ARTICLE - Nonfibrillar diffuse amyloid deposition due to a γ42

© 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 18 2589–2598

ARTICLE

Nonfibrillar diffuse amyloid deposition due to aγ42-secretase site mutation points to an essential rolefor N-truncated Aβ42 in Alzheimer’s disease

Samir Kumar-Singh, Chris De Jonghe, Marc Cruts, Reinhold Kleinert1, Rong Wang2,Marc Mercken3, Bart De Strooper4, Hugo Vanderstichele5, Ann Löfgren, Inge Vanderhoeven,Hubert Backhovens, Eugeen Vanmechelen5, Peter M. Kroisel6 andChristine Van Broeckhoven+

Laboratory of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, Born-Bunge Foundation,University of Antwerp, Universiteitsplein 1, B-2610, Antwerpen, Belgium, 1Laboratory of Neuropathology, Institute ofPathology and 6Laboratory of Human Genetics, Institute for Medical Biology and Human Genetics, University of Graz,Austria, 2Laboratory for Mass Spectrometry, The Rockefeller University, NY, USA, 3Janssen Research Foundation,Beerse, Belgium, 4Laboratory of Neuronal Cell Biology and Gene Transfer, University of Leuven and 5Innogenetics,Zwijnaarde, Belgium

Received 15 June 2000; Revised and Accepted 24 August 2000

Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 aminoacid long amyloid β-peptide (Aβ42) is considered central to Alzheimer’s disease (AD) pathology. However, it isgenerally believed that nonfibrillar pre-amyloid Aβ42 deposits have to mature in the presence of Aβ40 intofibrillar amyloid plaques to cause neurodegeneration. Here, we describe an aggressive form of AD caused bya novel missense mutation in APP (T714I) directly involving γ-secretase cleavages of APP. The mutation hadthe most drastic effect on Aβ42/Aβ40 ratio in vitro of ∼11-fold, simultaneously increasing Aβ42 and decreasingAβ40 secretion, as measured by matrix-assisted laser disorption ionization time-of-flight mass spectrometry.This coincided in brain with deposition of abundant and predominant nonfibrillar pre-amyloid plaques com-posed primarily of N-truncated Aβ42 in complete absence of Aβ40. These data indicate that N-truncated Aβ42 asdiffuse nonfibrillar plaques has an essential but undermined role in AD pathology. Importantly, inhibitingsecretion of full-length Aβ42 by therapeutic targeting of APP processing should not result in secretion of anequally toxic N-truncated Aβ42.

INTRODUCTION

The predominant protein component of the cortical and cerebro-vascular amyloid deposits of Alzheimer’s disease (AD) is theamyloid β-peptide (Aβ). Accumulating evidence suggests thatAβ production from amyloid precursor protein (APP) (1), itsaggregation into fibrils and its deposition are key etiologicalevents in AD (2). An understanding of these critical steps willbe crucial in devising therapeutic targets.

APP is processed by β-secretase (BACE) (3) and a yetunidentified α-secretase leading to soluble N-terminal APP frag-ments (APPsα and APPsβ) and membrane bound C-terminalfragments (α and β CTFs) (2). Whereas cleavages by β- andγ-secretases release 40–42 amino acids Aβ peptides (Aβ1–40and Aβ1–42), the major secretory pathway utilizes α-secretasethat cleaves the Aβ sequence between amino acids 16 and 17

of Aβ. Further processing of the αCTF by γ-secretase releasesN-terminally cleaved Aβ17–40 or Aβ17–42 peptides (p3). In additionto p3 (Aβ17-X), two other major peptides starting from aminoacids 5 (Aβ5-X) and 11 (Aβ11-X) are secreted by transfected cells(4–6) although their significance for the pathogenesis ofamyloid plaque formation is unclear (2). In contrast, the impor-tance of Aβ C-terminal heterogeneity is more apparent.Immunohistochemistry showed that, while Aβ42 deposits firstin diffuse plaques in AD and in Down’s syndrome (DS)patients (7,8), Aβ40 contributes to further growth of theamyloid plaques to form dense compact plaques (i.e. cored andneuritic plaques) (8,9). Aβ40 is also the predominant constituentof the amyloid deposits in blood vessel walls (congophilicamyloid angiopathy and dyshoric angiopathy) (8). Congophiliccored plaques or fibrillar amyloid deposits, composed chiefly

+To whom correspondence should be addressed. Tel: +32 3 820 2601; Fax:+32 3 820 2541; Email: [email protected]

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of full-length Aβ, are proposed to be pathogenic in AD and DSpatients (10–12). Recently, deposition of p3 in non-congophilicdiffuse plaques has also been recognized (7,13); however, theprecise significance of these amyloid deposits to disease patho-genesis and cognitive decline in AD and DS is poorly under-stood. p3 is considered to be relatively benign as it isnonfibrillar (13) and is proposed to lack domains involved inAPOE binding (14) and microglial and complement activation(15,16).

