Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10998-11002, December 1991 Medical Sciences Lysosomal of different classes are abnormally distributed 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, Harvard Medical School, Belmont, MA 02178; and tJuntendo University, Tokyo, Japan Communicated by Francis 0. Schmitt, August 15, 1991

ABSTRACT (3-Amyloid formation requires multiple ab- not be accessible to . Its generation implies either normal proteolytic cleavages of amyloid precursor protein proteolysis of APP molecules that are not inserted into the (APP), including one within its intramembrane doma. Ly- membrane or a cleavage that occurs after additional hydro- sosomes, which contain a wide variety ofprotes (cathepins) lases have exposed the intramembrane domain ofAPP during and other acid hydrolases, are major sites for the turnover of membrane turnover or membrane injury (5). membrane proteins and other cell constituents. Using uno We recently showed (6-8) that the lysosomal proteases cytochemistry, inmmunoelectron microscopy, and his- cathepsin B (CB) and cathepsin D (CD) in AD brain are tochemistry, we studied the expression and cellular distribu- present extracellularly in senile plaques at high levels. To tions of 10 lysosomal hydrolases, including 4 cathepins, in identify the source of extracellular cathepsins and to inves- neocortex from patients with Alzhemer d and control tigate the involvement of other lysosomal hydrolases in (non-Alzheimer-disease) individuals. In control brains, acid f-amyloid formation, we studied the cellular and subcellular hydrolases were localized exclusively to intracellular - distribution of a series of proteolytic and nonproteolytic related compartments. and 8 of the 10 pkedomlnated lysosomal enzymes, using enzyme histochemistry and im- in neurons. In Alzheimer-disease brains, strongly immuno- munocytochemistry at the light and electron microscopic reactive and lipofuscin granules accumulated mark- levels. Our findings show that many classes of lysosomal edly in the perikarya and proximal dendrites ofmany cortical hydrolases are abnormally localized extracellularly in rela- neurons, some of which were undergoing degeneration. More tion to the deposits of (-amyloid in AD brain, and these strikingly, these same hydrolases were present in equally high enzymes originate principally from degenerating neurons. or higher levels in senile plaques in Alzhemer disease, but they were not found extracellularly in control brains, inclading those from Parkinson or Huntngton disease patients. At the MATERIALS AND METHODS ultrastructural level, immunoreactivity in senile Tissue. Postmortem human brains from 10 individuals with plaques was localized to extracellular lipofuscin granules simn- a clinical diagnosis of probable AD, 10 age-matched neuro- iar in morphology to those within degenerating neurons. Two logically normal controls, and 6 brains each from patients cathepsins that were undetectable in neurons were absent from with Parkinson disease (PD) and stage III Huntington disease senile plaques. These results show that lysosome function is (HD) (all individuals were 62-78 yr old) were used. Brain altered in cortical neurons in Alzheimer-disease. The presence tissue was obtained from the Brain Tissue Resource Center, of a broad spectrum of acid hydrolases in senile plaques McLean Hospital (Belmont, MA). Brains from patients with indicates that lysosomes and their contents may be liberated no history of neuropsychiatric disease weighed 1200-1300 g from cells, principally neurons and their processes, as they and exhibited negligible microscopic histopathology (0 to 2 degenerate. Because cathepsns can cleave polypeptide sites on plaques per low-power field). Tissues were immersion-fixed APP relevant for (8-amyloid formation, their abnormal extra- in cold phosphate-buffered (0.15 M, pH 7.4) 10%o (vol/vol) cellular localization and dysregulation in Alzheiser disease can formalin. Postmortem intervals ranged from 30 min to 6 hr, account for the multiple hydrolytic events in 8-amylold for- and total fixation time was < 1 yr. Blocks (3 x 1 x 0.4 cm) mation. The actions of membrane-degrading acid hydrolases