²-Secretase Protein and Activity Are Increased in the Neocortex In

ORIGINAL CONTRIBUTION ␤-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease

Hiroaki Fukumoto, PhD; Bonnie S. Cheung, BS; Bradley T. Hyman, MD, PhD; Michael C. Irizarry, MD

Context: Amyloid plaques, a major pathological fea- activity increased by 63% in the temporal neocortex ture of Alzheimer disease (AD), are composed of an in- (P=.007) and 13% in the frontal neocortex (P=.003) in ternal fragment of amyloid precursor protein (APP): the brains with AD, but not in the cerebellar cortex. Activity 4-kd amyloid-␤ protein (A␤). The metabolic processing in the temporal neocortex increased with the duration of of APP that results in A␤ formation requires 2 enzy- AD (P=.008) but did not correlate with enzyme-linked im- matic cleavage events, a ␥-secretase cleavage dependent munosorbent assay measures of insoluble A␤ in brains with on presenilin, and a ␤-secretase cleavage by the aspartyl AD. Protein level was increased by 14% in the frontal cor- protease ␤-site APP-cleaving enzyme (BACE). tex of brains with AD (P=.004), with a trend toward a 15% increase in BACE protein in the temporal cortex (P=.07) Objective: To test the hypothesis that BACE protein and and no difference in the cerebellar cortex. Immunohisto- activity are increased in regions of the brain that de- chemical analysis demonstrated that BACE immunoreac- velop amyloid plaques in AD. tivity in the brain was predominantly neuronal and was found in tangle-bearing neurons in AD. Methods: We developed an antibody capture system to measure BACE protein level and BACE-specific ␤-secre- Conclusions: The BACE protein and activity levels are tase activity in frontal, temporal, and cerebellar brain ho- increased in brain regions affected by amyloid deposi- mogenates from 61 brains with AD and 33 control brains. tion and remain increased despite significant neuronal and synaptic loss in AD. Results: In the brains with AD, BACE activity and protein were significantly increased (PϽ.001). Enzymatic Arch Neurol. 2002;59:1381-1389

MYLOID-␤ PROTEIN (A␤) de- and colocalizes with Golgi and endosomal posits in the brain as senile markers within cells.1,3-5 It is modified dur- plaques in Alzheimer dis- ing and after translation by glycosylation, ease (AD). A␤ is produced sulfation, and furin-like proteolytic cleav- from the enzymatic cleavage of a prodomain within the Golgi, and ageA of amyloid precursor protein (APP) by it is enriched within lipid rafts.6-11 Sorting ␤-secretase and ␥-secretase. The process- and recycling of BACE are influenced by a ing of APP occurs by 2 major pathways, one C-terminal dileucine motif, the transmem- producing A␤(␤-secretase pathway) and the brane domain, and phosphorylation of ser- other producing a presumably nontoxic p3 ine at amino acid position 498.10,12,13 Al- fragment (␣-secretase pathway). In the though a homologous protein, BACE2, can ␤-secretase pathway, cleavage by the ␤-site also cleave APP, it is expressed in very low APP-cleaving enzyme (BACE) generates a levels in the adult brain.14-16 Because A␤ pro- truncated soluble ␤-cleaved APP fragment duction is abolished in neuronal cultures in acidic endosomal or secretory compart- and brains from BACE knockout mice, ments.1 The remaining membrane- BACE is considered to be the major ␤-secre- associated 11.5-kd fragment is cleaved by tase in neurons and the brain.17-19 presenilin-dependent ␥-secretase to yield A␤.2 We measured the protein level and en- For editorial comment zymatic activity of BACE, the primary brain ␤-secretase, in brains with AD and control see page 1367 brains to evaluate a role for increased A␤ production in the pathogenesis of AD. Although BACE is the primary The ␤-site APP-cleaving enzyme is a ␤-secretase, the level of BACE messenger From the Alzheimer Disease 501 amino acid aspartyl protease widely ex- RNA does not appear to be altered in AD 20-22 Research Unit, Department of pressed in brain, pancreas, and other tis- or transgenic mouse models of AD. Neurology, Massachusetts sues. In the brain, BACE is localized to neu- Messenger RNA may not reflect the BACE General Hospital, Charlestown. ronal cell bodies and proximal dendrites, protein level and activity, which are more

