Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10998-11002, December 1991 Medical Sciences Lysosomal hydrolases 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 proteases. 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 enzyme 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 lysosome- distribution of a series of proteolytic and nonproteolytic related compartments. and 8 of the 10 enzymes pkedomlnated lysosomal enzymes, using enzyme histochemistry and im- in neurons. In Alzheimer-disease brains, strongly immuno- munocytochemistry at the light and electron microscopic reactive lysosomes 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, hydrolase 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 protease 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 4 ..,# ., z.. .- `.v \ .11 41 , .d ., .; \ Jim A;e .. ..J" I 4 f a% .;Iso .s, a-~ 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 phosphatase (18), trimetaphosphatase The same five hydrolases were detected in neuronal lyso- (19), and aryl sulfatase (20) activities