Brain Research, 561 (1991) 177-180 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391
177
BRES 24864
Localization of calpain immunoreactivity in senile plaques and in neurones undergoing neurofibrillary degeneration in Alzheimer's disease Norihiko Iwamoto 1, Wipawan Thangnipon 1, Catherine Crawford 2 and Piers C. Emson 1 1MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.) and 2Laboratory of Molecular Biophysics, University of Oxford, Oxford, Oxford (U.K.) (Accepted 2 July 1991)
Key words: Alzheimer-type dementia; Senile plaque; Neurofibrillary tangle; Cytoskeleton; Membrane; Degradation; Calcium activated protease
An antibody raised against the calcium activated neutral protease (calpain) was used to investigate the possible involvement of this enzyme in the formation of plaques and tangles in Alzheimer-type dementia (ATD) brain. Our results revealed the presence of a number of strongly stained calpain positive neurones in the normal human cerebral cortex and a loss of calpain positive cells in ATD brain. Furthermore, double staining experiments revealed that calpain immunoreactivity was present in cells undergoing tangle formation, and was also present in senile plaques. These data suggest that activation of calpain may be an important factor in the abnormal proteolysis underlying the accumulation of plaques and tangles in ATD.
Recently, abnormal proteolysis has been reported in the brains of patients with Alzheimer-type dementia (ATD) 1'6. This aberrant proetolytic processing is thought to result in the accumulation of modified or abnormal fragments of common cellular structural proteins such as the microtubule associated protein tau, neurofilament and the amyloid protein precursor leading to the formation of neurofibrillary tangles 4"18 (NFTs) and the formation of extracellular senile plaques 16. If these two major diagnostic hallmarks of Alzheimer's disease (i.e. plaques and tangles) arise from aberrant proteolytic processing, it is of interest to consider which proteases may be involved. One possible candidate is the calcium protease calpain 11'2°'27"28 which is known to be involved in the degradation of both membrane 2'3'23'2s and cytoskeletal proteins14.24,25, 28. A calcium protease such as calpain is also an attractive candidate for a proteolytic enzyme implicated in ATD as it is activated by elevation of intracellular calcium 3'14'23'25. Indeed, both elevations in intracellular calcium 22 and reductions in intracellular calcium binding proteins 5'13 which may have a Ca 2+ buffering action 1°'21 in the neurone have been reported in ATD. In consequence it was of some interest to examine the distribution of calpain in the normal and ATD brain, and to consider if calpain expression was correlated with the presence of plaques or the occurrence of NFT markers
in damaged neurones. To determine the distribution of calpain, we immunostained sections from the cerebral frontal cortex of 5 pathologically verified ATD patients (age at death 78.3 + 6.7 years S.D.) and 6 control patients with no neuropathological abnormalities (age at death 83.2 - 7.5 years S.D.) with the anti-calpain antiserum. Antibodies against calpain were raised in rabbits. In brief, purified porcine renal calpain II (ref. 7) (200/zg) was emulsified with complete Freund's adjuvant and injected into New Zealand white rabbits, and rabbits were boosted every 4-6 weeks with 100 /~g of calpain II in incomplete Freund's adjuvant. Following the third boost, antiserum was collected and examined by Western blotting (Fig. 1). For immunostaining, 25-/~m-thick sections were cut from the frontal cortex of human brain samples which had been fixed with 10% formalin and cyroprotected with 30% sucrose. The sections were incubated with anticalpain antiserum 1:400 in 0.1 M PBS with 3% bovine serum albumin and 0.2% Triton X-100 for 48 h at 4 °C. After washing, sections were incubated with biotinylated anti-rabbit IgG and then avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Labs). Diaminobenzidine (DAB) was used as the peroxidase substrate to visualize sites of antibody binding. Some sections were also double stained with an anti-tau (z) antibody for identification of N F T 18. Sections were incubated with mouse
Correspondence: P.C. Emson, MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
178 Fig. 1. SDS-PAGE of purified calpain II and Western blots. Lane 1: molecular-mass markers. Lane 2: SDS-PAGE of purified calpain II from porcine kidney. Lane 3,4: Western blots of calpain II immunostained by the rabbit anti-calpain antiserum (lane 3) and normal rabbit serum (lane 4). This anti-calpain antiserum clearly stained the calpain II (lane 3) whereas normal rabbit serum showed no immunostaining (lane 4) (arrow, large subunit of calpain II; arrowhead, small subunit of calpain II).