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 protease-free bovine serum albumin (BSA) (Sigma, St Louis, Mo). Demographics of Cases* The homogenate was centrifuged at 15000 rpm (21000 ϫg) for 5 minutes. The supernatant fluid was used for the BACE and syn- Controls AD aptophysin assays. For synaptophysin, BACE activity, and BACE (n = 33) (n = 61) protein assays, increasing dilutions of an extract from a single Age, mean ± SD, y 79.5 ± 11.3 80.2 ± 9.0 control temporal cortex were used as a standard. Sex, M, % 39.4 36.1 For A␤ determinations, the adjacent cortex was homog- Postmortem interval, mean ± SD, h 14.6 ± 6.9 12.9 ± 6.2 enized in the Tris buffer without Triton X-100 and centri- APOE ⑀4 allele frequency† 0.09 0.40 fuged as described previously. The pellet was resuspended and homogenized in a solution of 70% formic acid and centrifuged *AD indicates Alzheimer disease. at 22000 rpm (44000 ϫg) for 5 minutes at 4°C to remove de- †PϽ.05. bris. The resulting supernatant fluid was neutralized with 1M Tris buffer (pH 11) and used for the assay of formic acid– extractable A␤ species. relevant to the physiological role of BACE. We developed quantitative assays of BACE protein and activity to BACE PROTEIN ASSAY address specific questions regarding the role of BACE in AD: Is BACE increased in brains with AD vs control brains The BACE protein assay (Figure 1A) is a sandwich enzyme- in areas vulnerable to AD neuropathological abnormali- linked immunosorbent assay (ELISA) using the capture antibody ties? How does BACE correlate with other clinical and MAB5308 (mouse monoclonal anti-BACE, C-terminus; Chemi- neuropathological measures of AD? We found that areas con, Temecula, Calif) and detector antibody PA1-756 (rabbit poly- with prominent amyloid deposition (eg, the neocortex) clonalanti-BACE,N-terminus;AffinityBioreagents,Golden,Colo). have selectively increased BACE protein and/or activity These antibodies are directed toward epitopes specific to BACE levels in brains with AD relative to controls; areas mini- and do not react to BACE2 by Western blot analysis. We coated 96-well microtiter plates (Greiner, Longwood, Fla) with MAB5308 mally affected by AD changes (eg, the cerebellum) do not in a 1:4000 ratio in carbonate buffer (100mM; pH 9.6) at 4°C demonstrate differences in BACE relative to controls. overnight.Theplateswerewashed3timeswithphosphate-buffered saline (PBS) (pH 7.0), then blocked with a blocking reagent (25% MATERIALS AND METHODS BlockAce; Dai-Nippon, Osaka, Japan) for 6 to 24 hours. The samples (50 µL of 0.004 wt/vol) were added to the wells con- CASE SELECTION taining 50 µL of blocking buffer (Super Block; Pierce, Rockford, Ill)inPBSandincubatedfor1hourat37°C.Theplateswerewashed Snap-frozen slices of the temporal cortex (Brodmann areas 20, 4 times with PBS, then incubated overnight at 4°C with PA1-756 21, and 22), frontal association cortex (Brodmann areas 9 and in a 1:750 ratio in incubation buffer (0.02M phosphate buffer; 10), and cerebellum were obtained from the Massachusetts Alz- 400 mM sodium chloride; 2mM EDTA; 1% BSA) containing 1% heimer Disease Research Center brain bank (Boston). These mouse serum. The plates were again washed with PBS 4 times, pathological specimens were from patients who had received fol- then incubated with horseradish peroxidase (HRP)–linked anti– low-up at the Memory Disorders Unit at Massachusetts General rabbit IgG (Jackson, West Grove, Pa) in a 1:3000 ratio in incu- Hospital (Boston), and therefore clinical and demographic in- bation buffer for 2 hours at room temperature. The plates were formation were available. Additional control specimens (n=7) then washed with PBS 6 times, and fluorometric measurements were obtained from the Harvard Brain Tissue Resource Center using a 320-nm excitation filter and 400-nm emission filter were (Belmont, Mass). We processed 61 brains with AD and 33 con- obtained after the addition of HRP substrate (QuantaBlu; Pierce). trol brains for pathological and biochemical analysis. Not all brain Increasing dilutions of an extract from the same control tempo- regions were available for all cases, however. For the temporal ral cortex were used as a standard for every plate; a best-fit log- cortex, there were 61 brains with AD and 18 controls; for the linear curve, at dilutions below saturation, was used as the stan- frontal cortex, 52 cases of AD and 22 controls; and for the cer- dard curve (Figure 2A). As negative controls, no signal greater ebellum, 57 brains with AD and 26 controls. All cases of AD were thanbackgroundfluorescence(eg,ELISAofhomogenizationbuffer diagnosed clinically using criteria from the National Institute of alone) was detected when the primary antibody was replaced with Neurological and Communicative Disorders and Stroke– mouse IgG, or when the secondary antibody was eliminated from Alzheimer’s Disease and Related Disorders Association23 and the assay, with samples more diluted than 0.01 wt/vol. pathologically with Reagan Institute Working Group/National Institute on Aging criteria (stages 5 and 6 according to Braak and BACE-SPECIFIC ENZYMATIC ACTIVITY ASSAY Braak24-26). Cases with additional neuropathological diagnoses were excluded (eg, dementia with Lewy bodies). Although there For the BACE activity assay (Figure 1B), we coated 96-well mi- was no significant difference in the age, sex, or postmortem in- crotiter plates with the capture antibody MAB5308, used the terval (range, 3-29 hours) between the AD and control groups blocking reagent, and added the samples (50 µL of 0.01 wt/vol) within the entire cohort and when separated by brain region ana- as described previously. Samples were incubated for 1 hour at lyzed, there was an increased frequency of the apolipoprotein E 37°C. The plates were washed 6 times with PBS, and the enzy- (APOE) ⑀4 allele in the AD group, as expected (Table). matic reaction was carried out by incubation with 10µM fluorogenic substrate for ␤-secretase. We used either substrate 1, (7- SPECIMEN COLLECTION methoxycoumarin-4-yl)acetyl [MOCA]-Ser-Glu-Val-Asn-Leu- Asp-Ala-Glu-Phe-Arg-N-(2,4-dinitrophenyl) [DNP]-Lys-Arg- For biochemical studies, a strip of the human cerebral or cere- Arg-NH2 (Peptides International, Louisville, Ky); or substrate bellar cortex was dissected at −20°C, taking care to avoid un- 2, Arg-Glu(5-[aminoethyl] aminonaphthalene sulfonate derlying white matter, and homogenized in 10 µL/µg (volume [EDANS])-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4Ј- per wet weight) of Tris buffer (50mM Tris; pH 7.2; 0.1% Triton dimethylaminoazo-benzene-4-carboxylate [DABCYL])-Arg (Cal- X-100; 200mM sodium chloride; 2mM EDTA) with a protease biochem, San Diego, Calif). Samples were incubated in acetate inhibitor cocktail (Complete; Roche, Indianapolis, Ind) and 2% buffer with 100mM sodium chloride (pH 4.1) containing 0.025%

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5. QuantaBlu Substrate HRP

4. Secondary Antibody HRP-α-rb 4. Fluorescence 3. Detector Antibody PA1-756

N-terminal N-terminal β-Secretase Cleavage

2. Sample 3. BACE Substrate BACE BACE

2. Sample C-terminal C-terminal

MAB5308 1. Capture Antibody MAB5308 1. Capture Antibody

ELISA Plate ELISA Plate

Figure 1. The ␤-site APP-cleaving enzyme (BACE) protein enzyme-linked immunosorbent assay (ELISA) (A) and activity assay (B). A, For the protein ELISA, BACE from brain homogenates is captured by the anti-BACE C-terminal antibody MAB5308 and detected by the anti-BACE N-terminal antibody PA1-756, followedby horseradish peroxidase (HRP)–linked anti–rabbit IgG (HRP-␣-rb) and activation of the QuantaBlu fluorescent substrate (Pierce, Rockford, Ill) by HRP. B, For the BACE-specific activity assay, BACE is also captured by MAB5308, and the quenched fluorescent substrate is added. Cleavage of the substrate releases the fluorescence, which can be quantitated by fluorimetry.

