Proc. Nati. Acad. Sci. USAVol. 88, pp. 10998-11002, December 1991Medical Sciences

Lysosomal hydrolases of different classes are abnormallydistributed in brains of patients with Alzheimer disease

(cytoehemisty/cell death/senile plaques/amyloid/lip sn)

ANNE M. CATALDO*t, PETER A. PASKEVICH*, EIKI KOMINAMI*, ANb RALPH A. NIXON*t§¶*Laboratones for Molecular Neuroscience, McLean Hospital, and tDepartments of Psychiatry and Neuropathology and §Program in Neuroscience, HarvardMedical School, Belmont, MA 02178; and tJuntendo University, Tokyo, Japan

Communicated by Francis 0. Schmitt, August 15, 1991

ABSTRACT (3-Amyloid formation requires multiple ab-normal proteolytic cleavages of amyloid precursor protein(APP), including one within its intramembrane doma. Ly-sosomes, which contain a wide variety ofprotes (cathepins)and other acid hydrolases, are major sites for the turnover ofmembrane proteins and other cell constituents. Using unocytochemistry, inmmunoelectron microscopy, and enzyme his-tochemistry, we studied the expression and cellular distribu-tions of 10 lysosomal hydrolases, including 4 cathepins, inneocortex from patients with Alzhemer d and control(non-Alzheimer-disease) individuals. In control brains, acidhydrolases were localized exclusively to intracellular lysosome-related compartments. and 8 of the 10 enzymes pkedomlnatedin neurons. In Alzheimer-disease brains, strongly immuno-reactive lysosomes and lipofuscin granules accumulated mark-edly in the perikarya and proximal dendrites ofmany corticalneurons, some of which were undergoing degeneration. Morestrikingly, these same hydrolases were present in equally highor higher levels in senile plaques in Alzhemer disease, but theywere not found extracellularly in control brains, incladingthose from Parkinson or Huntngton disease patients. At theultrastructural level, hydrolase immunoreactivity in senileplaques was localized to extracellular lipofuscin granules simn-iar in morphology to those within degenerating neurons. Twocathepsins that were undetectable in neurons were absent fromsenile plaques. These results show that lysosome function isaltered in cortical neurons in Alzheimer -disease. The presenceof a broad spectrum of acid hydrolases in senile plaquesindicates that lysosomes and their contents may be liberatedfrom cells, principally neurons and their processes, as theydegenerate. Because cathepsns can cleave polypeptide sites onAPP relevant for (8-amyloid formation, their abnormal extra-cellular localization and dysregulation in Alzheiser disease canaccount for the multiple hydrolytic events in 8-amylold for-mation. The actions of membrane-degrading acid hydrolasescould also explain how the intramembrane portion of APPcontaining the C terminus of (3-amyloid becomes accessible toproteases.

The formation of f8-amyloid in Alzheimer disease (AD) in-volves altered proteolytic processing of the amyloid precur-sor protein (APP), an .70-kDa transmembrane protein ex-pressed in various cell types, including neural cells (1, 2).More than one abnormal hydrolytic event appears necessaryto produce the -4-kDa ,B-amyloid peptide, including multipleatypical or abnormal proteolytic cleavages of APP. Genera-tion of the N terminus of the P-amyloid peptide implies thata normal cleavage is precluded at residue 667 of 751-residueAPP; normal cleavage forms the physiological polypeptideprotease nexin-2 (3, 4). The C terminus of (3-amyloid is partofthe intramembrane domain ofAPP, which normally would

not be accessible to proteases. Its generation implies eitherproteolysis of APP molecules that are not inserted into themembrane or a cleavage that occurs after additional hydro-lases have exposed the intramembrane domain ofAPP duringmembrane turnover or membrane injury (5).We recently showed (6-8) that the lysosomal proteases

cathepsin B (CB) and cathepsin D (CD) in AD brain arepresent extracellularly in senile plaques at high levels. Toidentify the source of extracellular cathepsins and to inves-tigate the involvement of other lysosomal hydrolases inf-amyloid formation, we studied the cellular and subcellulardistribution of a series of proteolytic and nonproteolyticlysosomal enzymes, using enzyme histochemistry and im-munocytochemistry at the light and electron microscopiclevels. Our findings show that many classes of lysosomalhydrolases are abnormally localized extracellularly in rela-tion to the deposits of (-amyloid in AD brain, and theseenzymes originate principally from degenerating neurons.

