0361-9230/9fl$3.00
Brain Research Bulletin. Vol. 24, pp. 543-549. 63Pergmon Press plc, 1990. Printed in the U.S.A.
+ .OO
Antigenic Expression of Cathepsin B in Aged Human Brain HANS-GERT BERNSTEIN,*’ HEIDRUN KIRSCHKE,? BERND WIEDERANDERS,t DIETMAR SCHMIDT$ AND AR1 RINNE$
*Institute of Anatomy, Medical Academy ~agdeb~rg, GDR ~~~tit~te of Biochemist, martin Luther universes Ha&e, CDR $District Hospital for Psychiatry, Brandenburg, GDR and $Institute of Pathology, University Tromsii, Norway Received 8 August 1989
H.-G., H. KIRSCHKE, B. WIBDERANDERS, D. SCHh%IDTAND A. RINSE. Antigenie expression ofcuthepsin B 1990.-The lysosomal thiol proteinase, cathepsin B, has been localizedin different regions of aged human brain by use of the peroxidase-antiperoxidase technique. Cathepsin B-immunoreactive material was detected in multiple neurons of human hippocampus, neocottical area A 10, prefrontal gyms and nut. basalis of Meynert as well as in single white matter astrocytes. In brains of Alzheimer disease-affected subjects cathepsin B was revealed in neuritic plaques too. Possible functional consequences with regard to normal aging, neuropeptide metabolism and pathological changes are discussed.
BERNSTEIN,
in aged human bruin. BRAIN RES BULL 24(4) 543-549,
Cathepsin B
Human brain
Aging
Alzheimer disease
hnmunohistochemistry
are accumulated at high concentrations in brains of Alzheimer patients (21). Keeping these findings in mind we have decided to study the dis~bution of one of the thiol proteinases, cathepsin B (E.C.3.4.22.1) in aged human brain in order to learn more about a possible function of this proteinase in nervous tissue. Furthermore, data are presented herein that show an appearance of cathepsin B-like material in lysosome-like vesicles accumulating in neuritic plaques found in the brains of Alzheimer patients.
THOUGH lysosome-associated acid proteinases (cathepsins) are known to be abundantly present in nervous tissue (Z-4, 14, 15) their functions relevance is not well und~t~ at present. There is, of course, convincing evidence for a paramount significance of cathepsins in the general protein turnover of the brain [for review, see (15)]. Besides this, biochemical data have been published in support of the idea that cathepsins are capable of splitting certain neuropeptides and/or their biologically less active precursors (2,14), thereby probably contributing to the neuropeptide metabolism of the brain. However, there is as yet no good neuroanatomical evidence in favor of a mo~ho~ction~ connection between the localization of cathepsins and the occurrence of peptides in neural structures (6,7). During past years some attention has been paid to brain cathepsins in relation to different neuropathological alterations, including irregular degradation of neurocytoskeieton proteins, myelin constituents and involvement of glial cells in epilepsy (4, 5, 13). Recently, a crucial role of certain cathepsins (namely thiol proteinases cathepsin B, H and L) in the process of aging of neuronal structures has been proposed (11). According to this interesting hypothesis, there occurs a decrease in thiol-protease activity due to the accumulation of enzymatically inactive (damaged?) molecules of thiol proteinases in nerve cells with age. This process is nought to result in a deposition of the aging pigment lipofuscin, dolichols (long-chain alcohols), neurofibrillary tangles and, finally, selective cell death. Moreover, recent investigations have demonstrated that dolichols
METHOD
This investigation is based on 18 human brains. Eleven of them were from persons aged between 55 and 84 years (mean age: 72 years). These human beings died from several diseases and their brains did not show neuropathological lesions. Two elderly persons without clinical signs of dementia, but who, on histological exa~nation, showed neuritic plaques in the neocortex, were excluded from furti&examination. Seven brains were taken from Alzheimer disease (AD) subjects. All AD subjects were clinically demented, and neuropathological examination (largely Bielschowsky silver staining technique) revealed both neuritic plaques and neurofibriilary tangles in neocortical areas and hippocampal formation. Ad~tion~ly, massive neuronal loss in the nut. basalis of Meynert was found in 5 of 7 patients using Nissl staining. Tissue pieces from parahippocampal gyrus, cortical area A 10, prefrontal gyms, and nut. basalis of Meynert were taken at
‘Requestsfor reprints should be addressedto H.-G. Bernstein, Institute of Neu~biolo~ and Brain Research,Acad. Sci., LeipzigerSu. 44, DDR-3O$N.l Magdeburg, GDR.