A 17 500 B 25 000 C 45 000 40 000 15 000 20 000 35 000 12 500 30 000 15 000 10 000 25 000 20 000 7500 10 000 15 000 5000 5000 10 000 BACE Protein, adj fl int BACE Activity, adj fl int 2500 BACE Activity, abs fl int 5000 0 0 0

0 0.0001 0.001 0.01 0 0.005 0.01 0.015 0.02 0.025 0.03 0 4 8 12 16 20 24 28 32 36 40 44 48 Dilution, wt/vol Dilution, wt/vol Time, h

D 16 000 E 30 000 F 10 000

14 000 25 000

12 000 20 000

10 000 1000 15 000 8000 10 000 BACE Activity, adj fl int BACE Activity, adj fl int BACE Activity, abs fl int 6000 100 5000 4000 0 0 0 1 10 100 100010 000 3.5 4 4.5 5 5.5 6 6.5 7 0101000 000 100 000 BACE Inhibitor, nM pH BACE Protein (adj fl int)

Figure 2. The ␤-site APP-cleaving enzyme (BACE) protein enzyme-linked immunosorbent assay (ELISA) and BACE enzymatic activity assay using fluorogenic substrate 1. Absolute fluorescence intensity (abs fl int) is not corrected for background fluorescence; adjusted fluorescence intensity (adj fl int) is corrected. A, Standard curve for BACE protein determination. B, Standard curve for BACE-specific enzymatic activity assay. There was a linear increase in BACE activity (as measured by fluorescence intensity of the cleavage product) with increasing concentration of the brain extract (solid circles). The BACE activity was completely abolished by 3µM peptidergic

inhibitor P10-P4Ј StatVal (Peptides International, Louisville, Ky) (open circles). C, Linear time-dependent increase in BACE substrate cleavage products (fluorescence intensity adjusted for background). The BACE cleavage products were measured at the indicated time points. D, Dose dependence of BACE peptidergic inhibitor P10-P4Ј StatVal on BACE activity. E, The pH dependency of BACE activity in our assay system. The pH optimum was approximately 4.0 to 4.5. F, In a serial dilution of brain extract, BACE activity was correlated with BACE protein. (Error bars are ± SE. Error bars not visible in C, D, and E are contained within the data points.)

BSA for 16 to 24 hours at 37°C, unless otherwise specified. The nor (MOCA or EDANS) from an acceptor (DNP or DABCYL), substrates are decapeptides flanking the ␤-secretase cleavage site which quenches the fluorescence by fluorescence resonance en- of the Swedish mutation of APP (APPKN670-1ML) with an auto- ergy transfer. Cleavage of the peptide bond within the substrate quenching fluorescent tag that is activated by ␤-secretase cleav- by ␤-secretase enzymatic activity leads to the separation of the age of the peptide.27 Each peptide separates the fluorescence do- donor-acceptor pair, resulting in an increase in fluorescence. The

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 fluorescence increase is proportional to the amount of peptide in TBS, and sequentially probed with primary antibody hydrolyzed (the enzymatic activity). The enzymatic product was (MAB5308, 1:500; rabbit polyclonal anti–glial fibrillary acidic pro- measured on a plate reader (Wallac Victor V2; Perkin-Elmer, tein [anti-GFAP], 1:500 [Dako, Carpinteria, Calif]; rabbit poly- Wellesley, Mass) using a 340-nm excitation filter and 400-nm clonal anti–A␤ R1282, 1:500 [D. Selkoe, MD, Harvard Medical emission filter for substrate 1, and 355 nm and 510 nm, respec- School, Boston, Mass]; and mouse monoclonal IgM anti– tively, for substrate 2. Increasing dilutions of an extract from the phospho-␶ TG3, 1:10 [P. Davies, PhD, Albert Einstein College same control temporal cortex was used as a standard for every of Medicine, Bronx, NY], in 1.5% normal goat serum in TBS) plate; a best-fit linear-linear curve, at dilutions below satura- and secondary antibody (biotinylated anti–mouse IgG1, 1:200; tion, was used as the standard curve (Figure 2B). For negative Cy5-linked anti–rabbit IgG, 1:200 [Jackson]; BODIPY- controls, no signal greater than background fluorescence was de- fluorescein-linked anti–rabbit IgG or anti–mouse IgM, 1:200 [Mo- tected when the capture antibody was replaced with mouse IgG, lecular Probes, Eugene, Ore]; and Cy3-linked streptavidin, 1:750 or when the N-terminal antibody PA1-756 was used as the cap- [Jackson]). Confocal images were obtained using a laser confo- ture antibody, with samples more diluted than 0.025 wt/vol. cal scanning system (MRC 1024; BioRad, Hercules, Calif).