of prefrontal cortex (area 10) were cut into 30-jum-thick could also explain how the intramembrane portion of APP Vibratome sections or were cryoprotected in 30% (wt/vol) containing the C terminus of (3-amyloid becomes accessible to sucrose overnight at 40C and cut by cryostat or wedge proteases. microtome into 10-,um-thick sections. Serial adjacent sec- tions were screened for histopathology by using Nissl and The formation of f8-amyloid in Alzheimer disease (AD) in- Bielschowsky silver stains. volves altered proteolytic processing of the amyloid precur- Antibodies. Immunocytochemistry involved polyclonal sor protein (APP), an .70-kDa transmembrane protein ex- antisera against 3-hexosaminidase A (HEX), a-glucosidase pressed in various cell types, including neural cells (1, 2). (GLU), cathepsin H (CH), cathepsin L (CL), cathepsin G More than one abnormal hydrolytic event appears necessary (CG), CB, and CD. Rabbit antisera to HEX and GLU were to produce the -4-kDa ,B-amyloid peptide, including multiple provided by Srinivasa Raghavan (Eunice Kennedy Shriver atypical or abnormal proteolytic cleavages of APP. Genera- Center, Waltham, MA). Previous characterizations of rabbit tion of the N terminus of the P-amyloid peptide implies that antisera to rat liver CH and CL (9-12), and sheep antiserum a normal cleavage is precluded at residue 667 of 751-residue to human brain CD (7) were reported previously. Antisera to APP; normal cleavage forms the physiological polypeptide nexin-2 (3, 4). The C terminus of (3-amyloid is part Abbreviations: AD, Alzheimer disease; PD, Parkinson disease; HD, ofthe intramembrane domain ofAPP, which normally would 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. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Laboratories for payment. This article must therefore be hereby marked "advertisement" Molecular Neuroscience, McLean Hospital, 115 Mill Street, Bel- in accordance with 18 U.S.C. §1734 solely to indicate this fact. mont, MA 02178. 10998 Downloaded by guest on September 26, 2021 Medical Sciences: Cataldo et al. Proc. Nati. Acad. Sci. USA 88 (1991) 10999

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I :A./ 1. n 1,.0 .. 1. ..,f ., .A 't A * 1. C FIG. 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 stained lysosomes 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 from tal cortices from normal control, PD, and HD brains. These ICN. A rabbit antiserum (13) raised against an uncoupled hydrolases included all major cathepsins (CD, CB, CL, and synthetic peptide corresponding to residues 1-40 of a-amy- CH) and two glycosidases (HEX and GLU). Five of these loid was provided by Dennis Selkoe (Harvard Medical antisera (CL, CD, CB, HEX, and GLU) intensely labeled School, Boston). lysosomes in neurons, particularly those in perikarya and Cytochemical Methods. Immunocytochemical studies em- proximal dendrites (Fig. 1). Lysosome density was also ployed the avidin-biotin technique of Hsu et al. (14) as higher in neurons than in other cell types. By contrast, the described (7). Immunocytochemical controls consisted of abundance of these hydrolases varied considerably in astro- tissue sections incubated in preimmune antisera or without cytes. Lysosomes in astrocytes were darkly stained by primary antisera. Human peripheral blood smears were used antisera to CD and CL but contained barely detectable HEX as positive controls in experiments using CG antiserum. and CB immunoreactivities. Oligodendroglia stained weakly Thioflavin-S histochemistry to identify (3-amyloid protein or not at all. was applied before immunocytochemical incubation to avoid Two other lysosomal hydrolases, CH and CG, were not masking histofluorescence by immunoreaction product (7). detected in neurons. CH antiserum strongly labeled lyso- Ultrastructural study of immunoreaction product involved a somes in astrocytes (Fig. 1). CG immunoreactivity was not pre-embedding staining technique (8, 15). Grids either were seen in any cell types in the brain, although the lysosomes of lightly poststained in 2% uranyl acetate and lead citrate (16, leukocytes in peripheral blood were intensely stained (Fig. 17) or were not poststained (negative