543
544
FIG. 1, Rocket monospecificity
BERNSTEIN
immunoelectrophoresis of the antiserum.
of anticathepsin
B
demonstrating
autopsy not later than 12 hr after the death, fixed in formalin and embedded in paraffin. The material was cut at 6 km and processed either for histological examination (Nissl) or for immunocytochemistry. In order to identify cathepsin B protein immunohistochemically the peroxidase-antiperoxidase technique (17) was used. The cathepsin B antiserum was produced in New Zealand rabbits against cathepsin B purified to homogeneity from human spleen. The monospecificity of antiserum was demonstrated by rocket immunoelectrophoresis (running anticathepsin B, diluted 1: 10000 against pure antigen, Fig. 1) and Western blot analysis. Immunoblotting Immunoblots
were prepared from human brain extracts (tem-
ET AL.
poral cortex) electrophoresed in 12% SDS-polyacrylamide gels. The electrotransfer to nitrocellulose was performed according to Towbin (20). The protein transfers were blocked in 10% nonfat dry milk for 1 hr before application of the primary antiserum (anti-human cathepsin B, diluted 1:2000 and I :4000). After incubation with anticathepsin B overnight at 4”C, the blots were rinsed and incubated with swine-anti-rabbit IgG (diluted 1: 150) (DAKO). After this procedure the blots were rinsed again and incubated with the soluble peroxidase-antiperoxidase complex (diluted 1: 100); 3,3’diaminobenzidine was used as chromogen. Molecular weights of the blotted proteins were calculated by transferred prestained markers (Sigma), Fig. 10. The experimental protocol for the immunohistochemical detection of cathepsin B was largely the same as described earlier (6). The primary antibody (rabbit-anti-human cathepsin B, final working dilution 1:200) was applied to the sections in a moist chamber for 12 hr at 4°C. The secondary antibody (swine-anti-rabbit IgG, Dakopatts, Denmark) and the soluble PAP-complex (Dakopatts) both diluted 150, were applied at room temperature. All dilution and rinsing procedures between the incubation steps were carried out in phosphate buffered saline (pH 7.4). The peroxidase activity was visualized with 3.3’-diaminobenzidine. Human liver sections providing a rich source of cathepsin B served as positive controls. In negative control experiments, the primary antibody was either replaced by buffer, by preimmune rabbit serum and by antiserum previously preabsorbed with pure antigen (Fig. 11). The sections were investigated under a light microscope (Leitz).
RESULTS
Cathepsin B-immunoreactive material was found to be widely distributed within the central nervous system of elderly persons. Its prevailing location was intraneuronally. Large pyramidal neurons of layers III and V of human neocortex stood out with a strong intracellular immunoreaction, whereas medium-sized intemeurons, small pyramids, and granular cells were less heavily stained (Figs. 2, 3 and 4). Concerning hippocampal formation it must be
FIG. 2. Immunoreactive pyramidal cells and medium-sized neurons in layer III of human cortical area A 10. Magnification ca. 80 X
CATHFPSIN B IN Ham
BRAlN
emphasized that cathepsin B immunoactivity showed a remarkable distribution. Most nerve cells of the hippocampal subfields CA,CA, were only weakly immunoreactive. However, single nerve cells situated in the band of pyramidal neurons (basket celis?) were intensely stained {Fig. 5). The same was true for the subictkm. Sohtary neurons (Fig. 6) expressed strong i~u~o~activi~, whereas most nerve cells did not show significant immunostaining. Hippocampal granular cells were found to be moderately stained. Sometimes, immunostaining was lacking at all in representatives of this cell type. Neurocytes belonging to the nut.