As yet, eight missense APP mutations have been identifiedin families with autosomal dominant early-onset AD (17,18).All these mutations clustering in close proximity to the secre-tase cleavage sites, affect APP metabolism in two distinctways. The Swedish double mutation (K670N/M671L) locatednear the β-secretase cleavage site (19), increases the productionof both Aβ40 and Aβ42 (20,21). In contrast, mutations locatednear the γ-secretase cleavage sites (17,18) result in anincreased production of Aβ42, in a similar manner to thatdescribed for presenilin gene (PS1 and PS2) mutations (22,23).Here we report a novel mutation that involves amino acid 43 ofAβ located directly at the γ42-secretase cleavage site and leadsto a severe AD pathology with unusual plaque compositionand morphology. Whereas Aβ40 was completely absent fromamyloid deposits in brain and formation of typical coredplaques was retarded, abundant diffuse non-congophilicamyloid plaques in association with dystrophic neurites andreactive gliosis were predominantly deposited.

RESULTS

APP T714I mutation

Family AD156 (Fig. 1A), an Austrian family consistent withautosomal dominant inheritance of early-onset AD, wasreferred for DNA diagnosis. The proband, her sister and theirmother were diagnosed as probable AD according toNINCDS–ADRDA criteria at age 38, 38 and 44 years respec-tively. However, signs of cognitive impairment and behaviourdisturbances were apparent several years earlier in all threepatients suggestive of a mean onset age of ∼34 years in thefamily. Genomic DNA of the proband was examined for muta-tions in APP, PS1 and PS2. In exon 17 of APP (Fig. 1B), aheterozygous C→T transition was identified at position 2208substituting Thr (T) at codon 714 by Ile (I) (T714I, numberingaccording to APP770 isoform). The mutation abolishes aTspRI restriction site that was used to confirm the presence ofthe mutation in the proband (156.1) and her sister (156.2)(Fig. 1C). The mutation was absent in the father as well as in50 healthy Austrian individuals. No other mutations weredetected.

The Austrian T714I mutation is the first APP mutationreported to date that involves amino acid 43 of Aβ locateddirectly at the γ42 secretase cleavage site. The early onset age,as well as rapid progression of the disease and early death, iscomparable with AD caused by mutations in PS1 (18).

Drastically altered APP processing in vitro

To understand the effect of the T714I mutation on the cleavagespecificity of γ42-secretase, we studied APP processing in trans-fected cells. Human embryonic kidney (HEK) 293T cells were

transiently transfected with the T714I APP cDNA and secretedAβ1–42 and Aβ1–40 levels were measured in conditionedmedium by enzyme-linked immunosorbent assay (ELISA)(24,25). Cells overexpressing wild-type and London V717I (26)APP cDNA were used as controls. The T714I mutationincreased Aβ42 and at the same time decreased Aβ40, resulting ina significantly increased Aβ1–42/Aβ1–40 ratio (P < 0.001), whichwas four times higher than in wild-type. In the same experi-ment, V717I resulted in a 1.8-fold increased Aβ1–42/Aβ1–40ratio, results that are comparable with previously publisheddata by Suzuki et al. (22). We also measured by ELISA Aβ1–42and Aβ1–40 in plasma of the patient (156.2), her unaffectedfather (156.3) and five unrelated age-matched controls. TheAβ1–42/Aβ1–40 ratio was 2.5-fold increased compared with theunaffected father and 1.7-fold compared with the controls.

The conditioned medium of the T714I and wild-type APPtransfected HEK293T cells was also analyzed by matrix-assistedlaser desorption ionization time-of-flight (MALDI-TOF) massspectrometry (4) (Fig. 2). This method allowed us to assess bothfull-length and N-truncated Aβ. Compared with wild-type, T714Ishowed a significant elevation of Aβ1–42 (3.5-fold) (P < 0.001),whereas at the same time Aβ1–40 decreased significantly by 68%

Figure 1. (A) Pedigree of AD156 segregating the APP T714I mutation. Solidsymbols indicate affected individuals; †, age at death; arrow, the probandwhere autopsy was performed. (B) Sequence analysis for 156.1 and 156.2showing a heterozygous C→T transition at position 2208 of the cDNA leadingto an amino acid substitution of threonine (T) to isoleucine (I) at codon 714 inexon 17 of APP (numbering according to APP770 isoform). (C) PCR–RFLPanalysis of PCR amplified APP exon 17 product followed by TspRI digestion.