monoclonal anti-tau antibody (1:5000 diluted, Sigma) at 4 °C overnight. A f t e r washing, the section was treated with a r h o d a m i n e conjugated sheep anti-mouse I g G (Derotec) for 3 h at r o o m temperature. In o r d e r to visualize senile plaques on the same sections were stained with 1% solution of thioflavin S for 10 min 8'17. Characterization of our anti-calpain II antibodies by Western blotting (Fig. 1) revealed that the antiserum contained antibodies recognizing the heavy and light chains of calpain II (Fig. 1, lane 3). In addition, immunological cross-reactivity b e t w e e n calpain I and II was observed as previously r e p o r t e d 9'29. Thus our antiserum should detect all calpain-like immunoreactivity in the brain. O u r antiserum revealed a n u m b e r of strongly stained neurones and glial cells in the n o r m a l human brain (Fig. 2a). This immunostaining pattern was essen-
a
kDa
1
2
3
4
98
67
~t
14
b
Fig. 2. The distribution of calpain immunoreactive cells in frontal cortex of human brain, a: in the normal control brain, calpain immunoreactive cells are observed in all layers except layer I. b: ATD brain shows decreased number of calpain immunoreactive neurones. Only a few positive neurones in layer V remain. Bar = 500 #m. c: high-power micrograph of calpain positive neurones in the normal control brain. The antiserum clearly stains both cell bodies and dendrites. Bar = 100 #m.
179
Fig. 3. Co-existence of calpain immunoreactivity and neurofibrillary tangles (NFF). a: pyramidal neurones with positive calpain immunoreactivity (arrow) and negative immunoreactivity (arrowhead). Bar = 50 gin. b: same section as in (a). NFTs are dearly immunostained by the anti-tau antibody (arrow and arrowhead), c: same section as in (a) and (b). Demonstration of co-expression of calpain and tau immunoreactivity. Calpain immunoreactivity located in a neurone with developing NFF, (arrow), whereas positive calpain-staining is not observed in a dead neurone where the NFT completely fills the cells intracellular space (arrowhead mature NFr).
tially similar to that seen by H a m a k u b o et al. 12 for rat brain, with calpain immunoreactivity concentrated in particular in the larger pyramidal cells (Fig. 2c). In the A T D cases (Fig. 2b) there was a clear loss of the larger calpain containing pyramidal neurones indicating that these cells are damaged in A T D . Further examination of the A T D cases using both anti-calpain and anti-tau antisera revealed that many of the larger calpain containing neurones also contained tau-like immunoreactivity (Fig. 3). Moreover, calpain immunoreactivity was concentrated on the edges of senile plaques (Fig. 4) when the plaques were identified by thioflavin S staining. The data presented here show that amongst the neurones affected in A T D there is a loss of those containing calpain. These calpain positive neurones include populations of the larger neurones which are particularly damaged in A T D 28 and are rich in amyloid precursor (APP) immunoreactivity 27. It was also particularly interesting to note that in the A T D cases, a number of cells with tau positive N F T were also calpain positive and that
Fig. 4. Frontal cortex (layers II-IV) immunostained with anticalpain antiserum showing positive plaques (a). In (b) and (c) confirmation of the coincidence of calpain immunoreactivity (b) is provided by the use of the amyloid marker thioflavin S (c). Note the clustering of strong calpain immunoreactivity around the core of the senile plaque (arrowhead in b). Bar = 20 #m.