basalis of Meynert showed a weak immunoreaction to cathepsin B antibody (Fig. 8). Though it is difficult to calculate what percentage of neurons is really cathepsin B-positive, it must be emphasized that by far not ah nerve cells were found to express the antigen. Besides neurons, cathepsin B-i~u~o~ctive material was identified in capillary endothelium of blood vessels, pericytes, and, occassionally, in white matter astrocytes (Fig. 7). Brain tissue of AD patients showed a distribution of cathepsin B ?iwhich was very similar to that described above for “normal”
FIG. 4. Single large-sized neuron located in layer V of area A 10 with apparent immunoreaction. Magnification ea. 297 x
FTO. 6. Single subicular nerve cell with intense immunostaining to catkpsin B antiserum. Magnification ca. 297 x
546
Ht:RNSTt;Jti
FIG. 7. White matter astrocyt~s
irhmunoveactive
aged brain. There were, however. some peculiarities to be mentioned here. First, due to the apparent loss of neurons that occurs in the nut. basalis of Meynert during dementia ( 1) , we were unable to reveal enzyme-positive neurocytes in this brain area. Second, and more remarkably, there was an accumulation of strongly cathepsin B-immunoreactive granules (seemingly lysosomes or lysosome-like vesicular bodies) in multiple netuitic plaques (Fig. 9). interestingly enough, areas with densely packed neuritic plaques did nearly not contain cathepsin B-positive
FIG. 8. Moderately reacting Magnification ca 300 x
fur
cathepsin
B. Magnfication
ET 41.
ca 250 x
neurocytes. often. cathepsin B-positive plaques were identified in close vicinity of small blood vessels. DISCLSSION
Howie and co-workers were the first to show that catbepsin B is immunohistochemically demonstrable in human brain nerve cells (10). The purpose of the present study was to get a more detailed information about the regional distribution and cellular
neurocytes in the nut. basalis of Meynen of a nondemented person.
CATHEPSIN
B IN HUMAN BRAIN
547
FIG. 9. Multiple neuritic plaques in the neocortexof a dementedperson. Note the granular nature of the
reaction product (lysosomes?).Magni~cation ca. 200 x
localization of the enzyme in the aged human CNS. As it has repeatedly been shown for the nonprimate brain (5-8) and the developing human brain (9), the main site of the antigenic expression of cathepsin B was the neuron. Our findings demonstrate the enzyme to be unevenly distributed throughout nerve cells of different areas of human brain. The hippocampal formation is an especially interesting brain area in this respect since the immunoreaction was confined to very few neurons. In search of a functional interpretation of our data we
FIG. 10. SDS electrophoresis of human brain extract and immunoblotting of cathepsin B, Proteins resolved on lane a transblotted on nitmcelluiose ~rnb~e and revealed by ~tica~epsin B, diluted 1:ZOOOand 1:4OW (lanes b and c). The molecular weights were calculated by standard proteins run in parallel. Besides a main fraction of about 30,000 D (human cerebral cathepsin B proper, I) three bands of lower molecular weight were found. Under reducing conditions there might be: the 25,000 D variant (II) the heavy chain (about 6000 D, III) and the light chain (about 4,000 D, IV). It coot, however, be excluded that the antiserum also recognizes some unknown component.