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(P <0.001). This brought down the secreted levels of Aβ1–40 toequal the amounts of secreted Aβ1–42 resulting in an increasedAβ1–42/Aβ1–40 ratio by 10.8-fold over wild-type. Similar effectswere seen on p3 with nearly equal levels of secreted Aβ17–40

and Aβ17–42 increasing Aβ17–42/Aβ17–40 ratio by 10.7-fold overwild-type. One other pronounced effect of the T714I mutationwas the significant increase of Aβ peptides ending at V39 andG38 irrespective of the N-terminal residue. Smaller effectswere also seen for full-length Aβ ending at G37. Thesepeptides were not artificially produced by proteolysis of full-length Aβ as synthetic Aβ1–40 and Aβ1–42 peptides were notdegraded when added to the medium of non-transfected cells(4). Compared with other APP mutations located close to theγ42-secretase cleavage site (22,26,27), T714I had the highestincrease in Aβ42/Aβ40 ratio. Interestingly an in vitro decrease inAβ40 was also reported recently for the French V715M muta-tion (27), one amino acid downstream of T714I. The mecha-nisms by which these mutations affect Aβ40 and Aβ42 secretionor lead to alternative cleavage to Aβ38 or Aβ39, is not yet under-stood. The transmembrane region of APP is in α-helicalconformation (28) and its interaction with PS1, the proposed γ-secretase (29,30), results in intramembranous proteolysis. It isplausible that PS1 might have different binding affinities orcleavage efficiencies to these mutated CTFs and a close spatialproximity of amino acids three or four positions apart in this α-helical conformation, might explain the effect of T714I orV715M on γ40-secretase activity as well.Prompted by a description of altered soluble APP for V715Mmutation (27), perhaps due to increased trafficking of the mutatedAPP into cellular compartments where α- and β-secretasesreside, we quantified APPsα and APPsβ for T714I. However,no significant changes over wild-type were noted (Fig. 3).

Unusual plaque morphology and composition

Neuropathological examination of the proband (156.1) showedextensive neuronal loss accompanied by diffuse gliosis,amyloid plaques and neurofibrillary tangles confirming thediagnosis of AD. We performed an in-depth analysis by histo-chemistry and immunohistochemistry on serial sections fromthree select brain areas: hippocampus including entorhinalcortex (ERC) and temporal and frontal cortices. Immuno-staining with an antibody recognizing both full-length Aβ andp3 (mAb 4G8), remarkably stained a huge plaque loadpredominantly as ‘cloudy’ diffuse plaques that sometimesenclosed a central lacuna. In the molecular layer of the dentategyrus, the amyloid plaques had the same non-neuritic ‘cottonwool’ appearance as described for PS1 ∆9 patients (31–33).We determined the fibrillar nature of the amyloid plaques bycongo-red and thioflavin staining. Cored plaques werecongophilic and fluorescent with thioflavin, whereas themajority of the diffuse plaques were non-congophilic (Fig. 4Aand B). In the few diffuse plaques faintly positive for thio-

Figure 2. (A) Representative MALDI-TOF mass spectra for APP wild-typeand T714I. Conditioned medium of HEK293T cells transfected with T714Iand wild-type APP were studied by IP/MS using mAb 4G8. Relative peakintensities were normalized with synthetic Aβ (12–28) peptide (marked std)and identities of the observed peaks were inferred as described in Materialsand Methods. Note that predominant peaks for peptides ending at residue 40 areclearly lost for T714I.(B) Effect on secreted Aβ with different N- and C- terminiwere analyzed in detail. These were peptides ending at Aβ42, Aβ40, Aβ39, Aβ38and Aβ37 and beginning at E1 (Aβ1-X), R5 (Aβ5-X), E11 (Aβ11-X) and L17(Aβ17-

X). Bars are equally scaled to allow interpanel comparison. Absence of fewbars in the wild-type is due to peaks too low to be measured. *, statistical signif-icance of at least P < 0.001 versus wild-type.


flavin, the fibrillar amyloid occupied only a small proportion oftotal amyloid recognized immunohistochemically. In the layerIII of ERC, the amyloid plaques were congophilic whereas in itssuperficial and deep layers, the diffuse plaques were againnonfibrillar. A strong neuritic pathology was evident in CA1and subicular fields of hippocampus and ERC. Neurites accu-mulated hyperphosphorylated tau (AT8), ubiquitin and APP,irrespective of plaque pathology (Fig. 4C and D). Usingendothelial cell markers (CD31 and CD34) with amyloidstaining, core plaques but not the diffuse plaques were closelyassociated with blood vessels. This feature was most remarkablein the endplate region where all fibrillar amyloid deposits wereassociated with blood vessels (Fig. 4E and F).

