318
Brain Research. 537 (1990) 318-322 Elsevier
BRES 24451
Amyloid I~/A4 protein precursor is bound to neuroflbrillery ~ Alzh~mer-type dementia
in
Haruyasu Yamaguchi 1'2, Koji Ishiguro 2, Mikio Shoji 2, Tsuneo Yamazaki 2, Yoichi Nakazato 3, Yasuo Ihara 4 and Shunsaku Hirai: 1College of Medical Care and Technology; and Departments of 2Neurology and ~Pathology, School of Medicine; Gunma University, Maebashi, Gunma 371 (Japan) 4Tokyo MetropolitanInstitute of Gerontology, Tokyo (Japan) (Accepted 18 September 1990)
Key words: Amyloid fl/A4 protein precursor; Tau protein; Alzheimer-typedementia; Neurofibriilary tangle; Immunoelectron microscopy; Paired helical filament
Localization of amyloid 13/A4protein precursor (APP) in Alzheimer's neurofibrillary tangles (NFT) was examined immunohistochcmically. Antiserum directed to N-terminal of APP intensely labeled intracellular NFT and some neuropil threads. The NFr, extracted from ~heimer brains by detergent treatments, were also immunoreactivewith this antiserum. Antisera to other parts of APP labeled NFI" after the formic acid pretreatment. However, Western blot analysis of NFT demonstrated no immunoreactivebands with APP antiserum. These findingssuggest that APP is a minor component of the NFT. In the brains of Alzheimer-type dementia, major histopathological hallmarks are senile plaques and neurofibrillary tangles (NFT). The latter consist of both paired helical filaments (PHF) and straight filaments (SF) 24. Recent studies revealed that the major component of senile plaque amyloid is derived from the larger amyloid fl/A4 protein precursor (APP) 9. Some immunohistochemical studies, demonstrating positive labeling of NFT with fl/A4 protein antisera 1'6'12'2°, raise a question whether fl/A4 protein is an essential 'core' component of PHF. In the present study, we demonstrated that APP is a minor component of the NF-I', and that this can explain the positive labeling of intraneuronal NFT with some fl/A4 antisera. Labeling of the extracellular NFT with fl/A4 antiserum will be discussed elsewhere 25. First, we examined autopsy brains from 3 patients with Alzheimer-type dementia (65, 81, and 82 years old), and from 4 control patients (59, 67, 74, and 82 years old) with no neurological diseases. These brains were removed within 2 h after death. Frontal and temporal specimens were fixed in a 4% paraformaldehyde and 0.3% picric acid solution for 6-12 h and sectioned using cryostat. Some specimens were fixed in ethanol-polyethylene glycol mixture (Kryofix, Merck, E R . G . ) for 12-24 h and embedded in paraffin. Sections, 2-8 /~m thick, were pretreated with 2% Triton X-100 or 75% formic acid and then immunostained with antisera to synthetic peptides of
(1) APP18_38 (N-terminal of APP, W63N) 9A9, (2) APP577_ 596 (pre-fl/A4 protein region, Z31pre), (3) fl/A4 protein 1_ 28 (amyloidogenic internal portion of APP, APPs97_624, fl 28K) 23, (4) APP666_695 (C-terminal of APP, W61C)191 and (5) microtubule associated protein tau320__329(tau-C6) 1°, and ABC kits (Vector Lab, CA). Two APP antisera, W63N and W61C, have been reported to recognize 106-135 kDa APP of human brain extract by immunoblot analysis 19. The Z31pre also recognized the same APP bands in Western blot analysis (data not shown), while fl 28K did not. Some sections were counterstained with Congo red. Control sections were reacted with preabsorbed antisera 19. In the control brains, both W63N and W61C principally immunostained almost all neurons in every area examined in all 4 cases (Fig. la), although some glial cells in both gray and white matter were also labeled, agreeing with previous immunohistochemical studies using rat brains 3"18. However, Z31pre did not label any cellular structures. The immunoreactivity of NF'I', neuropil threads, and degenerating neurites of the senile plaque of the Alzheimer brains is summarized in Table I. Double staining with W63N and Congo red demonstrated that almost all intraneuronal NFT was positive with W63N (Fig. lb), while extracellular NFT was usually negative with W63N. Moreover, some of the tau-C6-positive neuropil threads
Correspondence: H. Yamaguchi, College of Medical Care and Technology,Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
319
Fig. 1. a: W63N labels almost all neurons; cryostat section, control brain, b: W63N intensely labels NFT and less intensely some neuropii threads, and weakly non-NFT-bearing neurons; cryostat section, AIzheimer brain, c-g: serial ethanol-fixed and paraffin-embedded sections of the Alzheimer brain immunostained after formic acid pretreatment, demonstrating positive NFT with W61C (c), W63N (d), Z31pre (e), and tau-C6 (f). Z31pre showed diffuse background staining, fl 28K (g) labels the amyloid component of senile plaque and amyloid angiopathy, but not NFT. Arrow, arrowhead and asterisk indicate the same NFT, amyloidotic vessel and senile plaque, respectively, h: Sarkosyl-NFT, immunoreactive with W63N. Bar = 20/zm.