controls) (8). Enzyme 1C Inset). cytochemistry of acid (18), trimetaphosphatase The same five hydrolases were detected in neuronal lyso- (19), and aryl (20) activities used the lead capture somes in a series of 10 AD brains. The number oflysosomes technique of Gomori (21). Substrates included cytidine 5'- and their staining intensity were greatly increased in a monophosphate, trimetaphosphate, and p-nitrocatechol sul- substantial subpopulation ofneurons. Many ofthese neurons fate, respectively, which were obtained from Sigma. Tissue were otherwise normal appearing, and some exhibited mod- sections incubated in media containing no substrate or a erate to end-stage patterns of chromatolysis by Nissl stain nonspecific substrate served as negative controls to distin- (Fig. 2). Without exception, AD brains displayed a second guish nonspecific lead binding from the bona fide reaction prominent abnormality not observed in control, PD, or HD product. brains. Each of the five hydrolase antisera that labeled neuronal lysosomes also intensely stained numerous discrete extracellular areas within the brain parenchyma (Fig. 3). RESULTS These reactive areas were identified as senile plaques by the Six antisera against different hydrolases specifically labeled presence of 3-amyloid detected in the same section by intracellular lysosomal compartments in sections of prefron- thioflavin-S counterstaining or in adjacent serial sections

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FIG. 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 those in control brains are scattered among the abnormal intensely stained cells (B and C, arrowheads). (x300.) Downloaded by guest on September 26, 2021 11000 Medical Sciences: Cataldo et al. Proc. Nad. Acad. Sci. USA 88 (1991)

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) or lipid portion and an electron-dense linear matrix (pigment) stained by the Bielschowsky silver method (7). Immunore- component enclosed by a single continuous unit membrane. activity to each of the five hydrolases was abundant within Reactive lipofuscin granules were prominent in intact and virtually every thioflavin-positive deposit. Some "diffuse" degenerating neurons and in senile plaques. In the cell bodies plaques, which contained A4 immunoreactivity but no de- of normal-appearing neurons (Fig. 5), lipofuscin granules tectable thioflavin-positive material, displayed HEX immu- were discrete, well-confined, and often localized to one pole noreactivity above background levels. The immunostained ofthe cell, while in degenerating neurons, large aggregates of profiles in senile plaques were amorphous globular structures lipofuscin were often seen. Cell bodies of degenerating neu- ofvarious sizes (0.5-0.6 .um) distinct from the typical neuritic rons were frequently present in plaques, and remnants of profiles labeled with antibodies to r (22). The plaque material these cells that included immunoreactive lipofuscin and stained as intensely as degenerating neurons and consider- dense bodies were dispersed throughout the plaque. Analy- ably more darkly than normal-appearing neurons. HEX and ses ofskip serial sections through the entire plaque confirmed CB, the two hydrolases particularly enriched in neurons, that much of this lipofuscin was extracellular (Fig. 6). These highlighted senile plaques most effectively. CH and CG extracellular aggregates (Fig. 7) strongly resembled the li- antisera, by contrast, did not stain senile plaques (Fig. 3). pofuscin in degenerating neurons (Fig. 5). Lipofuscin gran- Three additional lysosomal hydrolases were identified in ules were not common within degenerating neurites of senile senile plaques and shown by in situ histochemical analyses to plaques; immunoreactivity in these structures was observed be enzymatically active. (Fig. 4A), trimeta- within 100- to 300-nm double-membrane-bound dense bod- phosphatase, and aryl sulfatase activities were detected within plaques identified by thioflavin-S counterstaining (Fig. ies. Hydrolase-positive lipofuscin granules were often 4B). The enzymatic activities measured in senile plaques closely apposed to amyloid "asters" (Fig. 7). ,3-Amyloid was were higher than the activities within surrounding neurons, not immunostained (Fig. 7). which were below the limit of detection in this assay. In freshly fixed mouse neocortex stained under the same his- tochemical conditions, neuronal lysosomes were the most prominently 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 or multiple closely attached granules containing a homogeneous