Next we examined whether in vivo the mutation had similareffects on APP processing. Reactivity to a C-terminal Aβ40-specific monoclonal antibody, JRF/cAb40/10, was completelyabsent in both blood vessel walls and amyloid plaques in theisocortex (Fig. 5A), while a faint immunoreactivity was occa-

Figure 3. Analysis of soluble APP (APPsα and APPsβ). Supernatants wereresolved on a NuPAGE gel and immunoblotted with mAb 6E10 for APPsα(A) or 53/4 for APPsβ (B). Bands for T714I and wild-type were quantifiedusing the NIH Image software package (data not shown).

Figure 4. Congophilicity of T714I amyloid deposits and their relation to neuritic pathology and blood vessels. (A) Amyloids in the end plate region werecongophilic and higher magnification (square) shown in (B) demonstrates that the majority of congophilic cored plaques were vascular in origin. (C) Neuriticpathology was either independent in CA1 area (AT8, black; 4G8, red), or was related to diffuse plaques (D). Note here dystrophic neurites (AT8, purple; ubiquitin,brown) showing close proximity to diffuse amyloid plaques (blue) in subicular area. Amyloid was also deposited in a halo around healthy looking neurons (arrow)not taking up stain for hyperphosphorylated tau and ubiquitin. (E) Most of the amyloid plaques in molecular layer of dentate gyrus showed no relation with bloodvessels, whereas cored plaques in the end plate region were mostly dyshoric angiopathic (F). Scale bars: (A) 500 µm; (B–E) 40 µm; (F) 200 µm.


sionally present in the endplate region of the hippocampus (2–3amyloid deposits per 10 fields of 0.7 mm2 each) (Figures 4Aand B and 5D). In contrast, a C-terminal Aβ42-specific mono-clonal antibody, JRF/cAb42/12, imparted a strong reactivity toall amyloid deposits including diffuse plaques, blood vesselwalls and ‘infrequent’ cored plaques (Fig. 5B). Sections werestained with 4G8 and compared with immunoreactivity forantibodies specific for Aβ40 and Aβ42 by image analysis (Fig.5A–C). In the hippocampus, Aβ40 constituted only 1–7% ofamyloid deposits while in neocortex Aβ40 was completelyabsent (Fig. 5D). Multi-spectral analysis by confocal laserscanning microscope (CLSM) also demonstrated the absenceof Aβ40 (Fig. 5E and F) and a full histochemical overlap for4G8 and Aβ42-specific antibody. These observations wereconfirmed using other Aβ40-specific [FCA3340 (34), R209 (35)]and Aβ42-specific [21F12 (36), FCA3542 (34), R226 (35)]antibodies (data not shown).

We studied the N-truncation of Aβ in these amyloid depositsand confirmed whether the diffuse plaques were capable ofinciting any glial reaction. Reactivity for a panel of antibodiesagainst N-terminal Aβ (6E10, 6F/3D and JRF/AβN/11), whencompared with 4G8 reactivity, indicated that diffuse plaquesconsisted of N-truncated Aβ while full-length Aβ wasconfined to blood vessel walls, dense amyloid cores andamyloid plaques in ERC layer III and remarkably negative fordeep cortical layers (Fig. 6A and B). Abundant glial andinflammatory pathology was noted in association with diffusebut mostly compact plaques using astroglial (GFAP), micro-glial (CD68, HLA–DR) and complement (C1q) markers. An

intense glial activation was noted in many regions withoutplaque pathology as in white matter (Fig. 6C–F).