these (some 30% of all tau positive cells) were those undergoing N F T formation. Calpain immunoreactivity was present in those cells undergoing NFT formation containing cell cytoplasm and a nucleus but absent in the dead cell 'ghosts' which contain only mature NFTs (Fig. 3c). These data suggest that calpain activation may both be involved in the formation of N F T and in the death of these cells 2°. The demonstration of calpain immunoreactivity associated with the senile plaque (Fig. 4) may also suggest a role for calpain in the formation of the plaque. It is reported that a protease inhibitor 1 and a lysosomal protease 6 have been found associated with senile plaques. This evidence indicates that abnormal protein processing may be occurring in the senile plaque. Since the amyloid protein precursor is a membrane spanning protein 16, its modification in the dying cell may alter its susceptibility to calpain proteolysis 2'3'23 which may produce the
180 accumulation of insoluble amyloid. The calpain immunoreactivity surrounding the plaque may perhaps represent calpain produced by glial cells, which include reactive astrocytes known to be associated with plaques and which play an important role in the formation of the plaque 15'26. However, irrespective of whether the calpain immunoreactivity in the plaque is n e u r o n a l or glial in origin t h e involvement of this enzyme in proteolytic processing of the constituents of both the plaques and tangles strongly suggests that this calcium activated protease is closely involved with the pathogenesis of A T D 14,24,25, 27. Indeed, modification of synaptic m e m b r a n e b o u n d
1 Abraham, C.R., Selkoe, D.J. and Potter, H., Immunoehemicai identification of the serine protease inhibitor a l-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease, Cell, 52 (1988) 487-501. 2 Ahkong, Q.E, Botham, G.M., Woodward, A.W. and Lucy, J.A., Calcium-activated thiolproteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol, Biochem. J., 192 (1980) 829-836. 3 Anderson, D.R., Davis, J.L. and Carraway, K.L., Calciumpromoted changes of the human erythrocyte membrane, involvement of spectrin, transglutaminase, and a membranebound protease, J. Biol. Chem., 252 (1977) 6617-6623. 4 Anderton, B.H., Breinburg, D., Downes, M.J., Green, EJ., Tomlinson, B.E., Ulrich, J., Wood, J.N. and Kahn, J., Monodonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants, Nature, 298 (1982) 84-86. 5 Arai, H., Emson, EC., Mountjoy, C.Q., Carassco, L.H. and Heizmann, C.W., Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia, Brain Research, 418 (1987) 164-169. 6 Cataldo, A.M., Thayer, C.Y., Bird, E.D., Wheelock, T.R. and Nixon, R.A., Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin, Brain Research, 513 (1990) 181-192. 7 Crawford, C., Brown, N.R. and Willis, A.C., Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate, Biochem. J., 265 (1990) 575579. 8 Dawbarn, D. and Emson, EC., Neuropeptide Y-like immunoreactivity in neuritic plaques of Alzheimer's disease, Biochem. Biophys. Res. Commun., 126 (1985)289-294. 9 Dayton, W.R., Scholimeyer, J.D., Lepley, R.A. and Cort6s, L.R., A calcium-activated protease possibly involved in myofibrillar protein turnover, isolation of a low-calcium requiring form of the protease, Biochem. Biophys. Acta, 659 (1981) 4861. 10 Feher, J.J., Measurement of facilitated calcium diffusion by a soluble calcium-binding protein, Biochem. Biophys. Acta, 773 (1984) 91-98. 11 Guroff, G., A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem., 239 (1964) 149155. 12 Hamakubo, T., Kannagi, R., Murachi, T. and Matus, A., Distribution of calpain I and II in rat brain, J. Neurosci., 6 (1986) 3103-3111. 13 Ichimiya, Y., Emson, EC., Mountjoy, C.Q., Lawson, D.E.M. and Heizmann, C.W., Loss of calbindin-28K immunoreactive neurones from the cortex in AIzheimer-type dementia, Brain Research, 475 (1988) 156-159. 14 Ishizaki, Y., Tashiro, T. and Kurokawa, M., A calcium-activated protease which preferentially degrades the 160-kDa component of the neurofilament triplet, Eur. J. Biochem., 131 (1983) 41-45. 15 Iwamoto, N., Suzuki, Y., Makino, Y., Haga, C., Kosaka, K. and Iizuka, R., Cell membrane changes in brains manifesting
proteins by activated calpain may also contribute to the memory disturbance characteristic of A T D 19 by influencing intraceUular signalling.