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O ., 4..". i, /I\- C s.-0 FIG. 4. In situ histochemical analyses identified acid phosphatase (A and C), trimetaphosphatase, and aryl sulfatase (data not shown) FIG. 5. Immunoelectron microscopy of two neurons from AD activities within cortical sections from human and mouse brains. cortex that displayed strong HEX immunoreactivity by immunocy- Senile plaques in AD tissue contained high amounts of hydrolase tochemistry at the light microscopic level. One relatively normal- reaction product (A, arrowhead). The coarser granularity surround- appearing neuron (lower right) contains increased amounts of well- ing the plaque is due to nonspecific lead staining. The identity of compartmentalized immunoreactivity in lipofuscin granules (ar- these lesions was confirmed by thioflavin-S histofluorescence (B). In rows). A second degenerating neuron (upper left) is filled with mouse neocortex, reactive lysosomes were prominent within neu- immunoreactive lipofuscin, some of which has formed large aggre- ronal perikarya and proximal dendrites (C, arrow; section in C is gates (thick arrows). This material is shown at higher magnification counterstained with Nissl stain). (A and B, x140; C, x960.) in Inset. Grids were not poststained. (x 1500; Inset, x27,000.) Downloaded by guest on September 26, 2021 Medical Sciences: Cataldo et al. Proc. Nadl. Acad. Sci. USA 88 (1991) 11001

FIG. 6. HEX immunoreactivity within senile plaques analyzed in skip serial semithin sections. A plastic-embedded 30-,m section of HEX-immunostained neocortex (A) displays intense immunoreactivity within a senile plaque (P) and several adjacent neurons (thick and thin arrows). 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 ofthe plaque (B, C, and D). (A, x360; B-D, x660.) DISCUSSION become affected in AD. Increased levels and altered intra- cellular distributions of acid hydrolases were seen in many These studies, summarized in Table 1, show that f-amyloid neurons that appear normal by other morphologic criteria, deposits in AD are associated with a wide range oflysosomal suggesting that these accumulations may be a relatively early hydrolases existing in an abnormal extracellular location. In marker of metabolic compromise. normal brain, neurons were identified as major sites of lysosomal processing activity as evidenced by the high den- Although lysosomal hydrolases are normally intracellular sity of lysosomes and abundance of acid hydrolases. These enzymes, eight different acid hydrolases were abundant results extend earlier studies (7, 8, 23, 24). We also observed extracellularly in association with 3-amyloid deposits in AD striking differences in the distribution of lysosomal hydro- brain. Moreover, every acid hydrolase that we observed to be lases between neurons and glia, confirming conclusions from abundant in neuronal lysosomes was also shown by histo- earlier biochemical analyses that lysosomes of different neu- chemistry or immunocytochemistry to be a prominent con- ral cell types are heterogeneous in enzyme composition (25, stituent of senile plaques. By contrast, an acid hydrolase 26). CB and HEX were particularly enriched in neurons present only in lysosomes of astrocytes (CH) was not de- compared with glial cells. CH, which is known to be in low tected in senile plaques. The possibility that neurons and abundance in brain (27), was detected only in astrocytes. theirprocesses are a principal source oflysosomal hydrolases That lysosomal processing may be particularly active in in plaques is supported by other evidence. Of the immuno- neurons accords with the suspected role of the lysosomal stained structures in the brain parenchyma, only the accu- system in basal protein metabolism, including the turnover of mulated hydrolase-laden lysosomes and