- Email: [email protected]
Cystatin a-like immunoreactivity is widely distributed in human brain and accumulates in neuritic plaques of alzheimer disease subjects
Brain Research Bulletin, Vol. 33, No. 5, pp. 477-481, 1994 Copyright 0 1994 ElsevierScienceLtd
Pergamon
Printedin the USA. All rights reserved 0361-9230/94$6.00 + .oO
Cystatin A-like Immunoreactivity Is Widely Distributed in Human Brain and Accumulates in Neuritic Plaques of ~zheimer Disease Subjects HANS-GERT BE~S~IN,*’ MIKKO J~VI~N,~
* Institute
RKITA RINNE,-/- HEIDRUN KIRSCHKE,$ BRIGITTE KN&EL~ AND AR1 RINNE#
forNeurobiology, P.0. Box 1860, D-39008 Magdeburg, Germany
f Masku Neurological Rehabilitation Center, Finland $ institute ofBiochemistry Martin Luther ~niversi~ Halle, Germany $ Institute of Patholo~, Universi~ of Gulu, Finland Y/Institute of Clinical Immunology, Friedrich Schiller University Jena, Germany #Institute of Pathology, University of Tromsii, Norway Received 28 December 1992; Accepted 1 September 1993 BERNSTEIN, H.-G., R. RINNE, H. KIRSCHKE, M. JARVINEN, B. KNGFEL AND A. RINNE. CystutinA-like immunoreuctivify is widely distributed in human brain and accumulates in neuriticplaques ofAlzheimer disease subjects. BRAIN RES BULL 33(5) 477-481, 1994.-The cellular localization of cystatin A, an endogeneously occurring inhibitor of lysosomal thiol proteases (cathepsins B, H, L and S), was studied immunohistoche~~y in human postmortem brain using the peroxidase-~ti~~xidase method. Both potyclonal and monoclonal antibodies to cystatin A were employed. Western blot analysis revealed one molecular form of the inhibitor in human brain extracts. Its molecular weight was about 13.000. Immunostaining appeared in a sizeable population of neurons and a few cells surrounding cerebral blood vessels (pericytes). In Alzheimer disease subjects cystatin A was found in many neuritic plaques. Possible functional consequences with regard to a role of cystatin A in the inhibition of the Alzheimer amyloid precursor protein (APP)-clipping enzyme, cathepsin B, are discussed. Cystatin A
Human brain
Alzheimer disease
Neuritic plaques
disease is an insidious and ultimately fatal form of dementia that affects a considerable portion of the elder human population. A characteristic neuropatholo~c feature of the disease is the deposition of amyloid in neuritic plaques and within the wall of the cerebral microvasculature (26,31,33,40). The formation of amyloid (A& in Alzheimer disease (AD) is thought to involve altered proteolytic processing of the amyloid precursor protein, an 105-120 kDa transmembrane protein expressed in neurons and other cells (12,20,21,25,26). However, aberrant splitting of APP to potentially amyloidogenic fragments does not necessarily lead to the formation of A/3. Moreover, work from several laboratories has demonstrated that potentially amyloidogenic fragments may be normal cellular products (8,30,34). Recently, a lysosomallendosomal pathway has been proposed to be involved in this process (3,10,13,16,36) and the presence of lysosomal proteinases in senile plaques has been demonstrated (4,6,9,27,28). Moreover, cathepsin B has recently been identified as a amyloid precursor secretase (32,35,36). Therefore, it cannot be ruled out that this enzyme (and perhaps other cathepsins) is actively involved in the normal processing of amyloid precursor protein precIuding A@ protein accumu-
lation which can lead to amyloid formation (22). Aberrant proteolytic cleavage of the amyloid precursor (as observed in amyfoid formations might be the result of an inhibition of this thiol proteinase (6,18,39). Cystatin C, an inhibitor of thiol proteinases known to exist in neural tissue ($24) is deposited in meningeal and grey matter blood vessels in a cerebral amyloid angiopathy known as cerebral haemorrhage with amyloidosis of the Iceland type, as well as in arteriolar walls and in some neuritic plaques of AD patients t&14,38,41). Although the cystatin C gene was found not to be linked to early onset familial AD (29), this inhibitor may well be involved in brain degeneration in other forms of AD. We were interested in investigating, if another well-known inhibitor of cathepsins, cystatin A (also called stefin A), is also present in nervous tissue and whether it appears in association with neuritic plaques to learn more about the subtle interaction between proteiases and their inhibitors in the formation of amyloid as a characteristic neuropathological hallmark in Alzheimer disease. In this study we demonstrate that cystatin A-like material is immunohist~hemically detectable in neurons of post mortem
ALZHEIMER
*To whom requests for reprints should be addressed. 477
478
BERNSTEIN
FIG. 1. Immunodetection of cyst&n A-like material in pyramidal neurons of human neocortex. Calibration bar = 2.5 urn. human brain and that the inhibitor antigen is concentrated
FIG. 3. Demonstration of cystatin A-immnnoreactive material in nerve cells of the hypothalamic ym.
NW. paraventri~l~is.
Calibration
bar = 31
in neu-
ritic plaques of AD patients.
and neurofibrillary tangles (especially in neocortical areas and hip~ampai formation). Tissue pieces from par~ip~campal gyrus, cortical area A 10, superior temporal gyrus. Nut. basalis of Meynert and hypothalamus were taken at autopsy not later than 12 h after death, fixed in formalin, embedded in paraffin and cut into 6pm thick sections. To identify cystatin A antigen immunohistochemically, we employed two different antibodies. Cystatin A was purified both neuritic plaques
METHOD The investigation is based on 13 human brains. Six brains were from persons between the ages 62 and 84 years (mean age: 7.5 years). The human beings died from several diseases and their brains did not show neuropathological lesions at necropsy. Seven brains were taken from AD subjects. AU AD subjects were clinically demented, and neuropathological examination revealed
FIG. 2. Neurons belonging tatin A antigen. Calibration
ET AL.
to the Nut. basalis of Meynert bar = 12 pm.
express cys-
FIG. 4. Cystatin A-containing neurons in the NW. ventromedialis hypothalami as revealed by use of the monoclonal antibody mAK6(1)HW E9/DI. Calibration bar = 20 pm.