compared shape and location of strongly immunoreactive hippocampal cells with findings published in the literature about the distribution of various neurotransmitter and peptide-containing neurocytes in this region. Surprisingly enough, the distribution pattern of the thiol proteinase resembles that of e~eph~iner~c neural elements as recentiy described by Kulmala (12). Though there is some biochemical and pharmacological evidence that thiol proteinases are able to split endogenous opiates and other neuropeptides (2, 14, 18) our finding must be regarded with great caution. First, nobody has conclusively shown that cathepsins are really involved in the in vivo metabolism of peptides occurring in the nervous tissue. Second, due to some delay between the death of the patients and our preparation procedure it cannot be excluded with ultimate certainty that the antiserum recognizes some unknown antigen (for example, cleavage products of a protein) that accidently accumulates in putatively peptide-containing neurons (see also results of Western blot). Concerning the postulated role of cysteine proteinases (and especially of cathepsin B) in normal brain aging (1 l), we feel that our findings provide only little new information to substantiate this hypothesis. Cathepsin B was found in multiple neocortical and some hippocampal neurons. Both neocortex and hippocampus are known to be seriously affected by alterations taking place during aging. It is, however, noteworthy to emphasize that our i~unohistochemic~ approach does not permit us to draw any conclusions with regard to the catalytic activity of cathepsin B in i~unost~n~ neurons, since our antibody recognizes the protein body only. Moreover, the aging pigment lipofuscin was found to be present also in those neurons, which were only weakly immunostained, or lacked an immunoreaction at all (data are not shown). A third interesting aspect of our work is the presence of cathepsin B-i~~o~active material in neuritic plaques. There is evidence for the involvement of an aspartic proteinase, cathepsin D in the formation of this characteristic hallmark bf Alzheimer disease (9,19). A possible mechanism by which cathepsin B might contribute to the growth of neuritic plaques has been proposed by Sahenk and Lasek (16), who conclusively showed that selective blockers of thiol proteinases disrupt the regular anterograde-
548
BERNSTEIN
ET AL.
FIG. 11. Human brain section prepared from an Alzheimer patient with identified neuritic plaques at consecutive sections. The section was exposed to anticathepsin B previously preabsorbed with pure antigen. Please note that there is only a very faint (unspecific) staining of some neurons. whereas no plaques are stained. Magnification ca. 1: 150.
retrograde conversion of membranous organelles in axons. It is imaginable that such an event happens when a plaque starts to grow. Perhaps, the naturally occurring inhibitors of cysteine proteinases (cystatins) play a role in this process. We have
preliminary evidence in favor of the occurrence of cystatin C in some plaques. Further investigations at the fine structural level are needed to better understand the role of cysteine proteinases during aging.
REFERENCES 1. Arendt, T.; Bigl, V.; Arendt, A.; Tennstedt, A. Loss of neurons in the nucleus basalis of Meynert in Alzheimer’s disease, paralysis agitans and Korsakoff’s disease. Acta Neuropathol. (Berl.) 61:101-108; 1983. 2. Azaryan, N.; Barkhudaryan, N.; Galoyan, A. Some properties of human and bovine brain cathepsin B. Neurochem. Res. lo:151 l1524; 1985. 3. Barrett, A. J.; Kirschke, H. Cathepsin B, cathepsin H and cathepsin L. In: Lorand, L., ed. Methods in enzymology. New York: Academic Press; 80:525-561. 4. Berlet, H. H.; Ilzehiifer, H. Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein. FEBS Lett. 179:299-302; 1985. B.; 5. Bernstein, H.-G.; Keilhoff, G.; Kirschke, H.; Wiederanders, Rinne, A.; Khudoerkov, R.; Dom, A. Cathepsins B and D in rat brain glia during experimentally induced neuropathological defects. An immunocytochemical approach. Biomed. Biochim. Acta 45:14611464; 1986. 6. Bernstein, H.-G.; Kirschke, H.; Wiederanders, B.; Kloss, P.; Rinne, A.; Dom, A. Cathepsin B immunoreactivity is widely distributed in the rat brain. J. Hirnforsch. 29:17-19; 1988. 7. Bernstein, H.-G.; Reichenbach, A.; Kirschke, H.; Wiederanders, B. Cell-type-specific distribution of cathepsin B and D immunoreactivity within the rabbit brain. Neurosci. Lett. 98:135-138; 1989. 8. Bernstein, H.-G.; Kirschke, H.; Klog, P.; Wiederanders, B .; Rinne, A.; Ftihlich, H. Cathepsin B during early human brain development. Acta Histochem., in press; 1989. 9. Bernstein, H.-G.; Bruszis, S.; Schmidt, S.; Wiederanders, B.; Dom, A. Immunodetection of cathepsin D in neuritic plaques found in brains of patients with dementia of Alzheimer type. J. Himforsch. 30: 613-618; 1989.
10. Howie, A. J.; Burnett, D.; Cracker,
Il.
12.
13.
14.
