction 4) was analyzed by mass spectrometryand amino acid sequencing. The mass spectrometry data(Fig.9B) shows the presence of Aβ1–40 (peak 7), with avery minor contamination of Aβ1–38 (peak 3), Aβ1–36(peak 1), and Aβ2–40 (peak 5). Peaks 2, 4, and 6 repre-sent the formylated forms. Peak 9 correspond to Aβ1–40with two formyl groups. When subjected to amino acidsequence analysis, fraction 4 rendered the amino acid se-quence DAEFRHDSGYEV, corresponding to the Aβ se-quence starting at position one. It is not surprising, giventhe difference in sensitivity between amino acid sequenc-ing and MALDI-MS that we were unable to obtain se-quence data starting at position 2 of the Aβ sequence (Ala),due to the low amount of Aβ2–40 present in the sample.

Genetic studies

We used HhaI restriction enzyme analysis of PCR prod-ucts to determine the APOE genotype of cases 1 and 2. Af-ter cleavage, a band of 72 bp characteristic of the ε4 allelewas observed in both patients. No bands of 82 or 91 bp,typical of the ε2 and ε3 alleles, were observed. These re-sults indicate the presence of homozygosity for the APOE-ε4 allele in both patients.

DNA sequence analysis failed to show the presence ofmutations in exon 16 or 17 of the APP770 gene or in themajor open-reading frame of the PS-1 molecule.

Discussion

We report here the clinicopathological and biochemicalanalyses of two individuals homozygous for the APOE-ε4allele affected with senile dementia. From a clinical stand-point, patients 1 and 2 are notable for a relatively rapidcourse between onset of symptoms and death, with bothhaving marked impairment in verbal short-term memoryand significant behavioral disturbances in the end stage.Patient 2 was notable for the presence of early personalitychanges and extrapyramidal symptoms. Neuropathologi-cally, their main features were widespread Aβ angiopathy,perivascular tau deposits, and NFT. Lewy bodies that wereα-synuclein immunopositive were present in both patientsbeing more abundant in patient 1 than in patient 2. Al-though the dementia might have been the result of multi-ple neuropathological lesions, the combination of Aβ an-giopathy and perivascular neurofibrillary pathology, inaddition to the NFT appeared to be the most notable neu-ropathological abnormality. In spite of the severe Aβ an-giopathy, there was no evidence of cerebral hemorrhage.Aβ angiopathy was present in leptomeningeal vessels, cere-bral medium-sized and small vessels, and capillaries; how-ever, senile plaques were not found in the brain parenchy-ma. While dystrophic neurites were prominent in the pa-renchyma surrounding affected vessels, numerous corticaland subcortical NFT were also present. The presence of a“crown” of small, punctate tau-immunopositive depositssurrounding vessels has been observed as a minor phe-

nomenon in sporadic AD; however, it was a prominentfeature in the cases reported here. The observations deriv-ing from the immunohistochemistry and confocal mi-croscopy suggest that at least some of the tau deposits areclosely associated with GFAP-positive astrocytic processes.It remains for ultrastructural studies to distinguish whethertau immunopositivity represents a close relationship be-tween a neuronal and an astrocytic process, a form of glialtau deposits, or a tau-positive structure engulfed by astro-cytes.

The vascular amyloid deposits observed in these caseswere consistently labeled by Ab against Aβ in tissue sec-tions, in isolated leptomeningeal vessels and in purifiedamyloid fractions. By Western blot, mass spectrometryand amino acid sequence analyses, we determined that theAβ, deposited in leptomeningeal and cortical vessels, iscomposed mainly of the Aβ1–40 isoform. Minor amountsof the Aβ1–38, 1–36 and 2–40 species were also present.These data are in agreement with most of the studies onthe composition of the cerebrovascular amyloid in AD andHCHWA-D [7, 17, 21, 27, 33, 36]. The low signal inMALDI-MS for Aβ1–42 and the lack of immunoreactiv-ity with anti-AβAla42 in the Western blot analysis indi-cates that this isoform is only a very minor component ofthese vascular amyloid deposits. This was also confirmedby the difference in the reactions on vessels walls usinganti-AβVal40 and anti-AβAla42. In fact, while very fewvessels were immunolabeled with the latter, the great ma-jority of them were strongly immunolabeled with the for-mer. We also found strong immunoreactivity for the amy-loid-associated proteins ApoE, ApoJ, P-component andvitronectin in amyloid-ladden vessels.