MATERIALS AND METHODSTissue. Postmortem human brains from 10 individuals with

a clinical diagnosis of probable AD, 10 age-matched neuro-logically normal controls, and 6 brains each from patientswith Parkinson disease (PD) and stage III Huntington disease(HD) (all individuals were 62-78 yr old) were used. Braintissue was obtained from the Brain Tissue Resource Center,McLean Hospital (Belmont, MA). Brains from patients withno history of neuropsychiatric disease weighed 1200-1300 gand exhibited negligible microscopic histopathology (0 to 2plaques per low-power field). Tissues were immersion-fixedin cold phosphate-buffered (0.15 M, pH 7.4) 10%o (vol/vol)formalin. Postmortem intervals ranged from 30 min to 6 hr,and total fixation time was < 1 yr. Blocks (3 x 1 x 0.4 cm)of prefrontal cortex (area 10) were cut into 30-jum-thickVibratome sections or were cryoprotected in 30% (wt/vol)sucrose overnight at 40C and cut by cryostat or wedgemicrotome into 10-,um-thick sections. Serial adjacent sec-tions were screened for histopathology by using Nissl andBielschowsky silver stains.

Antibodies. Immunocytochemistry involved polyclonalantisera against 3-hexosaminidase A (HEX), a-glucosidase(GLU), cathepsin H (CH), cathepsin L (CL), cathepsin G(CG), CB, and CD. Rabbit antisera to HEX and GLU wereprovided by Srinivasa Raghavan (Eunice Kennedy ShriverCenter, Waltham, MA). Previous characterizations of rabbitantisera to rat liver CH and CL (9-12), and sheep antiserumto human brain CD (7) were reported previously. Antisera to

Abbreviations: AD, Alzheimer disease; PD, Parkinson disease; HD,Huntington disease; APP, amyloid precursor protein; HEX, 3-hex-osaminidase A; GLU, a-glucosidase; CD, cathepsin D; CB, cathep-sin B; CG, cathepsin G; CH, cathepsin H; CL, cathepsin L.ITo whom reprint requests should be addressed at: Laboratories forMolecular Neuroscience, McLean Hospital, 115 Mill Street, Bel-mont, MA 02178.

10998

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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CFIG. 1. Tissue sections from the prefrontal cortex of control brains incubated in anti-CL antiserum (A and Inset) display prominent

immunoreactivity within lysosomes of neurons (arrows). Anti-CH antiserum did not recognize neuronal lysosomes but intensely stainedlysosomes of type II reactive astrocytes (B, arrows). Antiserum directed against CG did not stain any cell types within brain parenchyma (C)but labeled the lysosomes of peripheral blood neutrophils (C Inset). (A-C, x300; A Inset, x675; C Inset, x700.)

human macrophage CG and liver CB were purchased fromICN. A rabbit antiserum (13) raised against an uncoupledsynthetic peptide corresponding to residues 1-40 of a-amy-loid was provided by Dennis Selkoe (Harvard MedicalSchool, Boston).

Cytochemical Methods. Immunocytochemical studies em-ployed the avidin-biotin technique of Hsu et al. (14) asdescribed (7). Immunocytochemical controls consisted oftissue sections incubated in preimmune antisera or withoutprimary antisera. Human peripheral blood smears were usedas positive controls in experiments using CG antiserum.Thioflavin-S histochemistry to identify (3-amyloid proteinwas applied before immunocytochemical incubation to avoidmasking histofluorescence by immunoreaction product (7).Ultrastructural study of immunoreaction product involved apre-embedding staining technique (8, 15). Grids either werelightly poststained in 2% uranyl acetate and lead citrate (16,17) or were not poststained (negative controls) (8). Enzymecytochemistry of acid phosphatase (18), trimetaphosphatase(19), and aryl sulfatase (20) activities used the lead capturetechnique of Gomori (21). Substrates included cytidine 5'-monophosphate, trimetaphosphate, and p-nitrocatechol sul-fate, respectively, which were obtained from Sigma. Tissuesections incubated in media containing no substrate or anonspecific substrate served as negative controls to distin-guish nonspecific lead binding from the bona fide reactionproduct.