15. 16.
17. 18.
19.
20.
J. The distribution of cathepsin B in human tissues. J. Pathol. 145:307-314; 1985. Ivy, G. 0. A proteinase inhibitor model of aging: Implications for decreased neuronal plasticity. In: Neuroplasticity, learning and memory. New York Alan R. Liss Inc.; 1987:125-150. Kulmala, H. K. Some enkephalin-or VIP immunoreactive hippocampal pyramidal cells contain neurofibrillary tangles in the brains of aged human and persons with Alzheimer’s disease. Neurochem. Pathol. 3:41-51; 1985. Marks, N.; Grynbaum, A.; Lajtha, A. The breakdown of myelinbound proteins by intra- and extracellular proteases. Neurochem. Res. 1:93-111; 1976. Marks, N.; Berg, M. J.; Benuck, M. Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides. Arch. Biochem. Biophys. 249:489-499; 1986. Pope, A.; Nixon, R. A. Proteases of human brain. Neurochem. Res. 9:291-323; 1984. Sahenk, 2.; Lasek, R. J. Inhibition of proteolysis blocks anterograderetrograde conversion of axonally transported vesicles. Brain Res. 460:199-203; 1988. Stemberger, L. A. Immunocytochemistry 2nd ed. Englewood Cliffs, NJ: Prentice-Hall; 1979. Suhar, A.; Marks, N. Purification and properties of brain cathepsin B. Evidence for cleavage of pituitary lipotropin. Eur. J. Biochem. 101:23-30; 1979. Suzuki, D. H.; Takeda, M.; Kato, Y.; Yamashita, T.; Tada, K.; Hariguchi, S.; Nishimura, T. Cathepsin D activities in dementia brains and the degradation of neurofiIament proteins with cathepsin D. Neurochem. Res. 13:264; 1988 (abstract). Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitro-cellulose sheets. Procedure
CATHEPSIN
B IN HUMAN BRAIN
and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4353; 1979. 21. Wolfe, S. L.; Ng Ying Kin, N. M. K.; Palo, J.; Bergeron, C.; Kotila,
549
M.; Varonen, S. Dolichols are elevated in brain tissue from Alzheimer disease, but not in urinary sediment from Alzheimer disease and Down’s syndrome. Neorochem. Pathol. 3:213-221; 1985.
Brain Research Bulletin. Vol. 24, pp. 543-549. 63Pergmon Press plc, 1990. Printed in the U.S.A.
+ .OO
Antigenic Expression of Cathepsin B in Aged Human Brain HANS-GERT BERNSTEIN,*’ HEIDRUN KIRSCHKE,? BERND WIEDERANDERS,t DIETMAR SCHMIDT$ AND AR1 RINNE$
*Institute of Anatomy, Medical Academy ~agdeb~rg, GDR ~~~tit~te of Biochemist, martin Luther universes Ha&e, CDR $District Hospital for Psychiatry, Brandenburg, GDR and $Institute of Pathology, University Tromsii, Norway Received 8 August 1989
H.-G., H. KIRSCHKE, B. WIBDERANDERS, D. SCHh%IDTAND A. RINSE. Antigenie expression ofcuthepsin B 1990.-The lysosomal thiol proteinase, cathepsin B, has been localizedin different regions of aged human brain by use of the peroxidase-antiperoxidase technique. Cathepsin B-immunoreactive material was detected in multiple neurons of human hippocampus, neocottical area A 10, prefrontal gyms and nut. basalis of Meynert as well as in single white matter astrocytes. In brains of Alzheimer disease-affected subjects cathepsin B was revealed in neuritic plaques too. Possible functional consequences with regard to normal aging, neuropeptide metabolism and pathological changes are discussed.
BERNSTEIN,
in aged human bruin. BRAIN RES BULL 24(4) 543-549,
Cathepsin B
Human brain
Aging
Alzheimer disease
hnmunohistochemistry
are accumulated at high concentrations in brains of Alzheimer patients (21). Keeping these findings in mind we have decided to study the dis~bution of one of the thiol proteinases, cathepsin B (E.C.3.4.22.1) in aged human brain in order to learn more about a possible function of this proteinase in nervous tissue. Furthermore, data are presented herein that show an appearance of cathepsin B-like material in lysosome-like vesicles accumulating in neuritic plaques found in the brains of Alzheimer patients.