320 TABLE I lmmunolabeling of neurofibrillary tangles (NFT), neuropil threads and senile plaque neurites with antisera to amyloid fl/A4 protein precursor (A PP) and tau -
negative, + occasionally positive, + often positive, + + mostly positive. APP18_3s* (W63N)
Without formic acid Intraneuronal NFT Extracellular NFT Neuropil threads Senile plaque neurites
+ +
With formic acid*** Intraneuronal NFI' Neuropil threads Senile plaque neurites
++ + ++
APP577_596(Z31pre) APP597_624(f128K)
++
APP~,r,6_695(W61C)
Tau32o_329**(Tau-C6)
_+ ++
++ + ++ ++
+ + ++
++ ++ ++
-
++ + ++
* Kang's sequence 9, ** Lee's sequence 1°, *** deformity and decreased Congophilia. (or curly fibers) were positive with W63N (Fig. lb). The cytoplasm of both N F T bearing and non-bearing neurons was weakly labeled. The W61C labeled NFT-bearing neurons, although the NFT itself was often negative. The formic acid pretreatment enhanced the labeling of NFT both with W63N (Fig. ld) and W61C (Fig. lc). The Z31pre labeled N F T only after the formic acid pretreatment (Fig. le). The diffuse background staining of Z31pre was denser in the plaque areas of the formic acid treated sections. It was not easy to distinguish extracellular NFT after the formic acid pretreatment, because this treatment deformed the tissue structure and diminished the Congophilia of NFT. The fl 28K (Fig. lg) labeled senile plaque amyloid but was consistently negative with intraneuronal NFT, even after the formic acid. The tau-C6 showed neuropil threads and both intraneuronal and extraceUular NFT (Fig. If). Second, we examined the ultrastructural localization of W63N in 2 Alzheimer brains. Vibratome sections of
paraformaldehyde-fixed tissues were immunostained with either W63N or normal rabbit serum and then embedded in epoxy resin 23. Ultrathin sections, observed without heavy metal staining, showed intense immunoreaction to both intracellular N F T (Fig. 2a) and neuropil threads (Fig. 2b,c). Immunoreaction products were located on P H F and SF at a high magnification (Fig. 2c). The circular profiles of P H F and SF represent poor penetration of the antiserum. The cytoplasm of these neurons showed diffuse and weak labeling. Third, we examined the immunoreactivity of detergent-treated N F T in order to clarify whether A P P simply adheres to P H F and SF or is incorporated into them. NFT were extracted from 3 Alzheimer brains by homogenization and centrifugation in Tris-buffered saline (TBSNFF) 1°. In addition, the T B S - N F T fraction was treated with either 1% Sarkosyl for 1 h at 4 °C (Sarkosyl-NFT), 2% Triton X-100 for 30 rain at room temperature (Triton-NFT), 2% trypsin for 1 h at 37 °C (Trypsin-NFT),
O Fig. 2. Immunoelectron micrograph using W63N, demonstrating positive NFT (a) and neuropil threads (b,c). Reaction products localized on pathological filaments (c). Bars = 200 nm.