DISCUSSION

The unusual effect of T714I on Aβ40 secretion and depositionin brain allowed for the first time the study of the role of Aβ40in plaque formation and clears many tenets about ADpathophysiology. It is noteworthy that in contrast to in vitrostudies where Aβ42 was demonstrated to be more fibrillogenicand aggregated faster than Aβ40 (37), in vivo studies in rodentspoint otherwise. Synthetic soluble Aβ1–40, but not Aβ1–42, wheninjected in rat brain, form fibrillar congophilic amyloiddeposits (38) and transgenic mice overexpressing mutant APPand PS1, deposit predominantly Aβ40 and not Aβ42 in paren-chymal congophilic compact plaques (39). Similarly in ADand DS patients, in cored plaques and especially in thecongophilic amyloid cores, Aβ40 is the predominant constit-uent (8,9). Although Aβ40 deposition is later in the diseaseprocess (8,9), it is unlikely that a rapid disease progression inT714I patients prevented its deposition. The irrefutable argu-ment to support this is that Aβ40 was absent from vessels,where it is the predominant constituent at all disease stages(8,40–42). This is the first neuropathological proof in humanof the role of Aβ40 in plaque maturation where a reduced coredplaque load suggests a role for Aβ40 in formation of amyloidcores but certainly not in delaying disease progression.

Instead, the severity of the disease paralleled a massivedeposition of diffuse, ‘cloudy’ amyloid plaques, composedchiefly of N-truncated Aβ42. Only amyloid in dense amyloid

Figure 5. Aβ40 and Aβ42 immunohistochemistry. (A) Aβ40 reactivity was completely absent in temporal cortex from all amyloid deposits with mAb JRF/cAb40/10,but were recognized efficiently by JRF/cAb42/12 (B) and 4G8 (C). (D) In the end plate region, Aβ40 reactivity was also absent except in few blood vessels andamyloid cores. Note here the loss of Aβ40 reactivity in a vessel (arrows) while present in amyloid core. These amyloid deposits were recognized by Aβ42 on serialsections (data not shown). (E) The results were confirmed on a CLSM. Here in subicular layer Aβ40 (red) is completely absent while reactivity was present for Aβ42(green) (F). Scale bars: upper panels, 500 µm; lower panels, 30 µm.


cores, congophilic amyloid angiopathy and amyloid plaques inERC layer III stained for full-length Aβ. Release of p3 by APPexpressing cells during normal metabolism is long known butonly recently the contribution of N-truncated Aβ deposition indiffuse plaques is being appreciated (7,13). It has been recog-nized that p3 is relatively insoluble and highly aggregatable(43,44). Furthermore, analysis of T714I clearly demonstratesthat the activity of α-secretase might preclude the formation offull-length Aβ, it does not preclude the formation of amyloidas p3. Developments of drugs targeting secretases have toconsider that partially blocking BACE (3) might switch toincreased utilization of the α-secretase pathway, producing p3

which might be as neurotoxic. A partial co-blockade of α-secretase should be seriously considered.

The presence of neuritic plaques containing abundantAβ-derived amyloid fibrils in AD brain tissues supports theconcept that fibril accumulation per se underlies neuronaldysfunction in AD (11,12). However, recent observations havechallenged this assumption by suggesting that earlier Aβ assem-blies formed during the process of fibrillogenesis may also playa role in AD pathogenesis (45,46). Protofibrils, metastable inter-mediates in fibril formation, were shown to be toxic to culturedneurons (47). Although full electron-microscopical data arelacking but based on Congo-red and thioslavin-S binding, we

Figure 6. N-truncation of diffuse plaques and glial pathology. (A) 6E10 (purple) and 4G8 (red) co-staining in ERC layer III, considered as full-length Aβ. Note thatalthough superficial and deep layers contain the classical diffuse plaques of T714I, occasionally compact congophilic plaques, positive for 6E10 were present asshown here (arrow). Most of these plaques associated closely with blood vessels on serial sections. (B) The cored plaques were 6E10 positive (purple) in hippocampus, themajority of amyloid plaques in molecular layer of dentate gyrus were composed of N-truncated Aβ as they were only recognized by 4G8 (blue). In few of theseplaques focal 6E10 reactivity (arrowheads) was demonstrated. On serial sections again a close relation with the blood vessel was noted. Note a small blood vesselis also positive (arrow). (C) Besides neurons (arrow), microglia (CD68, red) also sometimes occupied the centre of the typical plaques with a central lacuna givingan appearance of a doughnut. Note that in the region of ERC the size disparity in microglia between a vessel related microglia (arrowhead) and activated microgliain the lacuna often exceeds 50 µm diameter. (D) Although microglia were associated with diffuse plaques as well as present in plaque-free white matter, the full-length Aβ containing plaques doubly stained with 6E10 (purple) and 4G8 (blue) were more strongly associated with microglias (CD68, brown). Note here theassociation of microglia with a core-containing plaque resembling dyshoric angiopathy containing full-length Aβ. (E) Complement C1q activation was present innon-congophilic diffuse plaques like here in a neocortical region. (F) Astroglial activation detected by GFAP staining (red) was associated with diffuse plaques,cored plaques or was independent of plaque pathology as here in a neocortical region. Scale bar, 40 µm.