We are grateful to Mr. T. Buss for photographic work, Mrs. B.A. Oakley for helping to prepare the manuscript and Mr. R. Hills (MRC Brain Bank, Department of Psychiatry, University of Cambridge) for preparation of brain samples. N.I. and W.T. are visiting research fellows from the Juntendo University in Japan and the Mahidol University in Thailand respectively. W.T. acknowledges the support of the Hereditary Disease Association and the Sandoz Foundation for Gerontological Research for this study. We are also indebted to Tropon-Bayer (Cologne) for their continued support.
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senile plaques: an immunohistoehemical study of GM 1 membranous ganglioside, Brain Research, 522 (1990) 152-156. Kang, H., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K. and MUller-Hill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. Kei~nyi, G., Thioflavin S fluorescent and congo red anisotropic stainings in the histologic demonstration of amyloid, Acta Neuropathol., 7 (1967) 336-348. Kosik, K.S., Joachima, C.L. and Selkoe, D.J., Microtubule-associated protein tau (r) is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4044-4048. Lynch, G. and Baudry, M., The biochemistry of memory: a new and specific hypothesis, Science, 224 (1984) 1057-1063. Murachi, T., Calpain and calpastain, Trends Biochem. Sci., 5 (1983) 167-169. Persechini, A., Moncrief, N.D. and Kretsinger, R.H., The EFhand family of calcium-modulated proteins, Trends Neurosci., 12 (1989) 462-467. Peterson, C. and Goldman, J.E., Alterations in calcium content and biochemical processes in cultured skin fibroblasts from aged and Alzheimer donors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2758-2762. Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M. and Horecker, B.L., A dual role for the Ca2+-requiring proteinase in the degradation of hemoglobin by erythrocyte membrane proteinases, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 67146717. Schlaepfer, W.W., Lee, C., Lee, V.M.-Y. and Zimmerman, U.J., An immunoblot study of neurofilament degradation in situ and during calcium-activated proteolysis, J. Neurochem., 44 (1985) 502-509. Schlaepfer, W.W. and Micko, S., Calcium-dependent alterations of neurofilament proteins of rat peripheral nerve, J. Neurochem., 32 (1979) 211-219. Siman, R., Card, J.P., Nelson, R.B. and Davis, L.G., Expression of fl-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. Siman, R., Card, J.P. and Davis, L.G., Proteolytic processing of fl-amyloid precursor by calpain I, J. Neurosci., 10 (1990) 2400-2411. Suzuki, K., Imajoh, S., Emori, Y., Kawasaki, H., Minami, Y. and Ohno, S., Calcium-activated neutral protease and its endogenous inhibitor. Activation at the cell membrane and biological function, FEBS Lett., 220 (1987) 271-277. Terry, R.D., Peck, A., DeTeresa, R., Schechten, R. and Horoupian, D.S., Some morphometric aspects of the brain in senile dementia of the AIzheimer type, Ann. Neurol., 10 (1981) 184-192. Wheelock, M.J., Evidence for two structurally different forms of skeletal muscle Ca2+-activated protease, J. Biol. Chem., 257 (1982) 12471-12474.
177
BRES 24864
Localization of calpain immunoreactivity in senile plaques and in neurones undergoing neurofibrillary degeneration in Alzheimer's disease Norihiko Iwamoto 1, Wipawan Thangnipon 1, Catherine Crawford 2 and Piers C. Emson 1 1MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.) and 2Laboratory of Molecular Biophysics, University of Oxford, Oxford, Oxford (U.K.) (Accepted 2 July 1991)
Key words: Alzheimer-type dementia; Senile plaque; Neurofibrillary tangle; Cytoskeleton; Membrane; Degradation; Calcium activated protease
An antibody raised against the calcium activated neutral protease (calpain) was used to investigate the possible involvement of this enzyme in the formation of plaques and tangles in Alzheimer-type dementia (ATD) brain. Our results revealed the presence of a number of strongly stained calpain positive neurones in the normal human cerebral cortex and a loss of calpain positive cells in ATD brain. Furthermore, double staining experiments revealed that calpain immunoreactivity was present in cells undergoing tangle formation, and was also present in senile plaques. These data suggest that activation of calpain may be an important factor in the abnormal proteolysis underlying the accumulation of plaques and tangles in ATD.