CYSTATIN A-LIKE IMMUNO~A~VI~
FIG. 5. Multiple neuritic plaques immunostained for cystatin A in neocortical area A10 of a clinically bar = 3.5 pm.
confirmed
Alzheimer
subject. Calibration
homogeneity from human epidermis (11) and a polyclonal antiserum against the inhibitor was raised in rabbits. This antiserum was shown to be monospecific by Western blot analysis. Monoclonal antibodies were prepared by intraperitoneal immunization of Balb/c mice with purified cystatin A. Spleen cells obtained from these mice were fused with a myeloma cell line (P3-x63-Ag8,653) and cultured. Selected cell clones were tested for production of anti-cystatin A. After gel electrophoresis and dot blot analysis on nitrocellulose, one clone was found to produce an antibody that recognizes cystatin A but not cystatin C.
FIG. 7. Neuritic plaque immunoreactive for cystatin A as revealed by application of the monoclonal antibody mAK6(1)H@E9/Dl. Calibration bar = 35 pm.
to
This antibody (mAIS 6(l) H8/E9/Dl) was proven to be suitable for immunohistochemical purposes on sections of stratified squamous epithelia and tonsilla palatina and subsequently used together with the polyclonal antiserum in human brain immunohistochemistry. Immunoblots were prepared from human brain extracts (temporal cortex, 11 h post mortem) electrophoresed in 12.5 % SDS-polyacryl amide gels. Further handling was as described earlier (6). By use of different marker molecules the molecular weight was determined to be about 13 kD.
A
FIG. 6. Cystatin A-immunoreactive senile plaques of the same patient. calibration bar = 15 pm.
in the hippocampus
B
C
0’
FIG. 8. Western blot analysis of “cerebral cystatin A.” Lane A shows immunoblotting of brain extracts against monoclonal antibody mAK6(1)H8/E9/Dl. Lane B demonstrates immunoblotting of polyclonal anti-cystatin A against extracts from human tonsilla. Lanes C and D are immunoblots of anti-cystatin A antiserum against brain homogenate. Lane E demonstrates immunobiotting against purified cystatin A. Far right (Lane F) shows three molecular markers: 1. lactalbumin (mol. wt 14,000); 2. carbonic anhydrase (mol. wt 30,000) and 3. serum albumin (mol. wt 67,000). Antiserum quenched (preabosrbed) with pure cystatin A did not show immunoreaction in the Western blot (not shown).
480
BERNSTEIN
ET’ AL
Preliminary data from double labeling experiments (application of cystatin A and cathepsin B antisera to consecutive sections) revealed that many senile plaques expressed both antigens (data not shown). Western blot analysis showed that the cystatin A immunoreactive material in the human brain does not differ from the molecular species from nonneuronal sources with regard to its molecular weight and some immunological properties (Fig. 8). DISCUSSION
FIG 9. Control section of human neocortex of an AD patient incubated with polyclonal cystatin A antiserum preabsorbed with affinity-purified cystatin A. No staining is visible.
The peroxidase-antiperoxidase technique was used to immunolocalize cystatin A in human brain sections. The primary (polyclonal) antiserum to cystatin A was used at a dilution of 1:200. The monclonal antibody was employed at a dilution ot 1:20. In negative control experiments, the monclonal antibody was replaced by either buffer or ascites fluid. The polyclonal antiserum was substituted by antiserum previously preabsorbed with the pure antigen. All control procedures resulted in a complete lack of immunostaining (Fig. 9). The sections were investigated under a light microscope (Olympus, Hamburg, Germany).
RESULTS
Cystatin A immunoreactivity was found to be widely distributed in control and Alzheimer-diseased brains. Its cellular localization was confined to nerve cells. In neocortical areas large pyramidal cells were highlighted by intense immunostaining for cystatin A antigen (Fig. 1). In the hippocampus a moderate staining appeared in some CA1 and CA2 pyramidal neurons. A very strong immunoreaction was observed in neurons of the Nut. basalis of Meynert (Fig. 2). Also, multiple neurons in the hypothalamus appeared to contain large amounts of cystatin A antigen (Figs. 3 and 4). In addition, a few nonneuronal cells were found to be cystatin A immunoreactive. Judging from their location near cerebral blood vessels and from their shape, these cells might be pericytes. In sections from Alzheirner brains cystatin A immunoreactivity was observed to be present in several neuronal types. The regional distribution of these nerve cells was similar to that observed in the brains of unaffected individuals. Extracellularly, cystatin A immunoreactive material appeared to be concentrated in many senile plaques with a granular appearance (Figs. 5 and 6). On application of the monoclonal antibody, the number of immunoreactive plaques was slightly lower than after the application of the polyclonal antiserum (Fig. 7).