321 or a combination of 2% sodium dodecyl sulfate and 0.05% mercaptoethanol solution for 10 min at 95 °C (SDS-NFT), and then washed. Aliquots of these 5 kinds of NFT samples were dropped on slides, air dried, and then immunostained. Even after the removal of soluble components from NFT by the Sarkosyl, Triton, or SDS treatment, W63N labeled NFT, as well as tau-C6 did (Fig. lh), while Z31pre, fl 28K, and W61C did not. The trypsin-NFT was negative with all APP antisera but was weakly positive with tau-C6. Fourth, we tried to demonstrate W63N-immunoreactive bands of NFT in the Western blot analysis. Sarkosylinsoluble fractions from the Alzheimer brains 14 were solubilized in a routine SDS/mercaptoethanol buffer by heating, then electrophoresed, blotted on nitrocellulose membranes, and immunostained for W63N or Z31pre. However, we could not detect any immunoreactive bands or smear patterns (data not shown), yet antiserum to PHF showed smear patterns for the same specimen 7. Here, we will summarize our results: (1) antiserum to N-terminal of APP labeled the PHF and SF of both NFT and neuropil threads. (2) This antiserum recognized the detergent-insoluble component of NFT. (3) The formic acid pretreatment exposed other APP epitopes of NFT. (4) Smear pattern was not recognized in the Western blot analysis. From these results, we conclude that small amounts of APP are bound to PHF and SF of both NFT and neuropil threads. Using APP antisera, we have already shown positive labeling of senile plaque neurites together with negative labeling of N F T 19. In this study, we used the same antisera but got a positive staining of NFT. This is because (1) paraformaldehyde-cryostat or ethanol-paraffin sections showed better immunoreactivity than the previously used paraformaldehyde-paraffin sections, and (2) pretreatment of the sections with either Triton X-100 or formic acid greatly enhanced the labeling of APP. We used 4 kinds of rabbit polyclonal antisera to the APP sequence. Three (W63N, Z31pre and W61C) recognized APP in the immunoblot analysis, and therefore labeled NFT. This agreed with Cras's report 4 demonstrating an occasional labeling of NFT and neuropil threads with antibodies to recombinantly expressed APP. In contrast, one antiserum (fl 28K) did not recognize APP and did not label NFT, like other
polyclonal antiserum to fl/A4 protein 17. It is likely that epitopes of amyloidogenic fl protein region are hidden in the large APP molecule because of its hydrophobicity. However, polyclonal and monoclonal antibodies directed to the synthetic peptide of fl/A4 protein1_11 or 1-12 have been shown to label NET 12'20'22, while these antibodies did not label amyloid deposits. We suppose that these antibodies recognize APP. Moreover, the sequence of fl/A4 protein1_12 is contained in the secreted form of APE and antisera to this part of the fl protein recognize the secreted form of APP 15. Recent direct chemical analysis of NFT has revealed that major components of NFT are the carboxyl third of tau 1°'21 and ubiquitin 13, but not APP 1°. Fragments of MAP5 and whole molecules of tau are also incorporated in the outer coat of PHF 5"7. These microtubule associated proteins are processed, fragmentated, and then aggregated to form P H E It is likely that APP is also processed and fragmentated in the P H E This would explain the variable patterns of NFT labeling with our 3 kinds of antisera that recognize APP. A ligation study of the peripheral nerve has revealed that APP is transported by fast axonal flow, which depends on the microtubular system 11. Because microtubular transportation system is disturbed in the NFTbearing neurons, APP may accumulate in their cytoplasm and processes, and subsequently, it may be fragmentated and incorporated into PHF and SF. In the Alzheimer brains, PHF and SF appears in 3 kinds of structures: NFT, senile plaque neurites, and neuropil threads 24. Some previous immunohistochemical studies have shown the presence of APP in senile plaque neurites 2'4's'19. In the Alzheimer brains, the senile plaque neurites were immunoreactive with both APP and tau antisera 19, while in the brains of non-demented subjects, they were negative with APP 2 and tau 16 antisera. In other words, senile plaque neurites, which lack PHF and SF, were negative with APP antiserum. These results, taken with ours, led us to stress co-localization of APP with PHF and SE Finally, we emphasize that APP is a minor, not a main, component of NFT. It may be bound to PHF and SF when they are formed in the cytoplasm (NFT), dendrites (neuropil threads), and degenerating neurites of senile plaques.