IMMUNOPRECIPITATION OF BACE STATISTICAL ANALYSIS FROM HUMAN BRAIN To analyze BACE activity and BACE protein, we performed analy- The temporal neocortex was dissected and homogenized with 10 sis of variance (ANOVA) according to diagnosis (brains with AD µL/µg (volume per wet weight) of extraction buffer containing vs controls) and brain region (temporal cortex, frontal cortex, protease inhibitors. The homogenate was centrifuged at 15000 or cerebellum). Significant effects of diagnosis with ANOVA were rpm for 5 minutes. The supernatant fluid (500 µL) was incu- followed up using the t test to determine which brain regions bated overnight at 4°C with protein G-Sepharose (20 µL of 50%, were significant. For significant results within brain regions, cor- vol/vol; ImmunoPure Plus Immobilized Protein G; Pierce) to re- relation analysis was used to correlate BACE activity and pro- move nonspecific protein binding. After centrifugation at 15000 tein with duration of illness, formic acid–extractable A␤, or syn- rpm for 5 minutes, the supernatant fluid was incubated for 6 hours aptophysin (StatView; Abacus Concepts, Berkeley, Calif). at 4°C with 10 µg of MAB5308 or PA1-756 covalently coupled to protein G–Sepharose (25 µL of 50%, vol/vol) according to the RESULTS manufacturer’s instructions (Seize-X Protein G Immunoprecipi- tation Kit; Pierce). The sample was washed 3 times with washing buffer by centrifugation, and the bound protein was eluted BACE ELISA with elution buffer. The eluted samples were boiled for 5 minutes in loading buffer. Proteins were separated by 10% to 20% To quantitate BACE changes in AD, we developed spe- Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electro- cific assays for BACE protein and enzymatic activity (Fig- phoresis (Novex/Invitrogen, Carlsbad, Calif) under denaturing, ure 1). The ELISA for full-length BACE protein uses cap- reducing conditions, transferred to a polyvinylidene fluoride ture antibody MAB5308 and detector antibody PA1-756 (PVDF) membrane, blocked with 5% nonfat dry milk in Tris- (Figure 1A). The assay exhibits sensitivity for the mea- buffered saline (TBS), pH 7.4, with 0.05% polyoxyethylenesor- surement of BACE protein in brain extracts (Figure 2A) bitan monolaurate (Tween-20 [TBS-T]) for 2 hours, and incu- and produces a plateau with samples more concentrated bated with MAB5308 (1 µg/mL of MAB5308 in TBS buffer than 0.01 wt/vol (data not shown). Specificity of the pro- containing 1.5% goat serum) overnight at 4°C. After washing with tein assay was confirmed by immunoprecipitation of BACE TBS-T, the membrane was incubated with HRP anti–mouse IgG (1:5000; Jackson) for 2 hours, and the BACE band was visual- from human brain extracts with either antibody PA1-756 ized using enhanced chemiluminescence (Perkin-Elmer). or MAB5308, separation by Western blot analysis, and probing with MAB5308, which detected diffuse bands of 22,29,30 ELISA FOR A␤40, A␤42, AND SYNAPTOPHYSIN approximately 52 kd and 70 kd (Figure 3).

Total formic acid A␤ was determined from the sum of ELISAs BACE-SPECIFIC ENZYMATIC ACTIVITY ASSAY for A␤40 and A␤42 using BNT77 (mouse monoclonal anti– A␤11-28; Takeda, Osaka, Japan) as the capture antibody and BA27 Our antibody capture assay for BACE-specific ␤-secretase (monoclonal anti-A␤40; Takeda) or BC05 (monoclonal anti- ␤ activity uses MAB5308 to capture BACE from specimens A 42; Takeda) conjugated with HRP as the detector antibody; added to a multiplate well and incubation with a ␤-secre- QuantaBlu was used as the fluorescent HRP substrate.28 Synaptophysin ELISA was performed using MAB329 (mouse tase substrate (a fluorescence-quenching decapeptide rep- monoclonal IgM anti-synaptophysin; 1:2000; Chemicon) as the resenting the cleavage site of the Swedish mutant of APP) capture antibody and biotinylated SY38 (mouse monoclonal IgG1 in acidic conditions (pH 4.1) optimal for BACE function. anti-synaptophysin; 1:2000; Progen Biotechnik GmbH, Heidel- Capture of the BACE C-terminus allows the more N- berg, Germany) followed by HRP-conjugated streptavidin as the terminal catalytic domain to cleave the substrate. Cleav- detector antibody. We used 3,3Ј,5,5Ј-tetramethylbenzidine age of the peptide releases fluorescence proportional to the (Kirkegaard & Perry, Gaithersburg, Md) as the colorimetric HRP amount of peptide hydrolyzed: the enzymatic activity (Fig- substrate; it was measured at 450 nm. ure 1B). The amount of substrate cleaved increases in a linear manner across time with exposure to captured BACE IMMUNOHISTOCHEMISTRY 2 Ͻ (r 26=0.96; P .001) (Figure 2C). For immunohistochemical studies, brains were fixed in 4% para- The assay exhibits high sensitivity and linearity for the formaldehyde for at least 2 days and cryoprotected in 10% glyc- measurement of BACE activity in brain extracts in the range erol in PBS for at least 2 days before being cut into 50-µm sec- of brain dilutions used for analysis, with a linear-linear curve tions on a freezing sledge microtome. Sections were permeabilized fit for the standard (Figure 2B, solid circles), and a plateau with 0.5% Triton X-100 in TBS, blocked with 3% nonfat dry milk in fluorescence with samples more concentrated than 0.05

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 creased in regions of the brain that develop amyloid plaques. ab Homogenates from the temporal cortex (Brodmann areas 20, 21, and 22), frontal cortex (Brodmann areas 9 and 10), and cerebellar cortex were analyzed. The temporal and frontal regions were selected because they are well- 168 delineated, high-order association areas affected by amyloid plaques in AD and they develop progressive neurofibrillary changes and neuronal loss during the course 98 of the illness. The temporal cortex is generally more affected than the frontal cortex.33,34 The cerebellum was cho- sen as an area with minimal involvement in AD. 64 Molecular Weight, kd Analysis of variance according to diagnosis and brain

50 region showed that BACE protein level is altered in AD, with significantly increased BACE protein levels in the frontal cortex and a trend toward increased BACE protein levels in the temporal cortex relative to control cases

36 (Figure 4A). Results of ANOVA showed a significant main Ͻ effect for diagnosis (F1,227=11.2; P .001). Post hoc comparisons showed that mean BACE protein levels increased by 14% in the frontal cortex in AD (t70=3.0; Figure 3. The ␤-site APP-cleaving enzyme (BACE) immunoprecipitation and P=.004), with a 15% increase in mean BACE protein lev- Western blot analysis. Immunoprecipitation of the brain extracts els in the temporal cortex that failed to reach statistical demonstrates bands at approximately 52 kd and 70 kd. The BACE was captured with anti-BACE C-terminal antibody MAB5308 (lane a) or anti-BACE significance (t76=1.9; P=.07) and no significant differ- N-terminal antibody PA1-756 (lane b) and probed with MAB5308. ence in the cerebellum (t81=1.5; P=.13).