Neuronal lysosomes seem to play a pivotal role in the pathogenesis of Alzheimer disease (28,36). Enzymatically active lysosomes accumulate in neocortical neurons of AD patients long before typical degenerative changes can be detected, indicating that altered lysosome function is an early event in AD (28). Typical senile plaques contain various lysosomal enzymes, amongst which thiol proteases are of special interest due to their potential function in the normal or aberrant processing of the amyloid precursor (2,32,35). Inhibitors of such proteases may be expected to influence the processing of APP. Indeed, artificial inhibitors of lysosomal proteases (chloroquine, leupeptin and E 64) have been demonstrated to enhance the production of (potentially amyloidogenic) C-terminal fragments from the amyloid precursor protein under experimental conditions (16,18). This study shows for the first time the presence of cystatin A, an naturally occurring inhibitor of thiol proteases, in nerve cells of human brain and in senile plaques of Alzheimer patients. Amyloid seems to be associated with different molecular species of proteases inhibitors. It is well-established that the amyloid precursor appears in multiple forms among which are those differing by a variable length insert within the exracellular domain. The insert has considerable sequence homology to the family of Kunitz protease inhibitor proteins (17,25,37). Senile plaques are known to contain serine proteinase inhibitors, ar-antichymotrypsin and a,-antitrypsin (1,lS). Further, we and others had previously reported the occurrence of thiol proteinase inhibitor cystatin C in few neuritic plaques (.5,14). Although the significance of these findings remains to be determined, it is reasonable to speculate that the occurrence of proteinase inhibitors in neuritic plaques reflects their involvement in the regulation of the proteolytic cleavage of certain components of the plaques including the amyloid precursor protein (39). Cystatins are known to form inactive complexes with cathepsins B, H, L and S by reversibly binding to them, thereby being good candidates to control their activity. Cathepsin D, an aspartic proteinase also known to appear in neuritic plaques (5,14,38,) is thought to upregulate thiol proteinase activities by degrading cystatins (23). A disruption of these regulatory components might be responsible for some of the pathologic mechanisms occurring during the formation of neuritic plaques. We now intend to investigate the regional distribution of cystatin A in normal and diseased human brain by in situ hybrization to obtain more information about the origin and the significance of this protein for the central nervous system. ACKNOWLEDGEMENTS
We thank Dr. G. Poeggel for valuable advice during Western blot studies and Mr. R. Brown for critically reading the manuscript. This research was supported by KAI e.V. Berlin (Grant 020049).
REFERENCES 1. Abraham, C. R.; Selkoe, D. J.; Potter, H. C. Immunochemical identification of the serine protease inhibitor aI-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52:487-501; 1988. 2. Banati, R. B.; Rothe, G.; Valet, G.; Kreutzberg, G. W. Detection of lysosomal cysteine proteinases in micoglia: Plow cytometric mea-
surement and histochemical localization of cathepsins B and L. Glia 7:183-191; 1993. 3. Benowitz, L. I.; Rodriguez, W.; Paskevich, P.; Mufson, E. J.; Schenk, D.; Neve, R. L. The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exper. Neural. 106:237-250; 1989.
CYSTATIN A-LIKE IMMUNOREACTIVITY
B.; Dorn, 4. Bernstein, H.-G.; Bruszis, S.; Schmidt, D.; Wiederanders, A. Immunodetection of cathepsin D in neuritic plaques found in brains of patients with dementia of Alzheimer type. J. Himforsch. 30:613-618; 1989. 5. Bernstein, H.-G.; Jlrvinen, M.; Poll&ten, R.; Schirpke, H.; Knofel, B.; Rinne, R. Cystatin C containing neurons in human postmortem hypothalamus. Neurosci. Lett. 88:131-134; 1988. B.; Schmidt, D.; 6. Bernstein, H.-G.; Kirschke, H.; Wiederanders, Rinne, A. Antigenic expression of cathepsin B in aged human brain. Brain Res. Bull. 24:543-549; 1990. 7. Bernstein, H:-G.; Kirschke, H.; Wiederanders, B.; Khudoerkov, R. M.; Hinz, W.; Rinne, A. Lysosomal proteinases as putative diagnostic tools in human neuropathology: Alzheimer disease (AD) and schizophrenia. Acta histochem. Suppl. XLII:19-24; 1992. 8. Busiglio, J.; Gabuzda, D. H.; Matsudaira, P.; Yankner, B. A. Generation of /?-amyloid in the secretory pathway in neuronal and nonneuronal cells. Proc. Natl. Acad. Sci. USA 90:2092-2096: 1993. 9. Cataldo, A. M.; Paskevich, P. A.; Kominami, E.; Nixon, R. A. Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease. Proc. Natl. Acad. Sci. USA 88:10998-11002; 1991. 10. Cole, G. M.; Huynh, T. V.; Saitoh, T. Evidence for lysosomal processing of amyloid p-protein precursor in cultured cells. Neurochem. Res. 14:933-939; 1989. 11. Cole, G. M.; Bell, L.; Tuong, Q. B.; Saitoh, T. An lysosomal-endosomal pathway for degradation of amyloid precursor protein. Ann. NY Acad. Sci. 674:138-148; 1992. 12. Estus, S.; Golde, T. E.; You&in, S. G. Normal processing of the Alzheimer’s disease amyloid precursor protein. Ann. NY Acad. Sci. 674:102-117; 1992. 13. Golde, T. E.; Estus, S.; Younkin, L. H.; Selkoe, D. J.; Younkin, S. G. Processing of the amyloid protein-precursor to potentially amyloidogenic derivates. Science 255:728-730; 1992. 14. Goldgaber, D.; Lerman, J. I.; MC Bride, 0. W.; Saffiotti, U.; Gajdusek, C. D. Characterization and chromosomal localization of a DNA encoding brain amyloid of Alzheimer’s disease. Science 235:877-880; 1987. 15. Gollin, P. A.; Kalaria, R. N.; Eikelenboom, P.; Rozemuller, A.; Perry, G. A,-antitrypsin and a,-antichymotrypsin are in lesions of Alzheimer disease. Neuroreport 3:201-203; 1992. 16. Haass, C.; Koo, E. H.; Helton, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface P-amyloid precursor protein to lysosomes: Alternative processing into amyloid-bearing fragments. Nature 98:6075-6079; 1992. 17. Hyman, B. T.; Tanzi, R. E.; Marzloff, K.; Barbour, R.; Schenk, D. Kunitz-protease inhibitor-containing amyloid-beta protein precursor immunoreactivity in Alzheimer disease. J. Neuropathol. Exper. Neurol. 51:76-83; 1992. 18. Ivy, G. 0. Protease inhibition causes some manifestations of aging and Alzheimer’s disease in rodent and primate brain. Ann. NY Acad. Sci. 674:89-102; 1992 19. Jarvinen, M.; Rinne, A. Human spleen cysteine proteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. Biochem. Biophys. Acta 708:210-217; 1982. 20. Joachim, C. L.; Selkoe, D. J. The seminal role of P-amyloid in the pathogenesis of Alzheimer disease. Alzheimer Disease and Related Disorders. 6:7-34; 1992. 21. Kawai, M.; Cras, P.; Richey, P.; Tabaton, M.; Lowery, D. E.; Gonzalez-Dewitt, P. A.; Greenberg, B. D.; Gambetti, P.; Perry, G. Subcellular localization of amyloid precursor protein in senile plaques of Alzheimer disease. Am. J. Pathol. 140:947-958; 1992. 22. Knauer, M. F.; Soreghan, B.; Burdick, D.; Kosmoski, J.; Glabe, C. G. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid wp-protein. Proc. Natl. A&d. Sci. USA 89:7437-7441; 1992.