ApoE has been shown to play an important role inamyloid deposition in vivo [2]. ApoE knockout micecrossed with transgenic mice overexpressing a humanmutant ΑPP gene showed a dramatic reduction in amy-loid burden. In contrast, ΑPP transgenic mice carrying themurine apoE gene had numerous Aβ deposits. Geneticanalysis indicates an association between the inheritanceof the APOE-ε4 allele and the development of CAA andlate-onset AD [28, 29, 35]. The frequency of the APOE-ε4allele in AD from living and autopsy series was reportedto be between 30% and 40%, which is approximately threetimes that in the Caucasian population and in neurologicaldisorders other than AD. The APOE-ε4 allele frequencywas also found to be increased in subjects who had ac-crued brain Aβ deposits subsequent to head injury [19,24]. In contrast, the APOE-ε4 haplotype has low preva-lence in the NFT predominant form of senile dementia[4]. These foregoing studies implicate an important func-tional role for ApoE isoforms in the pathophysiology ofAD. Additionally, the risk of dementia in stroke patients hasbeen reported to be increased nearly twofold for APOE-ε4heterozygotes and sevenfold for homozygotes [34], withthe ε4 allele being a risk factor for CAA and CAA-relatedhemorrhage independent of its association with AD [11].Therefore, we hypothesize that the presence of the APOE-ε4 allele homozygosity in our cases was an important com-ponent involved in the build-up of the extensive lep-

10

tomeningeal and cortical Aβ-CAA observed, and in thedevelopment of dementia.

In AD, the parenchymal deposition of Aβ1–42/43 inneuritic plaques, has been proposed as the initiating eventof the pathogenic cascade [32], with the other features ofthe disease (NFT, synapse and cell loss) being conse-quences of the critical initiating event, but all contributingto the dementia. In our cases, the presence of dystrophicneurites surrounding affected neocortical vessels, may rep-resent a neuritic reaction in areas adjacent to the Aβ-CAA.This is in agreement with previous observations showingperivascular dystrophic neurites in AD [25]. It is of inter-est that the pathological features observed in the two pa-tients reported here have similarities to those seen in twoforms of presenile dementia, one associated with a stopcodon 145 mutation in the Prion Protein gene and anotherwith a single base substitution at the stop codon of the BRIgene [8, 40]. In both conditions an amyloid angiopathycoexists with severe neurofibrillary pathology [8, 30]. Theassociation of cerebral vascular amyloid with neurofibril-lary pathology emphasizes the concept that tau depositsmay pathogenically related to amyloid deposition.

A definitive classification of our cases is not possibleat this time. Two patients neuropathologically similar tothose reported here have been recently described and di-agnosed as “vascular variant of Alzheimer’s disease” [45]and “sporadic amyloid angiopathy” [22]; however, theirAPOE genotypes are not known. The neuropathology ofthese atypical cases, including those presented here, is dif-ferent from that observed in HCHWA-D because of thepresence of dementia with neurofibrillary lesions and theabsence of cerebral hemorrhage, and from the pathologyfound in AD because of the absence of senile plaques.Furthermore, the neuropathology of these atypical casesof dementia is also different from that of amyloid angiopa-thy of the sporadic variety. Our patients had dementia as-sociated with CAA and neurofibrillary pathology in theabsence of hemorrhages. Dementia has been estimated tooccur in 40% of individuals with intracerebral hemorrhagesrelated to sporadic CAA [41]. We excluded a diagnosis ofamyloid angiopathy of the sporadic variety on account ofthe presence of neurofibrillary lesions. Concerning the lat-ter, it should be noted that finding NFT in patients of thisage range is frequent [5], and that only a small number ofvery old individuals characterized by senile dementia withpredominant NFT have been reported [3, 37].

Acknowledgements We gratefully acknowledge Brenda Dupree,Rosemarie Richardson, Bridget Dennis, Constance Alyea, andFrancine Epperson for technical help, Urs Küderli for photographicassistance; Linda Bailey and Bradley S. Glazier helped in prepar-ing the manuscript. This research was supported by PHS GrantsAR02594, AG08721, AG10973 and P30 AG10133. J.G. is a recip-ient of a NIDA from the American Heart Association, NYC affili-ated. M.C. is a recipient of a Fellowship from the Ministerio deEducacion y Cultura, Spain.

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Note added in proof Following the acceptance of this manu-script, we identified and studied a third patient with senile demen-tia who was ApoE ε4 homozygous and had the neuropathologicfeatures described in patient 1 and 2, except no Lewy bodies wereseen.