Recently, abnormal proteolysis has been reported in the brains of patients with Alzheimer-type dementia (ATD) 1'6. This aberrant proetolytic processing is thought to result in the accumulation of modified or abnormal fragments of common cellular structural proteins such as the microtubule associated protein tau, neurofilament and the amyloid protein precursor leading to the formation of neurofibrillary tangles 4"18 (NFTs) and the formation of extracellular senile plaques 16. If these two major diagnostic hallmarks of Alzheimer's disease (i.e. plaques and tangles) arise from aberrant proteolytic processing, it is of interest to consider which proteases may be involved. One possible candidate is the calcium protease calpain 11'2°'27"28 which is known to be involved in the degradation of both membrane 2'3'23'2s and cytoskeletal proteins14.24,25, 28. A calcium protease such as calpain is also an attractive candidate for a proteolytic enzyme implicated in ATD as it is activated by elevation of intracellular calcium 3'14'23'25. Indeed, both elevations in intracellular calcium 22 and reductions in intracellular calcium binding proteins 5'13 which may have a Ca 2+ buffering action 1°'21 in the neurone have been reported in ATD. In consequence it was of some interest to examine the distribution of calpain in the normal and ATD brain, and to consider if calpain expression was correlated with the presence of plaques or the occurrence of NFT markers
in damaged neurones. To determine the distribution of calpain, we immunostained sections from the cerebral frontal cortex of 5 pathologically verified ATD patients (age at death 78.3 + 6.7 years S.D.) and 6 control patients with no neuropathological abnormalities (age at death 83.2 - 7.5 years S.D.) with the anti-calpain antiserum. Antibodies against calpain were raised in rabbits. In brief, purified porcine renal calpain II (ref. 7) (200/zg) was emulsified with complete Freund's adjuvant and injected into New Zealand white rabbits, and rabbits were boosted every 4-6 weeks with 100 /~g of calpain II in incomplete Freund's adjuvant. Following the third boost, antiserum was collected and examined by Western blotting (Fig. 1). For immunostaining, 25-/~m-thick sections were cut from the frontal cortex of human brain samples which had been fixed with 10% formalin and cyroprotected with 30% sucrose. The sections were incubated with anticalpain antiserum 1:400 in 0.1 M PBS with 3% bovine serum albumin and 0.2% Triton X-100 for 48 h at 4 °C. After washing, sections were incubated with biotinylated anti-rabbit IgG and then avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Labs). Diaminobenzidine (DAB) was used as the peroxidase substrate to visualize sites of antibody binding. Some sections were also double stained with an anti-tau (z) antibody for identification of N F T 18. Sections were incubated with mouse
Correspondence: P.C. Emson, MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
178 Fig. 1. SDS-PAGE of purified calpain II and Western blots. Lane 1: molecular-mass markers. Lane 2: SDS-PAGE of purified calpain II from porcine kidney. Lane 3,4: Western blots of calpain II immunostained by the rabbit anti-calpain antiserum (lane 3) and normal rabbit serum (lane 4). This anti-calpain antiserum clearly stained the calpain II (lane 3) whereas normal rabbit serum showed no immunostaining (lane 4) (arrow, large subunit of calpain II; arrowhead, small subunit of calpain II).