present here novel pathological findings that diffuse,non-congophilic nonfibrillar Aβ plaques are associated withneuritic pathology. The ‘cloudy’ diffuse plaques as a predominantplaque type in AD partially resemble those reported for thePS1 ∆9 patients (31–33) that present with AD and spastic para-paresis. The prevalent, large, nonfibrillar, but non-neuritic‘cotton wool’ plaques described in these patients are alsocomposed primarily of N-truncated Aβ42 (33 and unpublisheddata), suggesting that a heavily increased load of truncatedAβ42 changes the plaque morphology. We also show here inT714I patients, with a primarily APP etiology, a completelyplaque-independent development of dystrophic neurites andneurofibrillary pathology in few brain regions. This suggeststhat while nonfibrillar pre-amyloid deposits might have thepotential to cause neuronal toxicity, development of neuriticand amyloid pathology might be dissociated at some stage ofthe disease.

In conclusion, our study demonstrates the possible role ofAβ40 in formation of dense cored plaques in AD and presentsfor the first time evidence that N-truncated Aβ42 even of pre-amyloid nature might play a necessary and sufficient role tocause neuronal toxicity and cognitive decline in AD patients.

MATERIALS AND METHODS

AD diagnosis

Patients in family AD156 were diagnozed with AD based onneurological examination, neuropsychological testing, neuro-imaging and neuropathology (R. Kleinert et al., manuscript inpreparation). The mother was diagnosed at 44 years old. Shehad progressive memory problems and was disoriented in time.EEG showed moderate but generalized unspecific changeswhile CT showed brain atrophy. The proband as well as hersister had a neurological examination at 38 years old. Theyboth suffered from severe depression. Mini metal state exami-nation (MMSE) confirmed the presence of dementia. Singlephoton emission tomography showed clear hypoactivity whileCT confirmed the presence of brain atrophy. In both sisters, thedementia was rapidly progressing as measured repeatedly byMMSE. For example at the age of 39 the proband scored 18/30and her sister 11/30, at the age of 40 the scores had alreadydropped to 10/30 and 3/30, respectively. The actual age ofonset of the symptoms was several years earlier according tothe neurologists who treated the patients. Onset age wasestimated 5–7 years earlier for the mother and 4–5 years earlierfor the daughters. Therefore mean onset age in family AD156was estimated to be ∼34 years. The APOE genotype of theproband was E3E3, that of the sister E2E3. The APOE geno-type of the mother E2E3 was inferred from that of the fatherand siblings. The proband died at 41 years of age and had brainautopsy. The sister is still alive and is 42 years. Macroscopicexamination of the brain showed gross atrophy weighing∼1000 g. Sections derived from the fore-, mid- and hindbrainwere stained with haematoxylin and eosin (H&E), Nissl,Congo-red and modified Bielshowsky. A definite diagnosis ofpresenile AD was made.

Genetic analysis

Exons 16 and 17 of APP were PCR amplified from genomicDNA of patient 156.1 using published primer sets and PCRconditions (48) and PCR fragments were sequenced using theReady Reaction Rhodamine Dye Terminator Cycle Sequencingkit (Applied Biosystems, Foster City, CA). The products wereanalyzed on an ABI310 capillary DNA sequencer (AppliedBiosystems). The APP T714I mutation was analyzed by TspRIdigestion of PCR amplified APP exon 17. Wild-type fragmentsof 354 bp were cut into two fragments of 232 and 122 bp,respectively, whereas the T714I mutant fragments were not cut(Fig. 1). All coding exons of PS1 and PS2 were PCR amplifiedfrom genomic DNA using published primer sets and screenedfor mutations by single strand conformational polymorphismanalysis as described by Cruts et al. (49). APOE genotype wasdetermined as described (50,51).