RESULTSSix antisera against different hydrolases specifically labeledintracellular lysosomal compartments in sections of prefron-

tal cortices from normal control, PD, and HD brains. Thesehydrolases included all major cathepsins (CD, CB, CL, andCH) and two glycosidases (HEX and GLU). Five of theseantisera (CL, CD, CB, HEX, and GLU) intensely labeledlysosomes in neurons, particularly those in perikarya andproximal dendrites (Fig. 1). Lysosome density was alsohigher in neurons than in other cell types. By contrast, theabundance of these hydrolases varied considerably in astro-cytes. Lysosomes in astrocytes were darkly stained byantisera to CD and CL but contained barely detectable HEXand CB immunoreactivities. Oligodendroglia stained weaklyor not at all.Two other lysosomal hydrolases, CH and CG, were not

detected in neurons. CH antiserum strongly labeled lyso-somes in astrocytes (Fig. 1). CG immunoreactivity was notseen in any cell types in the brain, although the lysosomes ofleukocytes in peripheral blood were intensely stained (Fig.1C Inset).The same five hydrolases were detected in neuronal lyso-

somes in a series of 10 AD brains. The number oflysosomesand their staining intensity were greatly increased in asubstantial subpopulation of neurons. Many ofthese neuronswere otherwise normal appearing, and some exhibited mod-erate to end-stage patterns of chromatolysis by Nissl stain(Fig. 2). Without exception, AD brains displayed a secondprominent abnormality not observed in control, PD, or HDbrains. Each of the five hydrolase antisera that labeledneuronal lysosomes also intensely stained numerous discreteextracellular areas within the brain parenchyma (Fig. 3).These reactive areas were identified as senile plaques by thepresence of 3-amyloid detected in the same section bythioflavin-S counterstaining or in adjacent serial sections

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AFIG. 2. Pyramidal neurons of control brains contain abundant HEX-immunoreactive lysosomes (A, arrows). In AD brains, many of these

neurons display more intense immunostaining with anti-HEX (B, arrows), and anti-CL (C, arrows) antisera. Neurons staining similarly to thosein control brains are scattered among the abnormal intensely stained cells (B and C, arrowheads). (x300.)

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FIG. 3. In AD brains, senile plaques were intensely stained with anti-CD (A), anti-CL (C), and anti-HEX (E) but not with anti-CG (G)antisera. The identity of senile plaques was confirmed in the same tissue section by thioflavin-S histofluorescence (B, D, F, and H). (x88.)

immunostained with antiserum to 8-amyloid (A4 peptide) orstained by the Bielschowsky silver method (7). Immunore-activity to each of the five hydrolases was abundant withinvirtually every thioflavin-positive deposit. Some "diffuse"plaques, which contained A4 immunoreactivity but no de-tectable thioflavin-positive material, displayed HEX immu-noreactivity above background levels. The immunostainedprofiles in senile plaques were amorphous globular structuresofvarious sizes (0.5-0.6 .um) distinct from the typical neuriticprofiles labeled with antibodies to r (22). The plaque materialstained as intensely as degenerating neurons and consider-ably more darkly than normal-appearing neurons. HEX andCB, the two hydrolases particularly enriched in neurons,highlighted senile plaques most effectively. CH and CGantisera, by contrast, did not stain senile plaques (Fig. 3).Three additional lysosomal hydrolases were identified in

senile plaques and shown by in situ histochemical analyses tobe enzymatically active. Acid phosphatase (Fig. 4A), trimeta-phosphatase, and aryl sulfatase activities were detectedwithin plaques identified by thioflavin-S counterstaining (Fig.4B). The enzymatic activities measured in senile plaqueswere higher than the activities within surrounding neurons,which were below the limit of detection in this assay. Infreshly fixed mouse neocortex stained under the same his-tochemical conditions, neuronal lysosomes were the mostprominently labeled (Fig. 4C).By immunoelectron microscopy, lysosomal hydrolase re-

activity was greatest in lipofuscin complexes. These struc-tures were irregularly shaped and consisted of single ormultiple closely attached granules containing a homogeneous

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lipid portion and an electron-dense linear matrix (pigment)component enclosed by a single continuous unit membrane.Reactive lipofuscin granules were prominent in intact anddegenerating neurons and in senile plaques. In the cell bodiesof normal-appearing neurons (Fig. 5), lipofuscin granuleswere discrete, well-confined, and often localized to one poleof the cell, while in degenerating neurons, large aggregates oflipofuscin were often seen. Cell bodies of degenerating neu-rons were frequently present in plaques, and remnants ofthese cells that included immunoreactive lipofuscin anddense bodies were dispersed throughout the plaque. Analy-ses of skip serial sections through the entire plaque confirmedthat much of this lipofuscin was extracellular (Fig. 6). Theseextracellular aggregates (Fig. 7) strongly resembled the li-pofuscin in degenerating neurons (Fig. 5). Lipofuscin gran-ules were not common within degenerating neurites of senileplaques; immunoreactivity in these structures was observedwithin 100- to 300-nm double-membrane-bound dense bod-ies. Hydrolase-positive lipofuscin granules were oftenclosely apposed to amyloid "asters" (Fig. 7). ,3-Amyloid wasnot immunostained (Fig. 7).