monoclonal anti-tau antibody (1:5000 diluted, Sigma) at 4 °C overnight. A f t e r washing, the section was treated with a r h o d a m i n e conjugated sheep anti-mouse I g G (Derotec) for 3 h at r o o m temperature. In o r d e r to visualize senile plaques on the same sections were stained with 1% solution of thioflavin S for 10 min 8'17. Characterization of our anti-calpain II antibodies by Western blotting (Fig. 1) revealed that the antiserum contained antibodies recognizing the heavy and light chains of calpain II (Fig. 1, lane 3). In addition, immunological cross-reactivity b e t w e e n calpain I and II was observed as previously r e p o r t e d 9'29. Thus our antiserum should detect all calpain-like immunoreactivity in the brain. O u r antiserum revealed a n u m b e r of strongly stained neurones and glial cells in the n o r m a l human brain (Fig. 2a). This immunostaining pattern was essen-
a
kDa
1
2
3
4
98
67
~t
14
b
Fig. 2. The distribution of calpain immunoreactive cells in frontal cortex of human brain, a: in the normal control brain, calpain immunoreactive cells are observed in all layers except layer I. b: ATD brain shows decreased number of calpain immunoreactive neurones. Only a few positive neurones in layer V remain. Bar = 500 #m. c: high-power micrograph of calpain positive neurones in the normal control brain. The antiserum clearly stains both cell bodies and dendrites. Bar = 100 #m.
179
Fig. 3. Co-existence of calpain immunoreactivity and neurofibrillary tangles (NFF). a: pyramidal neurones with positive calpain immunoreactivity (arrow) and negative immunoreactivity (arrowhead). Bar = 50 gin. b: same section as in (a). NFTs are dearly immunostained by the anti-tau antibody (arrow and arrowhead), c: same section as in (a) and (b). Demonstration of co-expression of calpain and tau immunoreactivity. Calpain immunoreactivity located in a neurone with developing NFF, (arrow), whereas positive calpain-staining is not observed in a dead neurone where the NFT completely fills the cells intracellular space (arrowhead mature NFr).
tially similar to that seen by H a m a k u b o et al. 12 for rat brain, with calpain immunoreactivity concentrated in particular in the larger pyramidal cells (Fig. 2c). In the A T D cases (Fig. 2b) there was a clear loss of the larger calpain containing pyramidal neurones indicating that these cells are damaged in A T D . Further examination of the A T D cases using both anti-calpain and anti-tau antisera revealed that many of the larger calpain containing neurones also contained tau-like immunoreactivity (Fig. 3). Moreover, calpain immunoreactivity was concentrated on the edges of senile plaques (Fig. 4) when the plaques were identified by thioflavin S staining. The data presented here show that amongst the neurones affected in A T D there is a loss of those containing calpain. These calpain positive neurones include populations of the larger neurones which are particularly damaged in A T D 28 and are rich in amyloid precursor (APP) immunoreactivity 27. It was also particularly interesting to note that in the A T D cases, a number of cells with tau positive N F T were also calpain positive and that
Fig. 4. Frontal cortex (layers II-IV) immunostained with anticalpain antiserum showing positive plaques (a). In (b) and (c) confirmation of the coincidence of calpain immunoreactivity (b) is provided by the use of the amyloid marker thioflavin S (c). Note the clustering of strong calpain immunoreactivity around the core of the senile plaque (arrowhead in b). Bar = 20 #m.