INCREASED BACE ACTIVITY IN BRAINS WITH AD wt/vol (data not shown). Specificity of the activity assay was confirmed as follows: (1) The specific peptidergic inhibi- In support of the BACE protein studies, we hypoth- tor of BACE activity (Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val- esized that BACE activity would be altered in AD, with Asn-Sta-Val-Ala-Glu-Phe [P10-P4Ј StatVal]; Peptides Inter- significantly increased activity in the frontal and tempo- national) eliminated the enzymatic activity with a 50% ral cortices but not the cerebellum. Analysis of variance inhibitory concentration (IC50) of approximately 10nM, for BACE activity showed a significant main effect for di- Ͻ consistent with the published literature using purified BACE agnosis (F1,229=14.2; P .001). Post hoc comparisons (IC50 approximately 30nM)4 (Figure 2B, open circles, and showed that mean BACE activity increased in brains with Figure 2D). (2) Captured BACE had an optimum pH in AD by 63% in the temporal cortex (t76=2.8; P=.007) and the acidic range (pH, 4.0-4.5) consistent with published 13% in the frontal cortex (t72=3.0; P=.003), but not in 1 data (Figure 2E) and supporting cell culture studies lo- the cerebellum (t81=1.4; P=.18) (Figure 4B). There was calizing ␤-secretase processing of APP to acidic subcellu- no association of BACE protein or activity level with post- lar compartments and the trans-Golgi network.31 In the ab- mortem interval in the range of 3 to 29 hours. sence of antibody capture, the fluorogenic substrate 1 used in the activity assay detects thimet oligopeptidase as a INCREASED RATIO OF BACE ACTIVITY ␤-secretase with a maximum pH in the neutral range27;in TO BACE PROTEIN IN THE TEMPORAL CORTEX contrast, our antibody capture assay does not demon- IN BRAINS WITH AD strate significant ␤-secretase activity at the neutral pH. (3) After immunoprecipitation with MAB5308 covalently BecausemeanBACEactivitylevelswereincreasedtoagreater coupled to IgG resin, brain homogenate separated by extent than mean BACE protein levels in the cerebral cor- Western blot analysis and probed with the same antibody tex in brains with AD vs controls, we evaluated the ratio of demonstrates BACE bands (Figure 3, lane a). BACE activity to BACE protein levels for each case (BACE By using antibody capture at the appropriate pH, the act/prt).AnalysisofvariancefortheBACEact/prtratioshowed ␤ BACE activity assay avoids the detection of other -secre- a main effect for diagnosis (F1,227=6.1; P=.01), reflecting a tase cleavage activities (eg, BACE2, cathepsin D,32 or thi- significant 45% increase in the mean BACE act/prt ratio in 27 met oligopeptidase ). Using a single human brain ex- the temporal cortex (t76=2.5; P=.02) but not in the frontal tract as a standard, BACE protein was correlated with cortex(P=.92)orcerebellum(P=.81)inbrainswithAD(Fig- 2 BACE activity at various dilutions (r 3=0.86; P=.02) (Fig- ure4E).ThissuggeststhatBACEactivitylevelsmaybemodu- ure 2F). Fluorogenic substrate 1 and fluorogenic sub- lated by other factors in addition to BACE protein levels. strate 2 gave similar results in this assay. TEMPORAL CORTEX BACE ACTIVITY INCREASES INCREASED BACE PROTEIN IN BRAINS WITH AD WITH DURATION OF ILLNESS IN AD

Our primary outcome measures in this study were BACE We tested the hypotheses that significant increases in BACE protein and BACE activity in brains with AD vs control protein and activity levels were correlated with clinical and brains to test the hypothesis that these levels are in- pathological parameters of dementia progression. The pri-

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∗ 150 150 100 ∗ ∗ ∗ ∗ 100 100

50 50 50 BACE Protein, % Control BACE Activity, % Control BACE prt/syn Ratio, % Control

0 0 0 Temporal Frontal Cerebellar Temporal Frontal Cerebellar Temporal Frontal Cerebellar Cortex Cortex Cortex Cortex Cortex Cortex Cortex Cortex Cortex Brain Region Brain Region Brain Region 350 200 3.0 D ANOVA (P<.001) E ANOVA (P = .01) F (P = .008)

300 2.5 150 250 2.0 ∗ 200 1.5 100 150 ∗ 1.0 ∗ 100 50 0.5

BACE act/syn Ratio, % Control 50 0 Ratio of BACE act/prt Ratio, % Control Temporal BACE Activity, Arbitrary Units 0 0 Temporal Frontal Cerebellar Temporal Frontal Cerebellar 01052015 Cortex Cortex Cortex Cortex Cortex Cortex Duration, y Brain Region Brain Region

Figure 4. The ␤-site APP-cleaving enzyme (BACE) protein and specific activity in brains with Alzheimer disease (AD) and controls and correlation with clinical markers. A, Diagnosis demonstrates a significant main effect on BACE protein by analysis of variance (ANOVA) (PϽ.001), with significantly increased BACE protein levels in the frontal cortex in brains with AD as determined by a post hoc t test (asterisk indicates P=.004). B, Diagnosis also demonstrates a significant effect on BACE activity by ANOVA (PϽ.001), with significantly increased BACE activity in the temporal cortex (asterisk indicates P=.007) and frontal cortex (asterisk indicates P=.003) in brains with AD as determined by a post hoc t test. C and D, Because BACE is a predominantly neuronal protein, we normalized BACE measures for synaptic and neuronal loss by determining the ratio of BACE protein to synaptophysin protein (BACE Prt/Syn) (C) and the ratio of BACE activity to synaptophysin protein (BACE Act/Syn) (D). Normalized BACE measures are significantly increased in the temporal and frontal cortices in brains with AD (ANOVA, PϽ.001; asterisks indicate P=.02 [temporal] and PϽ.001 [frontal] in C and P=.01 [temporal] and P=.001 [frontal] in D as determined by a post hoc t test.) E, The ratio of BACE activity (Act) to BACE protein (Prt) shows an ANOVA effect by diagnosis (ANOVA, P=.01), reflecting an increased ratio in the temporal cortex (asterisk indicates P=.02 as determined by a post hoc t test). F, The BACE activity increases with duration of illness (P=.008) in the temporal cortex in brains with AD. (Error bars are ± SE.)