CFIG. 4. In situ histochemical analyses identified acid phosphatase

(A and C), trimetaphosphatase, and aryl sulfatase (data not shown)activities within cortical sections from human and mouse brains.Senile plaques in AD tissue contained high amounts of hydrolasereaction product (A, arrowhead). The coarser granularity surround-ing the plaque is due to nonspecific lead staining. The identity ofthese lesions was confirmed by thioflavin-S histofluorescence (B). Inmouse neocortex, reactive lysosomes were prominent within neu-ronal perikarya and proximal dendrites (C, arrow; section in C iscounterstained with Nissl stain). (A and B, x140; C, x960.)

FIG. 5. Immunoelectron microscopy of two neurons from ADcortex that displayed strong HEX immunoreactivity by immunocy-tochemistry at the light microscopic level. One relatively normal-appearing neuron (lower right) contains increased amounts of well-compartmentalized immunoreactivity in lipofuscin granules (ar-rows). A second degenerating neuron (upper left) is filled withimmunoreactive lipofuscin, some of which has formed large aggre-gates (thick arrows). This material is shown at higher magnificationin Inset. Grids were not poststained. (x 1500; Inset, x27,000.)

Proc. Nad. Acad. Sci. USA 88 (1991)

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FIG. 6. HEX immunoreactivity within senile plaques analyzed in skip serial semithin sections. A plastic-embedded 30-,m section ofHEX-immunostained neocortex (A) displays intense immunoreactivity within a senile plaque (P) and several adjacent neurons (thick and thinarrows). In skip serial semithin (O.5-Itm) sections through the same plaque, the appearance of immunoreactivity in the same two neurons (B)is contrasted with extracellular HEX-positive lipofuscin aggregates (long arrows) diffusely scattered throughout the central region of the plaque(B, C, and D). (A, x360; B-D, x660.)

DISCUSSIONThese studies, summarized in Table 1, show that f-amyloiddeposits in AD are associated with a wide range of lysosomalhydrolases existing in an abnormal extracellular location. Innormal brain, neurons were identified as major sites oflysosomal processing activity as evidenced by the high den-sity of lysosomes and abundance of acid hydrolases. Theseresults extend earlier studies (7, 8, 23, 24). We also observedstriking differences in the distribution of lysosomal hydro-lases between neurons and glia, confirming conclusions fromearlier biochemical analyses that lysosomes of different neu-ral cell types are heterogeneous in enzyme composition (25,26). CB and HEX were particularly enriched in neuronscompared with glial cells. CH, which is known to be in lowabundance in brain (27), was detected only in astrocytes.That lysosomal processing may be particularly active inneurons accords with the suspected role of the lysosomalsystem in basal protein metabolism, including the turnover ofmembrane constituents. Neurons, particularly those withlong axons, maintain a very large cytoplasmic volume andmembrane surface area.A sizeable population of neurons in AD brain displayed

massive accumulations of lysosomal hydrolases. Similar pat-terns are seen in other neocortical areas and hippocampus(ref. 7; unpublished results). Each of the five hydrolases thatare normally prominent in neurons was greatly elevated. Inaddition to confirming reports of increased CB and CDimmunoreactivity in AD neurons (6-8), these results showthat the abnormality is not restricted to a few cathepsins orto a particular class of acid hydrolases. Although accumula-tions of lysosomes and acid hydrolases were most dramaticin the occasional overtly degenerating neurons, this patternwas more widespread among neurons in brain areas known to

become affected in AD. Increased levels and altered intra-cellular distributions of acid hydrolases were seen in manyneurons that appear normal by other morphologic criteria,suggesting that these accumulations may be a relatively earlymarker of metabolic compromise.Although lysosomal hydrolases are normally intracellular