In vitro mutagenesis

Site-directed mutagenesis was performed on wild-type APP695cDNA cloned in pCDNA3 (52) using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA).Primers app714s, 5′-CGGTGTTGTCATAGCGATAGTGAT-CGTCATCACC-3′, and app714as, 5′-GGTGATGACGAT-CACTATCGCTATGACAACACCG-3′ were used to insertthe APP T714I mutation into the construct. The sequence ofthe constructs was confirmed by direct PCR sequencing of theinsertion fragment using the Taq dye terminator sequenase IIsequencing kit (Applied Biosystems). The products wereanalyzed on an ABI373 automated DNA sequencer (AppliedBiosystems). Mutant APP V717I were constructed as describedpreviously by Hendriks et al. (52).

cDNA transfection

HEK293T cells were transiently transfected with pCDNA3vector containing the T714I, V717I or wild-type APP695 cDNAconstructs using Fugene (Roche Diagnostics, Nutley, NJ)according to the manufacturer’s instructions. The presence ofthe constructs in the cells was confirmed by western blotting.To normalize for APP expression, cells were lysed in 300 µlRIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40,0.5% sodium deoxycholate, 0.1% SDS plus complete proteaseinhibitors). A dilution series of a 5 µl aliquot was separated ona 4–12% NuPAGE polyacrylamide gel. Proteins were blotted ona PVDF membrane and immunodetection was performed withantibody B10/4 using the Western Star Chemiluminescencesystem (Tropix, Bedford, MA). The full-length APP immuno-reactive band was quantified using self written softwares forthe VIDAS image analysis system (Kontron, München,Germany) (53).

Aβ ELISA

HEK293T cells were transfected in triplicate with wild-type orT714I APP cDNA in a six-well plate. One day after transfec-tion, 1 ml OPTIMEM medium without additives was added tothe HEK293T cells and conditioned for 24 h. Medium wascollected and pooled from six transfections. A 1 ml aliquot wasused for each ELISA. Aβ42 concentrations were measured by aprototype version of the INNOTEST β-amyloid1–42 HS ELISAdetecting Aβ42 peptide (24). Aβ40 was measured by ELISA


using rabbit antiserum R209 (35) as capturing antibody andbiotinylated 3D6 (36) as detector antibody, as described(24,25). Each experiment was performed in triplicate and theresults were averaged. A two-tailed unpaired t-test was used tocompare the mean level of Aβ produced by the wild-type andmutant transfectants.

Mass spectrometric Aβ analysis

In a second aliquot of supernatant, collected as describedabove, proteinase inhibitors (2 mM EDTA–Na, 10 µMleupeptin, 1 µM pepstatin A, 1 mM PMSF, 0.1 mM TLCK, 0.2mM TPCK) were added. Aβ peptides were analyzed byimmunoprecipitation/mass spectrometric Aβ assay (IP/MS) asdescribed previously (4). The Aβ peptides were immuno-precipitated from 1.0 ml of conditioned media using mAb 4G8(Senetek, Maryland Heights, MO) and protein G Plus/ProteinA-agarose beads (Oncogene Science, Cambridge, MA) andanalyzed using a MALDI-TOF mass spectrometer (Voyager-DE STR BioSpectrometry Workstation, PE/PerSeptiveBiosystem, Foster City, CA). Each mass spectrum was aver-aged from 256 measurements and calibrated by using bovineinsulin as internal mass calibrant. For comparing the peptidelevels in the conditioned media, synthetic Aβ (12–28) peptide(10 nM) was used as internal standard and the relative peakintensity was used. Both ELISA and MALDI-TOF mass spec-trometric analysis were performed by experimenters blinded tosample identity.

Quantification of APPsα and APPsβ

Conditioned medium (5 µl) was separated on a 4–12% NuPagegel (Novex). Proteins were transferred to a PVDF membrane,the membrane was blocked in PBS + 0.2% I-block + 0.1%Tween-20, incubated overnight at 4°C with primary antibodies6E10 diluted 1:2000 (for APPsα) or 53/4 diluted 1:500 (forAPPsβ), incubated with alkaline phosphatase labeledsecondary antibody (1:4000 diluted), and detected with eitherWestern Star chemiluminescent reagent (Tropix) or peroxidaselabeled secondary antibody (Amersham Pharmacia, LittleChalfont, UK).

Immunohistochemistry

Monoclonal antibody JRF/cAb40/10 and JRF/cAb42/12specific for the C-terminus of Aβ40 and Aβ42, respectively,were raised by immunizing mice with synthetic peptides corre-sponding to Aβ residues 36–40 (VGGVV) or residues 33–42(GLMVGGVVIA) (54). Specificity of the Aβ40 and Aβ42 mAbswas validated by ELISA and western blotting showing no crossreactivity. Similarly mAb JRF/AβN/11 specific for N-terminusof Aβ was raised against Aβ residues 1–7 (DAEFRHD) andrecognizes full-length Aβ (54).