mary clinical parameter available for the AD cases was du- PϽ.001). Of note was a hierarchical distribution of formic ration of dementia. Within AD cases, temporal cortex BACE acid–extractable A␤ levels in AD, which were highest in activity levels increased with the duration of illness the temporal cortex, then the frontal cortex, and finally the 2 Ͻ (r 58=0.11; P=.008) (Figure 4D). Frontal BACE protein and cerebellum (P .001 between brain regions in AD). activity levels did not significantly correlate with the du- For synaptophysin, ANOVA demonstrated a signifi- Ͻ ration of illness. cant effect for diagnosis (F1,227=15.5; P .001), with a 38% Ͻ reduction in the temporal cortex (t74=−3.5; P .001), a 14% BIOCHEMICAL CHARACTERIZATION reduction in the frontal cortex (t72=−2.2; P=.03), and no OF PATHOLOGICAL SPECIMENS significant difference in the cerebellar cortex in brains with AD (Figure 5B). These data together with the A␤ data sug- To investigate correlations of AD severity with BACE mea- gest that in this cohort, the temporal cortex was more af- sures in the same brain regions, we determined biochemi- fected by AD pathological changes than the frontal cor- cal measures of amyloid deposition (formic acid– tex and that the cerebellum was relatively spared. extractable A␤) and synaptic integrity (synaptophysin) by In AD cases, there was no significant correlation of ELISA in the temporal, frontal, and cerebellar cortices of BACE protein or activity with formic acid–extractable A␤ AD and control cases. Levels of total formic acid– (Figure 5C and D). In AD brain regions, levels of BACE extractable A␤ were markedly increased in brains with AD, protein and activity did not correlate with synaptophysin with the highest absolute levels in the temporal and fron- measures. tal cortices (Figure 5A). The ANOVA for total formic acid ␤ A demonstrated a main effect for diagnosis (F1,228=153; BACE IS EXPRESSED IN NEURONS AND PϽ.001), with significantly increased A␤ in the temporal NEUROPIL IN BRAINS WITH AD AND CONTROLS Ͻ cortex by 52-fold (t76=7.6; P .001), the frontal cortex by Ͻ 10-fold (t71=13; P .001), and the cerebellum by 82-fold To determine whether the increased BACE protein and ac- (t81=3.4; P=.001) in AD relative to control cases. There was tivity levels were due to BACE expression in astrocytes as- Ͻ also a main effect for brain region (F2,228=38; P .001) and sociated with gliosis in AD, we performed immunostain- an interaction between brain region and diagnosis (F2,228=34; ing of the temporal cortex in 5 brains with AD and 5 controls

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10 000 100 ∗

, pmol/g 8000 β ∗ 6000

50 Formic Acid A 4000 Synaptophysin Protein, % Control 2000 ∗ ∗ ∗ 0 0 Temporal Frontal Cerebellar Temporal Frontal Cerebellar Cortex Cortex Cortex Cortex Cortex Cortex Brain Region Brain Region

C 30 000 D 15 000 AD Controls

20 000 10 000 , pmol/g , pmol/g β β

10 000 5000 Frontal A Temporal A

0 0

00.5 1.0 1.5 2.0 2.5 3.0 0.40.6 0.8 1.0 1.2 1.4 Temporal BACE Activity, Arbitrary Units Frontal BACE Activity, Arbitrary Units

Figure 5. Biochemical measures of pathological characteristics and ␤-site APP-cleaving enzyme (BACE) activity. A, Formic acid–extractable A␤ is markedly increased in brains with AD (analysis of variance [ANOVA], PϽ.001), especially in the temporal neocortex and frontal neocortex (asterisks indicate PϽ.001 as determined by a post hoc t test) as well as in the cerebellum (asterisk indicates P=.001). B, Synaptophysin is reduced in the temporal and frontal cortices in brains with AD (ANOVA, PϽ.001; asterisks indicate PϽ.001 (temporal) and P=.03 (frontal). C and D, Increasing BACE activity is not significantly correlated with increasing formic acid–extractable A␤ levels in brains with AD. Control brain values are also depicted. (Error bars are ± SE.)

(Figure 6). Immunostaining of the temporal cortex and prt/syn) in each case (Figure 4C and D). For both mea- hippocampus with anti-BACE antibody demonstrated dif- sures, ANOVA showed significant main effects for diag- Ͻ fuse staining of the neuropil and staining of neuronal cell nosis (BACE act/syn, F1,227=12.6; P .001; BACE prt/syn, Ͻ bodies and neurites in AD cases and controls; in brains with F1,225=19.0; P .001), reflecting increases in normalized AD, BACE staining occasionally colocalized with neurofi- BACE protein levels of 60% in the temporal cortex (t74=2.3; Ͻ brillary tangle–bearing neurons (Figure 6C). Double stain- P=.02) and 33% in the frontal cortex (t70=4.2; P .001) ing with GFAP did not reveal BACE immunoreactivity in and increases in normalized BACE activity levels of 156% activated astrocytes in brains with AD (Figure 6B). These in the temporal cortex (t74=2.6; P=.01) and 37% in the results suggest that BACE is predominantly expressed in frontal cortex (t72=3.3; P=.001) in brains with AD. neurons in the brain and argue against the possibility that elevated BACE levels and activity come from up- COMMENT regulation in astroglial elements. Amyloid plaques are a characteristic neuropathologic fea- BACE PROTEIN AND ACTIVITY NORMALIZED TO ture of AD. In autosomal dominant familial AD and Down SYNAPTOPHYSIN PROTEIN ARE INCREASED IN AD syndrome, A␤ deposition can be attributed to excessive A␤ production mediated by APP/presenilin mutations or APP Because BACE is primarily a neuronal protein1,30 and BACE gene dosage effects. However, the mechanism by which ex- immunoreactivity was observed mainly in neurons, we cessive A␤ deposition occurs in sporadic AD is unknown. sought to correct our BACE data for neuronal loss. We nor- The BACE is critical in A␤ biosynthesis. It is present in high malized BACE activity and protein for synaptophysin mea- levels in the brain, efficiently cleaves APP at the ␤-secre- sures (as a surrogate marker for cortical damage) by ana- tase cleavage site, and localizes to acidic compartments in lyzing the ratios of BACE activity to synaptophysin protein the secretory pathway where A␤ production occurs.1,3-5 A␤ (BACE act/syn) and BACE protein to synaptophysin (BACE production is abolished in BACE knockout mice,18,19 whereas