lipofuscin in degen- membrane constituents. Neurons, particularly those with erating neurons match in their staining intensities the amor- long axons, maintain a very large cytoplasmic volume and phous immunoreactive granules seen within senile plaques. membrane surface area. The morphologies of most ofthese granules corresponded to A sizeable population of neurons in AD brain displayed those in pyramidal neurons (28). Finally, intensely immuno- massive accumulations oflysosomal hydrolases. Similar pat- reactive neuronal perikarya in various stages ofdegeneration terns are seen in other neocortical areas and hippocampus were identified in many plaques. (ref. 7; unpublished results). Each ofthe five hydrolases that The abnormal localization of such a broad spectrum of are normally prominent in neurons was greatly elevated. In lysosomal hydrolases in senile plaques indicates a release of addition to confirming reports of increased CB and CD the entire lysosomal compartment or its contents from cells immunoreactivity in AD neurons (6-8), these results show rather than the selective secretion of a few lysosomal en- that the abnormality is not restricted to a few cathepsins or zymes. Acid phosphatase activity has previously been de- to a particular class of acid hydrolases. Although accumula- tected in senile plaques, and accumulation of the enzyme in tions of lysosomes and acid hydrolases were most dramatic dystrophic neurites was suggested (29-32). Our results are in the occasional overtly degenerating neurons, this pattern consistent with these findings; dystrophic neurites often was more widespread among neurons in brain areas known to contained numerous immunoreactive dense bodies. The

FIG. 7. Extracellular, HEX-positive lipofuscin aggregates found in the plaque in Fig. 6A resemble aggregates in the degenerating neurons shown 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.) Downloaded by guest on September 26, 2021 11002 Medical Sciences: Cataldo et al. Proc. Nad. Acad. Sci. USA 88 (1991) Table 1. Cytochemical localization of lysosomal 2. Ishiura, S. (1991) J. Neurochem. 56, 363-369. hydrolase activities 3. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, F. T., McClure, D. & Ward, P. J. Senile (1990) Science 248, 1122-1124. Hydrolase Neurons Astrocytes plaques 4. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H., CB +++ + +++ Farrow, J. S., Geddes, J. W., Cotman, C. W. & Cunningham, D. D. (1989) Nature (London) 341, 546-549. CL +++ + +++ 5. Dyrks, T. W., Ridemann, A., Multhanp, G., Salbaum, J. M., CD +++ ++ +++ Lemaitre, H.-G., Kang, J., Muller-Hill, B., Masters, C. L. & CH - +++- Beyruther, K. (1988) EMBO J. 7, 949-957. CG - 6. Cataldo, A. M., Nixon, R. A., Thayer, C. Y., Benes, F. M. & HEX +++ ++++ Wheelock, T. R. (1987) Soc. Neurosci. Abstr. 13, 1150. GLU ++ ++++ 7. Cataldo, A. M., Thayer, C. Y., Bird, E. D., Wheelock, T. R. Trimetaphosphatase +++ * - + + & Nixon, R. A. (1990) Brain Res. 513, 181-192. 8. Cataldo, A. M. & Nixon, R. A. (1990) Proc. Natl. Acad. Sci. Acid phosphatase ++ +* - + + USA 87, 3861-3865. Aryl sulfatase +++*- + + 9. Kominami, E. & Katunuma, N. (1982) J. Biochem. (Tokyo) 91, *Preservation and localization of hydrolase activity was optimal in 67-71. perfused-fixed mouse tissue. Activities in neurons of human brain 10. Bando, Y., Kominami, E. & Katunuma, N. (1986) J. Biochem. were below the limit of detection in this assay. (Tokyo) 100, 35-42. 11. Kominami, E., Tsukajara, T., Bando, Y. & Katunuma, N. prominent contribution of neuronal perikarya to the extra- (1982) J. Biochem. (Tokyo) 98, 87-93. cellular lipofuscin in amyloid deposits, however, was nota- 12. Katunuma, N. & Kominami, E. (1983) Curr. Top. Cell Regul. 22, 91-101. ble. The infrequent report of degenerating neuronal cell 13. Joachim, C. L., Mori, H. & Selkoe, D. J. (1989) Nature (Lon- bodies in plaques may reflect the fact that many cytoplasmic don) 341, 226-230. constituents used as neuronal markers decrease as neurons 14. Hsu, S.-M., Raine, L. & Fanger, H. (1981) J. Histochem. degenerate and antibodies to these would not be expected to Cytochem. 