enzymes, eight different acid hydrolases were abundantextracellularly in association with 3-amyloid deposits in ADbrain. Moreover, every acid hydrolase that we observed to beabundant in neuronal lysosomes was also shown by histo-chemistry or immunocytochemistry to be a prominent con-stituent of senile plaques. By contrast, an acid hydrolasepresent only in lysosomes of astrocytes (CH) was not de-tected in senile plaques. The possibility that neurons andtheir processes are a principal source oflysosomal hydrolasesin plaques is supported by other evidence. Of the immuno-stained structures in the brain parenchyma, only the accu-mulated hydrolase-laden lysosomes and lipofuscin in degen-erating neurons match in their staining intensities the amor-phous immunoreactive granules seen within senile plaques.The morphologies of most of these granules corresponded tothose in pyramidal neurons (28). Finally, intensely immuno-reactive neuronal perikarya in various stages ofdegenerationwere identified in many plaques.The abnormal localization of such a broad spectrum of

lysosomal hydrolases in senile plaques indicates a release ofthe entire lysosomal compartment or its contents from cellsrather than the selective secretion of a few lysosomal en-zymes. Acid phosphatase activity has previously been de-tected in senile plaques, and accumulation of the enzyme indystrophic neurites was suggested (29-32). Our results areconsistent with these findings; dystrophic neurites oftencontained numerous immunoreactive dense bodies. The

FIG. 7. Extracellular, HEX-positive lipofuscin aggregates found in the plaque in Fig. 6A resemble aggregates in the degenerating neuronsshown in Fig. 5. Many extracellular lipofuscin granules (B, arrow) were situated in close proximity to ,-amyloid fibrils (B and C, arrowheads),which were not immunoreactive. Grids were not poststained. (A, x6000; B, x28,000; C, x53,000.)

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Table 1. Cytochemical localization of lysosomalhydrolase activities

SenileHydrolase Neurons Astrocytes plaques

CB +++ + +++CL +++ + +++CD +++ ++ +++CH - +++-CG -HEX +++ ++++GLU ++ ++++Trimetaphosphatase +++ * - + +Acid phosphatase ++ +* - + +Aryl sulfatase +++*- + +

*Preservation and localization of hydrolase activity was optimal inperfused-fixed mouse tissue. Activities in neurons of human brainwere below the limit of detection in this assay.

prominent contribution of neuronal perikarya to the extra-cellular lipofuscin in amyloid deposits, however, was nota-ble. The infrequent report of degenerating neuronal cellbodies in plaques may reflect the fact that many cytoplasmicconstituents used as neuronal markers decrease as neuronsdegenerate and antibodies to these would not be expected tohighlight degenerating neurons in senile plaques. By contrast,acid hydrolases and lysosomes are particularly abundant inperikarya and become even more conspicuous in theperikarya of degenerating neurons. In addition, when cellintegrity is disrupted, these membranous structures may bemore resistant to removal from the extracellular space thanother cellular structures.The liberation oflysosomes and lysosomal hydrolases from

degenerating cells into the extracellular space would accountparsimoniously for the multiple abnormal hydrolytic eventsneeded to form (-amyloid from APP (7, 8, 33). We suggestthat, as neurons degenerate, lysosomes become increasinglyfragile and leak hydrolases into the cytoplasm, causing ab-normal proteolysis and contributing to the demise of theneuron. Disintegration of the neuronal plasmalemma alsoliberates lysosomes and lipofuscin into the extracellularspace. Proteins, including APP, are abnormally processed asmembrane degradation proceeds within extracellular lyso-some-related compartments (e.g., lipofuscin), which are nowfreed from normal intracellular control mechanisms (34).Hydrolases slowly released from these compartments act onthe exterior surface of intact and degenerating plasma mem-branes. Because lipases and other membrane-degrading en-zymes are among the hydrolases in lysosomes, their actionscould explain how the intramembrane portion of APP con-taining the C terminus of f-amyloid may be made accessibleto proteases.The association of a range of lysosomal endopeptidases

and exopeptidases with ,3-amyloid deposits raises the furtherpossibility that continued processing of ,B-amyloid by theseenzymes generates peptides that have toxic effects on neu-rons (35-37).

We thank Mary E. Johnson and Johanne H. Khan for secretarialassistance and Lisa Kanaly-Andrews for technical expertise. Thiswork was supported by Public Health Service Grants AG08278,AG05134, and MH/NS31862 (the latter one to Brain Tissue ResourceCenter, McLean Hospital).

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