481
23. Lenarcic, B.; Kos, J.; Dolenc, I.; Lucovnik, P.; Krizaj, I.; Turk, V. Cathepsin D inactivates cysteine proteinase inhibitors cystatins. Biochem. Biophys. Res. Commun. 154:765-772; 1988. 24. Liifberg, H.; Grubb, A. 0.; Brun, A. Human brain cortical neurons contain gamma trace. Rapid isolation, immunohistochemical and physicochemical characterization of human gamma trace. Biomed. Res. 2:298-306; 1981. 25. Marotta, C. A.; Majocha, R. C.; Tate, B. Molecular and cellular biology of AIzheimer amyloid. J. Mol. Neurosci. 3:111-125; 1992. 26. Miiller-Hill, B.; Beyreuther, K. Molecular biology of Alzheimer’s disease. Annu. Rev. Biochem. 58:287-307; 1989. 27. Nakamura, Y.; Takeda, M.; Suzuki, H.; Hattori, H.; Tada, S.; Hashimoto, S.; Nishimura, T. Abnormal distribution of cathepsins in the brain of patients with Alzheimer’s disease. Neurosci. Lett. 130:195198; 1991. 28. Nixon, R. A.; Cataldo, A. M.; Pakevich, P. A.; Hamilton, D. J.; Wheelock, T. R.; Kanaley-Andrews, L. The lysosomal system in neurons: Involvement at multiple stages of Alzheimer’s disease pathogenesis. Ann. NY Acad. Sci. 674:65-88; 1992. 29. Par&t, M.; Crook, R.; Roques, P.; Rossor, M.; Chattier-Harlin, M.-C. The cystatin-C gene is not linked to early onset familial Alzheimer’s disease. Neurosci. Lett. 154:81-83; 1993. 30. Pastemak, J. M.; Palmert, M. P.; Usiak, M.; Wang, R.; ZurcherNeely, H.; Gonzalez-De Whitt, P. A.; Fairbanks, M. B.; Cheung, T.; Blades, D.; Henrikson, R. L.; Greenberg, B. D.; Cotter, R. J.; Younkin, S. G. Alzheimer’s disease and control brains contain soluble derivates of the amyloid protein precursor that end within the pamyloid protein region. Biochemistry 31:10936-10940; 1992. 31. Price, D. L.; Walker, L. C.; Martin, L. J.; Sisodia, S. S. Amyloidosis in aging and Alzheimer’s disease. Am. J. Pathol. 141:767-727; 1992. 32. Schonlein, C.; Huber, G. Characterization of potential APP secretases in rat brain. Eur. J. Neurosci. Suppl. 5:(abstr.)1067; 1992. 33. Selkoe, D. J. Deciphering Alzheimer’s disease. The amyloid precursor protein yields new clues. Science 248:1058-1060; 1990. 34. Shoji, M.; Golde, T. E.; Ghiso, J.; Cheung, T. T.; Estus, S.; Shaffer, L. M.; Cai, X.-D.; MC Kay, D. M.; Tintner, R.; Frangione, B.; Younkin, S.G. Production of the Alzheimer amyloid P-protein by normal proteolytic processing. Science 258:126-129; 1992. 35. Tagawa, K.; Kunishita, T.; Maruyama, K.; Yoshikawa, K.; Kominami, E.; Tsuchiya, T.; Suzuki, K.; Tabira, T.; Sugita, H.; Ishiura, S. Alzheimer’s disease amyloid p-clipping enzyme (APP secretase): Identification, purification, and characterization of the enzyme. Biochem. Biophys. Res. Commun. 177:377-387; 1991. 36. Tagawa, K.; Maruyma, K.; Ishiura, S. Amyloid P/A4 precursor protein (APP) processing in lvsosomes. Ann. NY Acad. Sci. 674:129137;‘1992 . . 37. Van Nostrand, W. E.; Scmaier, A. H.; Wagner, S. L. Potential role of proteases nexin-uamyloid P-protein as a cerebral anticoagulant. Ann. NY Acad. Sci. 674~243-252; 1992. 38. Vinters, H. V.; Nishimura, G. S.; Secor, D. L.; Pardridge, W. M. Immunoreactive A4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer’s disease. Am. J. Pathol. 137:233-240; 1990. 39. Wisniewski, K. E.; Gordonmajszak, W.; Maslinski, S.; Heanevkieras, J. Altered protein-patterns in brains of children with neuronal cerooid lipofuscinosis. Am. J. Med. Genet. 42:568-574: 1992. 40. Yamaguchi, H.; Yamazaki, T.; Lemare, C. A.; Frosch, M.‘P.; Selkoe, D. J. Beta amyloid is focally deposited within the outer membrane in the amyloid angiopathy of Alzheimer disease - An immunoelectron microscopic study. Am. J. Pathol. 141:249-259; 1992. 41. Young, W. H.; Robert, M. E.; Secor, D. L.; Kleikamp, T. J.; Vinters, H. V. Cerebral hemorrhage with biopsy-proved amyloid angiopathy. Arch. Neurol. 49:51-58; 1992.