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Figure 6. Confocal images of ␤-site APP-cleaving enzyme (BACE) immunostaining (red) in the brain. A, Temporal cortex from a control case immunostained with Cy3-labeled anti-BACE C-terminal antibody MAB5308 demonstrates neuronal expression of BACE in red. B, Human hippocampus immunostained with Cy3-labeled MAB5308 in red and BODIPY fluorescein–labeled anti–glial fibrillary acidic protein (rabbit polyclonal) in green demonstrates BACE staining in neuronal cell bodies and proximal neurites (red) but not in astrocytes (green). C, Human hippocampus immunostained with Cy3-labeled MAB5308 (mouse IgG1) in red, BODIPY fluorescein–labeled phospho-␶ (TG3; mouse IgM) in green, and Cy5-labeled A␤ (rabbit polyclonal R1282) in blue. The BACE is expressed in non–tangle-bearing neurons in red, and tangle-bearing neurons in yellow (arrows, double-stained for BACE and TG3). Scale bars indicate 25 µm.

the overexpression of BACE in APP transgenic mice in- We find that BACE protein and/or enzymatic activity creases A␤formation.35 Studies have found increased BACE levels are increased in the frontal and temporal cortices in protein levels in the hippocampal CA1 subfield by immu- brains with AD, implying that mechanisms of increased A␤ nostaining30 and in the frontal cortex by semiquantitative production are operative in sporadic AD. A␤deposition oc- Western blot analysis22 in brains with AD. cursincharacteristicanddiscreteanatomicalpatternsinbrains We have developed 2 new tools to study BACE in neu- with AD, including the cortical and limbic regions.33 We fo- ropathological samples. The BACE protein assay is a 2-site cused on specific temporal (Brodmann areas 20, 21, and 22) enzyme immunoassay consisting of capture (MAB5308) and frontal (Brodmann areas 9 and 10) neocortical regions and detector (PA1-756) antibodies directed toward dis- because these are anatomically well-demarcated, high-order tinct epitopes of the antigen, C-terminal and N-terminal, association areas that receive multimodal sensory input and respectively. The ELISA system is specific for BACE, as con- are consistently affected by senile plaques and neurofibril- firmed by the immunoprecipitation results. Compared with lary tangles in AD. Increased BACE is specific to these areas. Western blot analysis, ELISA can analyze more samples The alterations in BACE do not occur in the cerebellum, a in a single plate with a quantitative standard curve and is region not significantly affected by AD changes. The increase therefore suitable for the analysis of many samples. in BACE activity is surprising given the predominant neu- The BACE activity assay is an antibody capture assay, ronal localization of BACE and significant progressive tem- with activity measured via fluorescence emission after the poral lobe neuronal loss in AD34; measures of BACE protein cleavage of a ␤-secretase substrate. This substrate has pre- and activity levels are even more pronounced when normal- viously been used to assess ␤-secretase activity with puri- izedfortheneuronalproteinsynaptophysin,amarkerofsyn- fied BACE and brain extracts4,27; however, the substrate can aptic loss in AD. Possible mechanisms of BACE changes in- be cleaved by other proteases, such as thimet oligopepti- clude increased BACE expression and activity in remaining dase.27 To eliminate these other ␤-secretase activities, we neurons, the effect of posttranslational modification of BACE first captured BACE protein via its C-terminal domain from on activity, or increased BACE expression in astrocytes. Our brain extracts on ELISA plates coated with anti-BACE an- immunohistochemical studies do not suggest a significant tibody, then assayed the enzymatic reaction from the more glial up-regulation of BACE, consistent with the results of N-terminal catalytic domain of the captured BACE. The Sun et al.30 The ratio of BACE activity to BACE protein is other proteases are not bound by the anti-BACE C-terminal– increased in the temporal cortex in cases of AD, suggesting specific antibody MAB5308, which was raised against BACE that a posttranslational modification of BACE resulting in epitopes distinct from BACE2; therefore, this assay elimi- increased ␤-secretase activity, an alteration in critical BACE nates other sources of substrate cleavage. The isolation of cofactors, or an increase in active vs inactive splice forms BACE through antibody capture can also eliminate com- of BACE may occur in AD36; inactive splice forms have not petition by endogenous APP fragments as the BACE sub- been detected in the human brain.37 strate. Furthermore, the assay is run at an acidic pH opti- The BACE activity does not significantly correlate with mal for BACE ␤-secretase activity. The bound enzymatic the amount of A␤in the temporal cortex in brains with AD. activity measured from the human brain is inhibited in a This is probably because the amount of A␤ in the brains is dose-dependent manner by a BACE peptidergic inhibitor also modulated by ␣-secretase and ␥-secretase and by pro- at an IC50 of approximately 10nM, in agreement with pre- cesses of A␤ fibrillization, deposition, uptake, and catabo- vious studies of purified BACE protein.4 The assay can be lism. Despite the complicated pathways involved in A␤me- used to evaluate a large number of pathological speci- tabolism, recent data demonstrate that the overexpression mens and can also be adapted for screening inhibitors of of BACE in cell culture and in a transgenic mouse model BACE activity without the requirement for purified BACE. lead to elevated steady-state A␤ levels, suggesting that lev-