29, 557-580. highlight degenerating neurons in senile plaques. By contrast, 15. Broadwell, R. D. (1982) Strategies for Studying the Roles of acid hydrolases and lysosomes are particularly abundant in Peptides in Neuronal Function, Short Course Syllabus (Soc. perikarya and become even more conspicuous in the Neurosci. Washington), pp. 27-40. 16. Reynolds, E. S. (1963) J. Cell Biol. 17, 208. perikarya of degenerating neurons. In addition, when cell 17. Venable, J. H. & Coggeshall, R. (1965) J. Cell Biol. 25, 407 integrity is disrupted, these membranous structures may be (abstr.). more resistant to removal from the extracellular space than 18. Novikoff, A. B. (1963) in Ciba Foundation Symposium on other cellular structures. Lysosomes, eds. deReuck, A. V. S. & Cameron, M. P. (Little, The liberation oflysosomes and lysosomal hydrolases from Brown, Boston), pp. 36-73. degenerating cells into the extracellular space would account 19. Berg, G. G. (1960) J. Histochem. Cytochem. 8, 92-101. parsimoniously for the multiple abnormal hydrolytic events 20. Goldfischer, S. (1965) J. Histochem. Cytochem. 13, 520-523. needed to form (-amyloid from APP (7, 8, 33). We suggest 21. Gomori, G. (1952) Microscopic Histochemistry: Principles and Practice (Univ. of Chicago Press, Chicago), p. 273. that, as neurons degenerate, lysosomes become increasingly 22. Kosik, K. S., Joachim, C. L. & Selkoe, D. J. (1986) Proc. Natl. fragile and leak hydrolases into the cytoplasm, causing ab- Acad. Sci. USA 83, 4044-4048. normal proteolysis and contributing to the demise of the 23. Bernstein, H.-G., Sormunen, R., Jarvinen, M., Kloss, P., neuron. Disintegration of the neuronal plasmalemma also Kirschke, H. & Rinne, A. (1989) J. Hirnforsch. 30, 313-317. liberates lysosomes and lipofuscin into the extracellular 24. Bernstein, H.-G., Kirschke, H., Roskoden, T. & Wiederand- space. Proteins, including APP, are abnormally processed as ers, B. (1990) Acta Histochem. Cytochem. 23, 203-207. membrane degradation proceeds within extracellular lyso- 25. Bowen, D. M., Flack, R. H. A., Martin, R. O., Smith, C. B., some-related compartments (e.g., lipofuscin), which are now White, P. & Davidson, A. N. (1974) J. Neurochem. 22, 1099- freed from normal intracellular control mechanisms (34). 1107. 26. Hirsch, H. E., Duquette, P. & Parks, M. E. (1976) J. Neuro- Hydrolases slowly released from these compartments act on chem. 26, 505-512. the exterior surface of intact and degenerating plasma mem- 27. Kominami, E., Tsukahara, T., Bando, Y. & Katunuma, N. branes. Because and other membrane-degrading en- (1985) J. Biochem. 98, 87-93. zymes are among the hydrolases in lysosomes, their actions 28. Boellaard, J. W. & Schlote, W. (1986) Acta Neuropathol. 71, could explain how the intramembrane portion of APP con- 285-294. taining the C terminus of f-amyloid may be made accessible 29. Friede, R. L. (1965) J. Neuropathol. Exp. Neurol. 24, 477-491. to proteases. 30. Gonatas, N. K., Anderson, W. & Evangelista, I. (1967) J. The association of a range of lysosomal endopeptidases Neuropathol. Exp. Neurol. 26, 25-39. and exopeptidases with raises the further 31. Krigman, M. R., Feldman, R. G. & Bensch, K. (1965) Lab. ,3-amyloid deposits Invest. 14, 381-396. possibility that continued processing of ,B-amyloid by these 32. Suzuki, K. & Terry, R. D. (1967) Acta Neuropathol. 8, 276- enzymes generates peptides that have toxic effects on neu- 284. rons (35-37). 33. Nixon, R. A. & Cataldo, A. M. (1991) in Frontiers ofAlzheimer Research, eds. Ishii, T., Allsop, D. & Selkoe, D. J. (Elsevier, We thank Mary E. Johnson and Johanne H. Khan for secretarial Amsterdam), pp. 133-146. assistance and Lisa Kanaly-Andrews for technical expertise. This 34. Tapper, H. & Sundler, R. (1990) Biochem. J. 272, 407-414. work was supported by Public Health Service Grants AG08278, 35. Whitson, J. S., Selkoe, D. J. & Cotman, C. W. (1989) Science AG05134, and MH/NS31862 (the latter one to Brain Tissue Resource 243, 1488-1490. Center, McLean Hospital). 36. Yankner, B. A., Duffy, L. K. & Kirschner, D. A. (1990) Sci- ence 250, 279-282. 1. Ishiura, S., Nishikawa, T. & Tsukahara, T. (1990) Neurosci. 37. Koh, J.-Y., Yang, L. L. & Cotman, C. W. (1990) Brain Res. Lett. 115, 329-334. 533, 315-320. Downloaded by guest on September 26, 2021