In addition, other antibodies used in this study were thefollowing: mAb 6E10 (Senetek, Maryland Heights, MO; raisedagainst Aβ 1–17, recognizes Aβ 5–11), mAb 4G8 (Senetek; Aβresidues 17–24), 6F3D (Dako, Glostrup, Denmark; raisedagainst Aβ residues 7–17), mAb 21F12 (for Aβ42, Innogenetics,Gent, Belgium), rabbit antisera FCA3542 and FCA3340 (34),rabbit antisera R209 (35) (Aβ40) and R226 (35) (Aβ42), rabbitanti-Aβ1–40 (Sigma, St Louis, MO), mAb 22C11 (N-terminusAPP; Roche), mAb AT8 (abnormally phosphorylated tau; Inno-

genetics) and anti-glial fibrillary acidic protein (GFAP; Dako),CD68 (macrophage; Dako), rabbit anti-ubiquitin (Dako), C1qcomplement (Dako), HLA–DR [HLA–DP,DQ,DR (Dako)],CD31 (Dako) and CD34 [QBEnd (Dako)].

Antigen retrieval for Aβ immunohistochemistry wasperformed on sections treated with 98% formic acid for 10 minand for other antibodies as recommended by the supplier.Staining for single antigen was performed using streptavidin–biotin–horse radish peroxidase (ABC/HRP) or peroxidase–anti-peroxidase, utilizing 3′3′diaminobenzidine as a chromogen asdescribed by Kumar-Singh et al. (55). Immunohistochemistryinvolving detection of more than one antigen was performedusing species-specific or IgG subtype-specific secondaryantibodies conjugated directly with biotin, HRP, alkalinephosphatase or galactosidase (Southern Biotechnology,Birmingham, AL). This was followed by colour developmentusing one of the following chromogens (Roche): DAB,3-amino-9-ethylcarbazole (AEC), Fast-red, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium soluton(BCIP/NBT) or 5-bromo-4-chloro-3-indolyl-D-galactopyrano-side (X–gal). For Aβ40 immunohistochemistry, a sensitivetyramide amplification system (DuPont NEN, Boston, MA)was utilized.

Densitometric analysis

Densitometric analysis was performed for 5 µm thick serialsections stained with 21F12, JRF/cAb42/12 and FCA3542 (forAβ42), JRF/cAb40/10, R209 and FCA3340 (for Aβ40) and 4G8,was performed using the VIDAS image analysis system. Theobtained results were compared with staining of similar brainregions of patients with sporadic AD cases and PS1 (I143T)related familial AD. Pixels representing the immuno-cytochemical stain were counted to calculate the size of eachplaque. Also, the relative area occupied by Aβ staining in fivefields from hippocampus or ERC with most intense staining andenvisaging an area of 0.7 mm2, was determined as described byKumar-Singh et al. (53).

Fluorescent microscopy

For multiple labeling on a CLSM, 10 µm sections were incu-bated overnight with mAb 21F12 and a JRF/cAb42/12, washedand labeled with an anti-mouse TRITC-conjugated and an anti-rabbit FITC-conjugated antibody (Molecular Probes, Eugene,OR). Images were acquired with a Zeiss CLM-410 using either488 nm line of argon single laser or 632 nm helium–neon doublelaser for excitation. One micrometer thick consecutive opticalslices were captured for both fluorochromes separately and therelative Aβ42/Aβ40 content and ratios in amyloid plaques weredetermined.

ACKNOWLEDGEMENTS

The authors are grateful to F. Checler for FCA3340 andFCA3542 antisera, P. Mehta for R209 and R226 antisera, M.Savage for providing 53/4, E. Van Marck for the use of theimage analysis system, J.-P. Timmermans for the use of CLSMand B. Dermaut for translating the patients’ medical records.This work was supported by the Fund for Scientific Research-Flanders (FWO-F), the Interuniversity Attraction Poles (IUAPP4/17), the International Alzheimer Research Foundation


(IARF), Belgium, the FWF project S7403-MOB, Austria, andAlzheimer’s Association Grant (RG1-96–070 to R.W.), USA.C.D.J. and M.C. are postdoctoral fellows of the FWO-F.

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ARTICLE - Nonfibrillar diffuse amyloid deposition due to a γ42

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