Pergamon
Printedin the USA. All rights reserved 0361-9230/94$6.00 + .oO
Cystatin A-like Immunoreactivity Is Widely Distributed in Human Brain and Accumulates in Neuritic Plaques of ~zheimer Disease Subjects HANS-GERT BE~S~IN,*’ MIKKO J~VI~N,~
* Institute
RKITA RINNE,-/- HEIDRUN KIRSCHKE,$ BRIGITTE KN&EL~ AND AR1 RINNE#
forNeurobiology, P.0. Box 1860, D-39008 Magdeburg, Germany
f Masku Neurological Rehabilitation Center, Finland $ institute ofBiochemistry Martin Luther ~niversi~ Halle, Germany $ Institute of Patholo~, Universi~ of Gulu, Finland Y/Institute of Clinical Immunology, Friedrich Schiller University Jena, Germany #Institute of Pathology, University of Tromsii, Norway Received 28 December 1992; Accepted 1 September 1993 BERNSTEIN, H.-G., R. RINNE, H. KIRSCHKE, M. JARVINEN, B. KNGFEL AND A. RINNE. CystutinA-like immunoreuctivify is widely distributed in human brain and accumulates in neuriticplaques ofAlzheimer disease subjects. BRAIN RES BULL 33(5) 477-481, 1994.-The cellular localization of cystatin A, an endogeneously occurring inhibitor of lysosomal thiol proteases (cathepsins B, H, L and S), was studied immunohistoche~~y in human postmortem brain using the peroxidase-~ti~~xidase method. Both potyclonal and monoclonal antibodies to cystatin A were employed. Western blot analysis revealed one molecular form of the inhibitor in human brain extracts. Its molecular weight was about 13.000. Immunostaining appeared in a sizeable population of neurons and a few cells surrounding cerebral blood vessels (pericytes). In Alzheimer disease subjects cystatin A was found in many neuritic plaques. Possible functional consequences with regard to a role of cystatin A in the inhibition of the Alzheimer amyloid precursor protein (APP)-clipping enzyme, cathepsin B, are discussed. Cystatin A
Human brain
Alzheimer disease
Neuritic plaques
disease is an insidious and ultimately fatal form of dementia that affects a considerable portion of the elder human population. A characteristic neuropatholo~c feature of the disease is the deposition of amyloid in neuritic plaques and within the wall of the cerebral microvasculature (26,31,33,40). The formation of amyloid (A& in Alzheimer disease (AD) is thought to involve altered proteolytic processing of the amyloid precursor protein, an 105-120 kDa transmembrane protein expressed in neurons and other cells (12,20,21,25,26). However, aberrant splitting of APP to potentially amyloidogenic fragments does not necessarily lead to the formation of A/3. Moreover, work from several laboratories has demonstrated that potentially amyloidogenic fragments may be normal cellular products (8,30,34). Recently, a lysosomallendosomal pathway has been proposed to be involved in this process (3,10,13,16,36) and the presence of lysosomal proteinases in senile plaques has been demonstrated (4,6,9,27,28). Moreover, cathepsin B has recently been identified as a amyloid precursor secretase (32,35,36). Therefore, it cannot be ruled out that this enzyme (and perhaps other cathepsins) is actively involved in the normal processing of amyloid precursor protein precIuding A@ protein accumu-
lation which can lead to amyloid formation (22). Aberrant proteolytic cleavage of the amyloid precursor (as observed in amyfoid formations might be the result of an inhibition of this thiol proteinase (6,18,39). Cystatin C, an inhibitor of thiol proteinases known to exist in neural tissue ($24) is deposited in meningeal and grey matter blood vessels in a cerebral amyloid angiopathy known as cerebral haemorrhage with amyloidosis of the Iceland type, as well as in arteriolar walls and in some neuritic plaques of AD patients t&14,38,41). Although the cystatin C gene was found not to be linked to early onset familial AD (29), this inhibitor may well be involved in brain degeneration in other forms of AD. We were interested in investigating, if another well-known inhibitor of cathepsins, cystatin A (also called stefin A), is also present in nervous tissue and whether it appears in association with neuritic plaques to learn more about the subtle interaction between proteiases and their inhibitors in the formation of amyloid as a characteristic neuropathological hallmark in Alzheimer disease. In this study we demonstrate that cystatin A-like material is immunohist~hemically detectable in neurons of post mortem
ALZHEIMER
*To whom requests for reprints should be addressed. 477
478
BERNSTEIN
FIG. 1. Immunodetection of cyst&n A-like material in pyramidal neurons of human neocortex. Calibration bar = 2.5 urn. human brain and that the inhibitor antigen is concentrated
FIG. 3. Demonstration of cystatin A-immnnoreactive material in nerve cells of the hypothalamic ym.
NW. paraventri~l~is.
Calibration
bar = 31
in neu-
ritic plaques of AD patients.
and neurofibrillary tangles (especially in neocortical areas and hip~ampai formation). Tissue pieces from par~ip~campal gyrus, cortical area A 10, superior temporal gyrus. Nut. basalis of Meynert and hypothalamus were taken at autopsy not later than 12 h after death, fixed in formalin, embedded in paraffin and cut into 6pm thick sections. To identify cystatin A antigen immunohistochemically, we employed two different antibodies. Cystatin A was purified both neuritic plaques
METHOD The investigation is based on 13 human brains. Six brains were from persons between the ages 62 and 84 years (mean age: 7.5 years). The human beings died from several diseases and their brains did not show neuropathological lesions at necropsy. Seven brains were taken from AD subjects. AU AD subjects were clinically demented, and neuropathological examination revealed
FIG. 2. Neurons belonging tatin A antigen. Calibration
ET AL.
to the Nut. basalis of Meynert bar = 12 pm.
express cys-
FIG. 4. Cystatin A-containing neurons in the NW. ventromedialis hypothalami as revealed by use of the monoclonal antibody mAK6(1)HW E9/DI. Calibration bar = 20 pm.