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 elsofBACEproteinandactivityaffectA␤generationinvivo.35 7. Capell A, Steiner H, Willem M, et al. Maturation and pro-peptide cleavage of beta- secretase. J Biol Chem. 2000;275:30849-30854. The elimination of BACE in knockout mice dramatically 8. Haniu M, Denis P, Young Y, et al. Characterization of Alzheimer’s beta-secretase reducesA␤withoutanytoxicphenotypiceffects,18,19 sothera- protein BACE. J Biol Chem. 2000;275:21099-21106. pies aimed at reducing BACE may be less toxic than those 9. Creemers JW, Ines Dominguez D, Plets E, et al. Processing of beta-secretase by 38 furin and other members of the proprotein convertase family. J Biol Chem. 2001; targeting␥-secretase. ThepersistenceofhighlevelsofBACE 276:4211-4217. activity in AD cases of long duration supports the idea that 10. Huse JT, Pijak DS, Leslie GJ, Lee VM, Doms RW. Maturation and endosomal ␤ targeting of beta-site amyloid precursor protein-cleaving enzyme: the Alzhei- BACE inhibitors could be effective in reducing A produc- mer’s disease beta-secretase. J Biol Chem. 2000;275:33729-33737. tion, even in advanced AD. 11. Riddell DR, Christie G, Hussain I, Dingwall C. Compartmentalization of ß- To our knowledge, this is the first report quantitat- secretase (Asp2) into low-buoyant density, non-caveolar lipid rafts. Curr Biol. 2001;11:1288-1293. ing the up-regulation of BACE activity in clinically and 12. Walter J, Fluhrer R, Hartung B, et al. Phosphorylation regulates intracellular traf- pathologically characterized brains with sporadic AD and ficking of beta-secretase. J Biol Chem. 2001;276:14634-14641. delineating the anatomical pattern of BACE protein and 13. Yan R, Han P, Miao H, Greengard P, Xu H. The transmembrane domain of the Alz- heimer’s ß-secretase (BACE1) determines its late Golgi localization and access to functional dysregulation. The BACE activity is increased ß-amyloid precursor protein (APP) substrate. J Biol Chem. 2001;276:36788-36796. in a hierarchical neuroanatomical pattern consistent with 14. Saunders AJ, Kim T-W, Tanzi RE. BACE maps to chromosome 11 and a BACE Ͼ homolog, BACE2, reside in the obligate Down syndrome region of chromosome the extent of AD abnormalities (temporal cortex fron- 21. Science. 1999;286:1255a. tal cortex Ͼ cerebellum) and remains elevated in the tem- 15. Bennett BD, Babu-Khan S, Loeloff R, et al. Expression analysis of BACE2 in brain poral cortex throughout the course of the illness. These data and peripheral tissue. J Biol Chem. 2000;275:20647-20651. 16. Farzan M, Schnitzler CE, Vasilieva N, Leung D, Choe H. BACE2, a beta-secretase support BACE as an important target for therapeutics in homolog, cleaves at the beta site and within the amyloid-beta region of the amyloid- AD and imply that biochemical and pathological factors beta precursor protein. Proc Natl Acad Sci U S A. 2000;97:9712-9717. modulate BACE activity in sporadic AD. 17. Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neu- rosci. 2001;4:233-234. Accepted for publication June 19, 2002. 18. Luo Y, Bolon B, Kahn S, et al. Mice deficient in BACE1, the Alzheimer’s beta- secretase, have normal phenotype and abolished beta-amyloid generation. Nat Author contributions: Study concept and design (Drs Neurosci. 2001;4:231-232. Fukumoto, Hyman, and Irizarry); acquisition of data (Drs 19. Roberds SL, Anderson J, Basi G, et al. BACE knockout mice are healthy despite Fukumoto and Irizarry and Ms Cheung); analysis and in- lacking the primary beta-secretase activity in brain: implications for Alzheimer’s disease therapeutics. Hum Mol Genet. 2001;10:1317-1324. terpretation of data (Drs Fukumoto, Hyman, and Irizarry); 20. Yasojima K, McGeer EG, McGeer PL. Relationship between beta amyloid pep- drafting of the manuscript (Drs Fukumoto, Hyman, and tide generating molecules and neprilysin in Alzheimer disease and normal brain. Brain Res. 2001;919:115-121. Irizarry and Ms Cheung); critical revision of the manuscript 21. Irizarry MC, Locascio JJ, Hyman BT. Beta-site APP cleaving enzyme mRNA ex- for important intellectual content (Drs Fukumoto, Hyman, pression in APP transgenic mice. Am J Pathol. 2001;158:173-177. and Irizarry); statistical expertise (Drs Hyman and Irizarry); 22. Holsinger D, McLean CA, Beyreuther K, Masters CL, Evin G. Increased expression of amyloid precursor ß-secretase in Alzheimer’s disease. Ann Neurol. 2002;51:783-786. obtained funding (Drs Hyman and Irizarry); administra- 23. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical tive, technical, and material support (Drs Fukumoto and diagnosis of Alzheimer’s disease. Neurology. 1984;34:939-944. Irizarry and Ms Cheung); study supervision (Drs Fuku- 24. Braak H, Braak E. Neuropathological staging of Alzheimer-related changes. Acta Neuropathol (Berl). 1991;82:239-259. moto, Hyman, and Irizarry). 25. Consensus recommendations for the postmortem diagnosis of Alzheimer’s dis- This study was supported by grants AG00793, ease. Neurobiol Aging. 1997;18(suppl 1):S1-S2. 26. Hyman BT, Trojanowski JQ. Consensus recommendations for the postmortem di- AG05134 (Massachusetts Alzheimer Disease Research Cen- agnosis of Alzheimer disease from the National Institute on Aging and the Reagan ter, Boston), and PHS MH/NS 31862 (Harvard Brain Tis- Institute Working Group on diagnostic criteria for the neuropathological assess- sue Resource Center, Belmont, Mass) from the National In- ment of Alzheimer disease. J Neuropathol Exp Neurol. 1997;56:1095-1097. 27. Koike H, Seki H, Kouchi Z, et al. Thimet oligopeptidase cleaves the full-length stitutes of Health, Bethesda, Md, and the Walters Family Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS Foundation, New York, NY. Dr Fukumoto is supported by cells. J Biochem (Tokyo). 1999;126:235-242. Takeda Chemical Industries, Osaka, Japan. 28. Suzuki N, Cheung TT, Cai XD, et al. An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (APP717) mu- Antibodies BNT77, BA27, and BC05 were kindly pro- tants. Science. 1994;264:1336-1340. vided by Takeda. 29. Charlwood J, Dingwall C, Matico R, et al. Characterization of the glycosylation profiles of Alzheimer’s beta-secretase protein Asp-2 expressed in a variety of cell Corresponding author and reprints: Michael C. Irizarry, lines. J Biol Chem. 2001;276:16739-16748. MD, Alzheimer Disease Research Unit, Center for Aging, 30. Sun A, Koelsch G, Tang J, Bing G. 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Correction

Error in Materials and Methods. In the Original Contribution by Fukumoto et al titled “␤-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease,” published in the September 2002 issue of the ARCHIVES (2002;59:1381-1389), an error occurred in the “Materials and Methods” sec- tion. On page 1382, the homogenization procedure was incorrectly described as being performed at “10 µL/µg (volume per wet weight),” and the BACE ELISA at “0.004 wt/vol.” The correct protocol should have read that the tissue was “homogenized in 10 µL/mg volume per wet weight (=0.1 mg wt/µL vol) of Tris buffer” and that the samples for the BACE ELISA were loaded as “50 µL of 0.001 wt/vol.” All dilutions are in the following units: milligrams of wet weight per microliters of buffer volume.

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