these (some 30% of all tau positive cells) were those undergoing N F T formation. Calpain immunoreactivity was present in those cells undergoing NFT formation containing cell cytoplasm and a nucleus but absent in the dead cell 'ghosts' which contain only mature NFTs (Fig. 3c). These data suggest that calpain activation may both be involved in the formation of N F T and in the death of these cells 2°. The demonstration of calpain immunoreactivity associated with the senile plaque (Fig. 4) may also suggest a role for calpain in the formation of the plaque. It is reported that a protease inhibitor 1 and a lysosomal protease 6 have been found associated with senile plaques. This evidence indicates that abnormal protein processing may be occurring in the senile plaque. Since the amyloid protein precursor is a membrane spanning protein 16, its modification in the dying cell may alter its susceptibility to calpain proteolysis 2'3'23 which may produce the
180 accumulation of insoluble amyloid. The calpain immunoreactivity surrounding the plaque may perhaps represent calpain produced by glial cells, which include reactive astrocytes known to be associated with plaques and which play an important role in the formation of the plaque 15'26. However, irrespective of whether the calpain immunoreactivity in the plaque is n e u r o n a l or glial in origin t h e involvement of this enzyme in proteolytic processing of the constituents of both the plaques and tangles strongly suggests that this calcium activated protease is closely involved with the pathogenesis of A T D 14,24,25, 27. Indeed, modification of synaptic m e m b r a n e b o u n d
1 Abraham, C.R., Selkoe, D.J. and Potter, H., Immunoehemicai identification of the serine protease inhibitor a l-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease, Cell, 52 (1988) 487-501. 2 Ahkong, Q.E, Botham, G.M., Woodward, A.W. and Lucy, J.A., Calcium-activated thiolproteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol, Biochem. J., 192 (1980) 829-836. 3 Anderson, D.R., Davis, J.L. and Carraway, K.L., Calciumpromoted changes of the human erythrocyte membrane, involvement of spectrin, transglutaminase, and a membranebound protease, J. Biol. Chem., 252 (1977) 6617-6623. 4 Anderton, B.H., Breinburg, D., Downes, M.J., Green, EJ., Tomlinson, B.E., Ulrich, J., Wood, J.N. and Kahn, J., Monodonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants, Nature, 298 (1982) 84-86. 5 Arai, H., Emson, EC., Mountjoy, C.Q., Carassco, L.H. and Heizmann, C.W., Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia, Brain Research, 418 (1987) 164-169. 6 Cataldo, A.M., Thayer, C.Y., Bird, E.D., Wheelock, T.R. and Nixon, R.A., Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin, Brain Research, 513 (1990) 181-192. 7 Crawford, C., Brown, N.R. and Willis, A.C., Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate, Biochem. J., 265 (1990) 575579. 8 Dawbarn, D. and Emson, EC., Neuropeptide Y-like immunoreactivity in neuritic plaques of Alzheimer's disease, Biochem. Biophys. Res. Commun., 126 (1985)289-294. 9 Dayton, W.R., Scholimeyer, J.D., Lepley, R.A. and Cort6s, L.R., A calcium-activated protease possibly involved in myofibrillar protein turnover, isolation of a low-calcium requiring form of the protease, Biochem. Biophys. Acta, 659 (1981) 4861. 10 Feher, J.J., Measurement of facilitated calcium diffusion by a soluble calcium-binding protein, Biochem. Biophys. Acta, 773 (1984) 91-98. 11 Guroff, G., A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem., 239 (1964) 149155. 12 Hamakubo, T., Kannagi, R., Murachi, T. and Matus, A., Distribution of calpain I and II in rat brain, J. Neurosci., 6 (1986) 3103-3111. 13 Ichimiya, Y., Emson, EC., Mountjoy, C.Q., Lawson, D.E.M. and Heizmann, C.W., Loss of calbindin-28K immunoreactive neurones from the cortex in AIzheimer-type dementia, Brain Research, 475 (1988) 156-159. 14 Ishizaki, Y., Tashiro, T. and Kurokawa, M., A calcium-activated protease which preferentially degrades the 160-kDa component of the neurofilament triplet, Eur. J. Biochem., 131 (1983) 41-45. 15 Iwamoto, N., Suzuki, Y., Makino, Y., Haga, C., Kosaka, K. and Iizuka, R., Cell membrane changes in brains manifesting
proteins by activated calpain may also contribute to the memory disturbance characteristic of A T D 19 by influencing intraceUular signalling.
We are grateful to Mr. T. Buss for photographic work, Mrs. B.A. Oakley for helping to prepare the manuscript and Mr. R. Hills (MRC Brain Bank, Department of Psychiatry, University of Cambridge) for preparation of brain samples. N.I. and W.T. are visiting research fellows from the Juntendo University in Japan and the Mahidol University in Thailand respectively. W.T. acknowledges the support of the Hereditary Disease Association and the Sandoz Foundation for Gerontological Research for this study. We are also indebted to Tropon-Bayer (Cologne) for their continued support.
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