CYSTATIN A-LIKE IMMUNO~A~VI~
FIG. 5. Multiple neuritic plaques immunostained for cystatin A in neocortical area A10 of a clinically bar = 3.5 pm.
confirmed
Alzheimer
subject. Calibration
homogeneity from human epidermis (11) and a polyclonal antiserum against the inhibitor was raised in rabbits. This antiserum was shown to be monospecific by Western blot analysis. Monoclonal antibodies were prepared by intraperitoneal immunization of Balb/c mice with purified cystatin A. Spleen cells obtained from these mice were fused with a myeloma cell line (P3-x63-Ag8,653) and cultured. Selected cell clones were tested for production of anti-cystatin A. After gel electrophoresis and dot blot analysis on nitrocellulose, one clone was found to produce an antibody that recognizes cystatin A but not cystatin C.
FIG. 7. Neuritic plaque immunoreactive for cystatin A as revealed by application of the monoclonal antibody mAK6(1)H@E9/Dl. Calibration bar = 35 pm.
to
This antibody (mAIS 6(l) H8/E9/Dl) was proven to be suitable for immunohistochemical purposes on sections of stratified squamous epithelia and tonsilla palatina and subsequently used together with the polyclonal antiserum in human brain immunohistochemistry. Immunoblots were prepared from human brain extracts (temporal cortex, 11 h post mortem) electrophoresed in 12.5 % SDS-polyacryl amide gels. Further handling was as described earlier (6). By use of different marker molecules the molecular weight was determined to be about 13 kD.
A
FIG. 6. Cystatin A-immunoreactive senile plaques of the same patient. calibration bar = 15 pm.
in the hippocampus
B
C
0’
FIG. 8. Western blot analysis of “cerebral cystatin A.” Lane A shows immunoblotting of brain extracts against monoclonal antibody mAK6(1)H8/E9/Dl. Lane B demonstrates immunoblotting of polyclonal anti-cystatin A against extracts from human tonsilla. Lanes C and D are immunoblots of anti-cystatin A antiserum against brain homogenate. Lane E demonstrates immunobiotting against purified cystatin A. Far right (Lane F) shows three molecular markers: 1. lactalbumin (mol. wt 14,000); 2. carbonic anhydrase (mol. wt 30,000) and 3. serum albumin (mol. wt 67,000). Antiserum quenched (preabosrbed) with pure cystatin A did not show immunoreaction in the Western blot (not shown).
480
BERNSTEIN
ET’ AL
Preliminary data from double labeling experiments (application of cystatin A and cathepsin B antisera to consecutive sections) revealed that many senile plaques expressed both antigens (data not shown). Western blot analysis showed that the cystatin A immunoreactive material in the human brain does not differ from the molecular species from nonneuronal sources with regard to its molecular weight and some immunological properties (Fig. 8). DISCUSSION
FIG 9. Control section of human neocortex of an AD patient incubated with polyclonal cystatin A antiserum preabsorbed with affinity-purified cystatin A. No staining is visible.
The peroxidase-antiperoxidase technique was used to immunolocalize cystatin A in human brain sections. The primary (polyclonal) antiserum to cystatin A was used at a dilution of 1:200. The monclonal antibody was employed at a dilution ot 1:20. In negative control experiments, the monclonal antibody was replaced by either buffer or ascites fluid. The polyclonal antiserum was substituted by antiserum previously preabsorbed with the pure antigen. All control procedures resulted in a complete lack of immunostaining (Fig. 9). The sections were investigated under a light microscope (Olympus, Hamburg, Germany).
RESULTS
Cystatin A immunoreactivity was found to be widely distributed in control and Alzheimer-diseased brains. Its cellular localization was confined to nerve cells. In neocortical areas large pyramidal cells were highlighted by intense immunostaining for cystatin A antigen (Fig. 1). In the hippocampus a moderate staining appeared in some CA1 and CA2 pyramidal neurons. A very strong immunoreaction was observed in neurons of the Nut. basalis of Meynert (Fig. 2). Also, multiple neurons in the hypothalamus appeared to contain large amounts of cystatin A antigen (Figs. 3 and 4). In addition, a few nonneuronal cells were found to be cystatin A immunoreactive. Judging from their location near cerebral blood vessels and from their shape, these cells might be pericytes. In sections from Alzheirner brains cystatin A immunoreactivity was observed to be present in several neuronal types. The regional distribution of these nerve cells was similar to that observed in the brains of unaffected individuals. Extracellularly, cystatin A immunoreactive material appeared to be concentrated in many senile plaques with a granular appearance (Figs. 5 and 6). On application of the monoclonal antibody, the number of immunoreactive plaques was slightly lower than after the application of the polyclonal antiserum (Fig. 7).