THOUGH lysosome-associated acid proteinases (cathepsins) are known to be abundantly present in nervous tissue (Z-4, 14, 15) their functions relevance is not well und~t~ at present. There is, of course, convincing evidence for a paramount significance of cathepsins in the general protein turnover of the brain [for review, see (15)]. Besides this, biochemical data have been published in support of the idea that cathepsins are capable of splitting certain neuropeptides and/or their biologically less active precursors (2,14), thereby probably contributing to the neuropeptide metabolism of the brain. However, there is as yet no good neuroanatomical evidence in favor of a mo~ho~ction~ connection between the localization of cathepsins and the occurrence of peptides in neural structures (6,7). During past years some attention has been paid to brain cathepsins in relation to different neuropathological alterations, including irregular degradation of neurocytoskeieton proteins, myelin constituents and involvement of glial cells in epilepsy (4, 5, 13). Recently, a crucial role of certain cathepsins (namely thiol proteinases cathepsin B, H and L) in the process of aging of neuronal structures has been proposed (11). According to this interesting hypothesis, there occurs a decrease in thiol-protease activity due to the accumulation of enzymatically inactive (damaged?) molecules of thiol proteinases in nerve cells with age. This process is nought to result in a deposition of the aging pigment lipofuscin, dolichols (long-chain alcohols), neurofibrillary tangles and, finally, selective cell death. Moreover, recent investigations have demonstrated that dolichols
METHOD
This investigation is based on 18 human brains. Eleven of them were from persons aged between 55 and 84 years (mean age: 72 years). These human beings died from several diseases and their brains did not show neuropathological lesions. Two elderly persons without clinical signs of dementia, but who, on histological exa~nation, showed neuritic plaques in the neocortex, were excluded from furti&examination. Seven brains were taken from Alzheimer disease (AD) subjects. All AD subjects were clinically demented, and neuropathological examination (largely Bielschowsky silver staining technique) revealed both neuritic plaques and neurofibriilary tangles in neocortical areas and hippocampal formation. Ad~tion~ly, massive neuronal loss in the nut. basalis of Meynert was found in 5 of 7 patients using Nissl staining. Tissue pieces from parahippocampal gyrus, cortical area A 10, prefrontal gyms, and nut. basalis of Meynert were taken at
‘Requestsfor reprints should be addressedto H.-G. Bernstein, Institute of Neu~biolo~ and Brain Research,Acad. Sci., LeipzigerSu. 44, DDR-3O$N.l Magdeburg, GDR.
543
544
FIG. 1, Rocket monospecificity
BERNSTEIN
immunoelectrophoresis of the antiserum.
of anticathepsin
B
demonstrating
autopsy not later than 12 hr after the death, fixed in formalin and embedded in paraffin. The material was cut at 6 km and processed either for histological examination (Nissl) or for immunocytochemistry. In order to identify cathepsin B protein immunohistochemically the peroxidase-antiperoxidase technique (17) was used. The cathepsin B antiserum was produced in New Zealand rabbits against cathepsin B purified to homogeneity from human spleen. The monospecificity of antiserum was demonstrated by rocket immunoelectrophoresis (running anticathepsin B, diluted 1: 10000 against pure antigen, Fig. 1) and Western blot analysis. Immunoblotting Immunoblots
were prepared from human brain extracts (tem-
ET AL.