Neuronal lysosomes seem to play a pivotal role in the pathogenesis of Alzheimer disease (28,36). Enzymatically active lysosomes accumulate in neocortical neurons of AD patients long before typical degenerative changes can be detected, indicating that altered lysosome function is an early event in AD (28). Typical senile plaques contain various lysosomal enzymes, amongst which thiol proteases are of special interest due to their potential function in the normal or aberrant processing of the amyloid precursor (2,32,35). Inhibitors of such proteases may be expected to influence the processing of APP. Indeed, artificial inhibitors of lysosomal proteases (chloroquine, leupeptin and E 64) have been demonstrated to enhance the production of (potentially amyloidogenic) C-terminal fragments from the amyloid precursor protein under experimental conditions (16,18). This study shows for the first time the presence of cystatin A, an naturally occurring inhibitor of thiol proteases, in nerve cells of human brain and in senile plaques of Alzheimer patients. Amyloid seems to be associated with different molecular species of proteases inhibitors. It is well-established that the amyloid precursor appears in multiple forms among which are those differing by a variable length insert within the exracellular domain. The insert has considerable sequence homology to the family of Kunitz protease inhibitor proteins (17,25,37). Senile plaques are known to contain serine proteinase inhibitors, ar-antichymotrypsin and a,-antitrypsin (1,lS). Further, we and others had previously reported the occurrence of thiol proteinase inhibitor cystatin C in few neuritic plaques (.5,14). Although the significance of these findings remains to be determined, it is reasonable to speculate that the occurrence of proteinase inhibitors in neuritic plaques reflects their involvement in the regulation of the proteolytic cleavage of certain components of the plaques including the amyloid precursor protein (39). Cystatins are known to form inactive complexes with cathepsins B, H, L and S by reversibly binding to them, thereby being good candidates to control their activity. Cathepsin D, an aspartic proteinase also known to appear in neuritic plaques (5,14,38,) is thought to upregulate thiol proteinase activities by degrading cystatins (23). A disruption of these regulatory components might be responsible for some of the pathologic mechanisms occurring during the formation of neuritic plaques. We now intend to investigate the regional distribution of cystatin A in normal and diseased human brain by in situ hybrization to obtain more information about the origin and the significance of this protein for the central nervous system. ACKNOWLEDGEMENTS
We thank Dr. G. Poeggel for valuable advice during Western blot studies and Mr. R. Brown for critically reading the manuscript. This research was supported by KAI e.V. Berlin (Grant 020049).
REFERENCES 1. Abraham, C. R.; Selkoe, D. J.; Potter, H. C. Immunochemical identification of the serine protease inhibitor aI-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52:487-501; 1988. 2. Banati, R. B.; Rothe, G.; Valet, G.; Kreutzberg, G. W. Detection of lysosomal cysteine proteinases in micoglia: Plow cytometric mea-
surement and histochemical localization of cathepsins B and L. Glia 7:183-191; 1993. 3. Benowitz, L. I.; Rodriguez, W.; Paskevich, P.; Mufson, E. J.; Schenk, D.; Neve, R. L. The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exper. Neural. 106:237-250; 1989.
CYSTATIN A-LIKE IMMUNOREACTIVITY
B.; Dorn, 4. Bernstein, H.-G.; Bruszis, S.; Schmidt, D.; Wiederanders, A. Immunodetection of cathepsin D in neuritic plaques found in brains of patients with dementia of Alzheimer type. J. Himforsch. 30:613-618; 1989. 5. Bernstein, H.-G.; Jlrvinen, M.; Poll&ten, R.; Schirpke, H.; Knofel, B.; Rinne, R. Cystatin C containing neurons in human postmortem hypothalamus. Neurosci. Lett. 88:131-134; 1988. B.; Schmidt, D.; 6. Bernstein, H.-G.; Kirschke, H.; Wiederanders, Rinne, A. Antigenic expression of cathepsin B in aged human brain. Brain Res. Bull. 24:543-549; 1990. 7. Bernstein, H:-G.; Kirschke, H.; Wiederanders, B.; Khudoerkov, R. M.; Hinz, W.; Rinne, A. Lysosomal proteinases as putative diagnostic tools in human neuropathology: Alzheimer disease (AD) and schizophrenia. Acta histochem. Suppl. XLII:19-24; 1992. 8. Busiglio, J.; Gabuzda, D. H.; Matsudaira, P.; Yankner, B. A. Generation of /?-amyloid in the secretory pathway in neuronal and nonneuronal cells. Proc. Natl. Acad. Sci. USA 90:2092-2096: 1993. 9. Cataldo, A. M.; Paskevich, P. A.; Kominami, E.; Nixon, R. A. Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease. Proc. Natl. Acad. Sci. USA 88:10998-11002; 1991. 10. Cole, G. M.; Huynh, T. V.; Saitoh, T. Evidence for lysosomal processing of amyloid p-protein precursor in cultured cells. Neurochem. Res. 14:933-939; 1989. 11. Cole, G. M.; Bell, L.; Tuong, Q. B.; Saitoh, T. An lysosomal-endosomal pathway for degradation of amyloid precursor protein. Ann. NY Acad. Sci. 674:138-148; 1992. 12. Estus, S.; Golde, T. E.; You&in, S. G. Normal processing of the Alzheimer’s disease amyloid precursor protein. Ann. NY Acad. Sci. 674:102-117; 1992. 13. Golde, T. E.; Estus, S.; Younkin, L. H.; Selkoe, D. J.; Younkin, S. G. Processing of the amyloid protein-precursor to potentially amyloidogenic derivates. Science 255:728-730; 1992. 14. Goldgaber, D.; Lerman, J. I.; MC Bride, 0. W.; Saffiotti, U.; Gajdusek, C. D. Characterization and chromosomal localization of a DNA encoding brain amyloid of Alzheimer’s disease. Science 235:877-880; 1987. 15. Gollin, P. A.; Kalaria, R. N.; Eikelenboom, P.; Rozemuller, A.; Perry, G. A,-antitrypsin and a,-antichymotrypsin are in lesions of Alzheimer disease. Neuroreport 3:201-203; 1992. 16. Haass, C.; Koo, E. H.; Helton, A.; Hung, A. Y.; Selkoe, D. J. Targeting of cell-surface P-amyloid precursor protein to lysosomes: Alternative processing into amyloid-bearing fragments. Nature 98:6075-6079; 1992. 17. Hyman, B. T.; Tanzi, R. E.; Marzloff, K.; Barbour, R.; Schenk, D. Kunitz-protease inhibitor-containing amyloid-beta protein precursor immunoreactivity in Alzheimer disease. J. Neuropathol. Exper. Neurol. 51:76-83; 1992. 18. Ivy, G. 0. Protease inhibition causes some manifestations of aging and Alzheimer’s disease in rodent and primate brain. Ann. NY Acad. Sci. 674:89-102; 1992 19. Jarvinen, M.; Rinne, A. Human spleen cysteine proteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. Biochem. Biophys. Acta 708:210-217; 1982. 20. Joachim, C. L.; Selkoe, D. J. The seminal role of P-amyloid in the pathogenesis of Alzheimer disease. Alzheimer Disease and Related Disorders. 6:7-34; 1992. 21. Kawai, M.; Cras, P.; Richey, P.; Tabaton, M.; Lowery, D. E.; Gonzalez-Dewitt, P. A.; Greenberg, B. D.; Gambetti, P.; Perry, G. Subcellular localization of amyloid precursor protein in senile plaques of Alzheimer disease. Am. J. Pathol. 140:947-958; 1992. 22. Knauer, M. F.; Soreghan, B.; Burdick, D.; Kosmoski, J.; Glabe, C. G. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid wp-protein. Proc. Natl. A&d. Sci. USA 89:7437-7441; 1992.
481
23. Lenarcic, B.; Kos, J.; Dolenc, I.; Lucovnik, P.; Krizaj, I.; Turk, V. Cathepsin D inactivates cysteine proteinase inhibitors cystatins. Biochem. Biophys. Res. Commun. 154:765-772; 1988. 24. Liifberg, H.; Grubb, A. 0.; Brun, A. Human brain cortical neurons contain gamma trace. Rapid isolation, immunohistochemical and physicochemical characterization of human gamma trace. Biomed. Res. 2:298-306; 1981. 25. Marotta, C. A.; Majocha, R. C.; Tate, B. Molecular and cellular biology of AIzheimer amyloid. J. Mol. Neurosci. 3:111-125; 1992. 26. Miiller-Hill, B.; Beyreuther, K. Molecular biology of Alzheimer’s disease. Annu. Rev. Biochem. 58:287-307; 1989. 27. Nakamura, Y.; Takeda, M.; Suzuki, H.; Hattori, H.; Tada, S.; Hashimoto, S.; Nishimura, T. Abnormal distribution of cathepsins in the brain of patients with Alzheimer’s disease. Neurosci. Lett. 130:195198; 1991. 28. Nixon, R. A.; Cataldo, A. M.; Pakevich, P. A.; Hamilton, D. J.; Wheelock, T. R.; Kanaley-Andrews, L. The lysosomal system in neurons: Involvement at multiple stages of Alzheimer’s disease pathogenesis. Ann. NY Acad. Sci. 674:65-88; 1992. 29. Par&t, M.; Crook, R.; Roques, P.; Rossor, M.; Chattier-Harlin, M.-C. The cystatin-C gene is not linked to early onset familial Alzheimer’s disease. Neurosci. Lett. 154:81-83; 1993. 30. Pastemak, J. M.; Palmert, M. P.; Usiak, M.; Wang, R.; ZurcherNeely, H.; Gonzalez-De Whitt, P. A.; Fairbanks, M. B.; Cheung, T.; Blades, D.; Henrikson, R. L.; Greenberg, B. D.; Cotter, R. J.; Younkin, S. G. Alzheimer’s disease and control brains contain soluble derivates of the amyloid protein precursor that end within the pamyloid protein region. Biochemistry 31:10936-10940; 1992. 31. Price, D. L.; Walker, L. C.; Martin, L. J.; Sisodia, S. S. Amyloidosis in aging and Alzheimer’s disease. Am. J. Pathol. 141:767-727; 1992. 32. Schonlein, C.; Huber, G. Characterization of potential APP secretases in rat brain. Eur. J. Neurosci. Suppl. 5:(abstr.)1067; 1992. 33. Selkoe, D. J. Deciphering Alzheimer’s disease. The amyloid precursor protein yields new clues. Science 248:1058-1060; 1990. 34. Shoji, M.; Golde, T. E.; Ghiso, J.; Cheung, T. T.; Estus, S.; Shaffer, L. M.; Cai, X.-D.; MC Kay, D. M.; Tintner, R.; Frangione, B.; Younkin, S.G. Production of the Alzheimer amyloid P-protein by normal proteolytic processing. Science 258:126-129; 1992. 35. Tagawa, K.; Kunishita, T.; Maruyama, K.; Yoshikawa, K.; Kominami, E.; Tsuchiya, T.; Suzuki, K.; Tabira, T.; Sugita, H.; Ishiura, S. Alzheimer’s disease amyloid p-clipping enzyme (APP secretase): Identification, purification, and characterization of the enzyme. Biochem. Biophys. Res. Commun. 177:377-387; 1991. 36. Tagawa, K.; Maruyma, K.; Ishiura, S. Amyloid P/A4 precursor protein (APP) processing in lvsosomes. Ann. NY Acad. Sci. 674:129137;‘1992 . . 37. Van Nostrand, W. E.; Scmaier, A. H.; Wagner, S. L. Potential role of proteases nexin-uamyloid P-protein as a cerebral anticoagulant. Ann. NY Acad. Sci. 674~243-252; 1992. 38. Vinters, H. V.; Nishimura, G. S.; Secor, D. L.; Pardridge, W. M. Immunoreactive A4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer’s disease. Am. J. Pathol. 137:233-240; 1990. 39. Wisniewski, K. E.; Gordonmajszak, W.; Maslinski, S.; Heanevkieras, J. Altered protein-patterns in brains of children with neuronal cerooid lipofuscinosis. Am. J. Med. Genet. 42:568-574: 1992. 40. Yamaguchi, H.; Yamazaki, T.; Lemare, C. A.; Frosch, M.‘P.; Selkoe, D. J. Beta amyloid is focally deposited within the outer membrane in the amyloid angiopathy of Alzheimer disease - An immunoelectron microscopic study. Am. J. Pathol. 141:249-259; 1992. 41. Young, W. H.; Robert, M. E.; Secor, D. L.; Kleikamp, T. J.; Vinters, H. V. Cerebral hemorrhage with biopsy-proved amyloid angiopathy. Arch. Neurol. 49:51-58; 1992.