poral cortex) electrophoresed in 12% SDS-polyacrylamide gels. The electrotransfer to nitrocellulose was performed according to Towbin (20). The protein transfers were blocked in 10% nonfat dry milk for 1 hr before application of the primary antiserum (anti-human cathepsin B, diluted 1:2000 and I :4000). After incubation with anticathepsin B overnight at 4”C, the blots were rinsed and incubated with swine-anti-rabbit IgG (diluted 1: 150) (DAKO). After this procedure the blots were rinsed again and incubated with the soluble peroxidase-antiperoxidase complex (diluted 1: 100); 3,3’diaminobenzidine was used as chromogen. Molecular weights of the blotted proteins were calculated by transferred prestained markers (Sigma), Fig. 10. The experimental protocol for the immunohistochemical detection of cathepsin B was largely the same as described earlier (6). The primary antibody (rabbit-anti-human cathepsin B, final working dilution 1:200) was applied to the sections in a moist chamber for 12 hr at 4°C. The secondary antibody (swine-anti-rabbit IgG, Dakopatts, Denmark) and the soluble PAP-complex (Dakopatts) both diluted 150, were applied at room temperature. All dilution and rinsing procedures between the incubation steps were carried out in phosphate buffered saline (pH 7.4). The peroxidase activity was visualized with 3.3’-diaminobenzidine. Human liver sections providing a rich source of cathepsin B served as positive controls. In negative control experiments, the primary antibody was either replaced by buffer, by preimmune rabbit serum and by antiserum previously preabsorbed with pure antigen (Fig. 11). The sections were investigated under a light microscope (Leitz).
RESULTS
Cathepsin B-immunoreactive material was found to be widely distributed within the central nervous system of elderly persons. Its prevailing location was intraneuronally. Large pyramidal neurons of layers III and V of human neocortex stood out with a strong intracellular immunoreaction, whereas medium-sized intemeurons, small pyramids, and granular cells were less heavily stained (Figs. 2, 3 and 4). Concerning hippocampal formation it must be
FIG. 2. Immunoreactive pyramidal cells and medium-sized neurons in layer III of human cortical area A 10. Magnification ca. 80 X
CATHFPSIN B IN Ham
BRAlN
emphasized that cathepsin B immunoactivity showed a remarkable distribution. Most nerve cells of the hippocampal subfields CA,CA, were only weakly immunoreactive. However, single nerve cells situated in the band of pyramidal neurons (basket celis?) were intensely stained {Fig. 5). The same was true for the subictkm. Sohtary neurons (Fig. 6) expressed strong i~u~o~activi~, whereas most nerve cells did not show significant immunostaining. Hippocampal granular cells were found to be moderately stained. Sometimes, immunostaining was lacking at all in representatives of this cell type. Neurocytes belonging to the nut.
basalis of Meynert showed a weak immunoreaction to cathepsin B antibody (Fig. 8). Though it is difficult to calculate what percentage of neurons is really cathepsin B-positive, it must be emphasized that by far not ah nerve cells were found to express the antigen. Besides neurons, cathepsin B-i~u~o~ctive material was identified in capillary endothelium of blood vessels, pericytes, and, occassionally, in white matter astrocytes (Fig. 7). Brain tissue of AD patients showed a distribution of cathepsin B ?iwhich was very similar to that described above for “normal”
FIG. 4. Single large-sized neuron located in layer V of area A 10 with apparent immunoreaction. Magnification ea. 297 x
FTO. 6. Single subicular nerve cell with intense immunostaining to catkpsin B antiserum. Magnification ca. 297 x
546
Ht:RNSTt;Jti
FIG. 7. White matter astrocyt~s
irhmunoveactive
aged brain. There were, however. some peculiarities to be mentioned here. First, due to the apparent loss of neurons that occurs in the nut. basalis of Meynert during dementia ( 1) , we were unable to reveal enzyme-positive neurocytes in this brain area. Second, and more remarkably, there was an accumulation of strongly cathepsin B-immunoreactive granules (seemingly lysosomes or lysosome-like vesicular bodies) in multiple netuitic plaques (Fig. 9). interestingly enough, areas with densely packed neuritic plaques did nearly not contain cathepsin B-positive
FIG. 8. Moderately reacting Magnification ca 300 x
fur
cathepsin
B. Magnfication
ET 41.
ca 250 x
neurocytes. often. cathepsin B-positive plaques were identified in close vicinity of small blood vessels. DISCLSSION
Howie and co-workers were the first to show that catbepsin B is immunohistochemically demonstrable in human brain nerve cells (10). The purpose of the present study was to get a more detailed information about the regional distribution and cellular
neurocytes in the nut. basalis of Meynen of a nondemented person.
CATHEPSIN
B IN HUMAN BRAIN
547
FIG. 9. Multiple neuritic plaques in the neocortexof a dementedperson. Note the granular nature of the
reaction product (lysosomes?).Magni~cation ca. 200 x
localization of the enzyme in the aged human CNS. As it has repeatedly been shown for the nonprimate brain (5-8) and the developing human brain (9), the main site of the antigenic expression of cathepsin B was the neuron. Our findings demonstrate the enzyme to be unevenly distributed throughout nerve cells of different areas of human brain. The hippocampal formation is an especially interesting brain area in this respect since the immunoreaction was confined to very few neurons. In search of a functional interpretation of our data we
FIG. 10. SDS electrophoresis of human brain extract and immunoblotting of cathepsin B, Proteins resolved on lane a transblotted on nitmcelluiose ~rnb~e and revealed by ~tica~epsin B, diluted 1:ZOOOand 1:4OW (lanes b and c). The molecular weights were calculated by standard proteins run in parallel. Besides a main fraction of about 30,000 D (human cerebral cathepsin B proper, I) three bands of lower molecular weight were found. Under reducing conditions there might be: the 25,000 D variant (II) the heavy chain (about 6000 D, III) and the light chain (about 4,000 D, IV). It coot, however, be excluded that the antiserum also recognizes some unknown component.
compared shape and location of strongly immunoreactive hippocampal cells with findings published in the literature about the distribution of various neurotransmitter and peptide-containing neurocytes in this region. Surprisingly enough, the distribution pattern of the thiol proteinase resembles that of e~eph~iner~c neural elements as recentiy described by Kulmala (12). Though there is some biochemical and pharmacological evidence that thiol proteinases are able to split endogenous opiates and other neuropeptides (2, 14, 18) our finding must be regarded with great caution. First, nobody has conclusively shown that cathepsins are really involved in the in vivo metabolism of peptides occurring in the nervous tissue. Second, due to some delay between the death of the patients and our preparation procedure it cannot be excluded with ultimate certainty that the antiserum recognizes some unknown antigen (for example, cleavage products of a protein) that accidently accumulates in putatively peptide-containing neurons (see also results of Western blot). Concerning the postulated role of cysteine proteinases (and especially of cathepsin B) in normal brain aging (1 l), we feel that our findings provide only little new information to substantiate this hypothesis. Cathepsin B was found in multiple neocortical and some hippocampal neurons. Both neocortex and hippocampus are known to be seriously affected by alterations taking place during aging. It is, however, noteworthy to emphasize that our i~unohistochemic~ approach does not permit us to draw any conclusions with regard to the catalytic activity of cathepsin B in i~unost~n~ neurons, since our antibody recognizes the protein body only. Moreover, the aging pigment lipofuscin was found to be present also in those neurons, which were only weakly immunostained, or lacked an immunoreaction at all (data are not shown). A third interesting aspect of our work is the presence of cathepsin B-i~~o~active material in neuritic plaques. There is evidence for the involvement of an aspartic proteinase, cathepsin D in the formation of this characteristic hallmark bf Alzheimer disease (9,19). A possible mechanism by which cathepsin B might contribute to the growth of neuritic plaques has been proposed by Sahenk and Lasek (16), who conclusively showed that selective blockers of thiol proteinases disrupt the regular anterograde-
548
BERNSTEIN
ET AL.
FIG. 11. Human brain section prepared from an Alzheimer patient with identified neuritic plaques at consecutive sections. The section was exposed to anticathepsin B previously preabsorbed with pure antigen. Please note that there is only a very faint (unspecific) staining of some neurons. whereas no plaques are stained. Magnification ca. 1: 150.
retrograde conversion of membranous organelles in axons. It is imaginable that such an event happens when a plaque starts to grow. Perhaps, the naturally occurring inhibitors of cysteine proteinases (cystatins) play a role in this process. We have
preliminary evidence in favor of the occurrence of cystatin C in some plaques. Further investigations at the fine structural level are needed to better understand the role of cysteine proteinases during aging.
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