1 Allsop, D., Haga, S., Bruton, C., Ishii, T. and Roberts, G.W., Neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid fl-protein of Alzheimer's disease, Am. J. Pathol., 136 (1990) 255-260. 2 Arai, H., Lee, V.M.-Y., Otvos, L., Greenberg, B.D., Lowery, D.E., Sharma, S.K., Schmid, M.L. and Trojanowski, J.Q., Defined neurofilament, r, and fl-amyloid precursor protein epitopes distinguish Alzheimer from non-AIzheimer senile plaques, Proc. Natl. Acad. Sci. U.S.A, 87 (1990) 2249-2253.
3 Card, J.P., Meade, R.P. and Davis, L.G., Immunocytochemical localization of the precursor protein for ~0-amyloid in the rat central nervous system, Neuron, 1 (1988) 835-846. 4 Cras, P., Kawai, M., 8iedlak, S., Mulvihill, P., Gambetti, P.,
Lowery, D., Gonzalez-DeWhitt, P., Greenberg, B. and Perry, G., Neuronal and microglial involvement in fl-amyloid protein deposition in Alzheimer disease, Am. J. Pathol., 137 (1990) 241-246. 5 Hasegawa, M., Arai, T. and Ihara, Y., Immunochemical
322
6
7
8
9
10 11
12
13 14 15
evidence that fragments of phosphorylated MAP5 (MAPIB) are bound to neurofibrillary tangles in Alzheimer disease, Neuron, 4 (1990) 909-918. Hyman, B.T., Van Hoesen, G.W., Beyreuther, K. and Masters, C.L., A4 amyloid protein immunoreactivity is present in Alzheimer's disease neurofibrillary tangles, Neurosci. Lett., 101 (1989) 352-355. Ihara, Y., Kondo, J., Miura, R. Nakagawa, Y., Mori, H. and Honda, T., Characterization of antisera to paired helical filaments and tau. An implication for the extent of tau tightly bound to paired helical filaments, Gerontology, 36, Suppl. l (1990) 15-24. Ishii, T., Kametani, F., Haga, S. and Sato, M., The immunohistochemical demonstration of subsequences of the precursor of the amyloid A4 protein in senile plaques in Alzheimer's disease, Neuropathol. Appl. Neurobiol., 15 (1989) 135-147. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K. and M011er-Hill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. Kondo, J., Honda, T., Mori, H., Hamada, Y., Miura, R., Ogawara, M. and Ihara, Y., The carboxyl third of tau is tightly bound to paired helical filaments, Neuron, 1 (1988) 827-834. Koo, E.H., Sisodia, S.S., Archer, D.R., Martin, L.J., Weidemann, A,, Beyreuther, K., Fischer, P., Masters, C.L. and Price, D.L., Precursor of amyloid protein in Alzheimer disease undergoes fast antegrade axonal transport, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 1561-1565. Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.N. and Beyreuther, K., Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque core and blood vessels, EMBO J., 4 (1985) 2757-2763. Mori, H., Kondo, J. and Ihara, Y., Uniquitin is a component of paired helical filaments in Alzheimer's disease, Science, 235 (1987) 1641-1644. Nukina, N. and Ihara, Y., Proteolytic fragments of Alzheimer's paired helical filaments, J. Biochem., 98 (1985) 1715-1718. Palmert, M.R., Siedlak, S.L., Podlisny, M.B., Greenberg, B., Shelton, E.R., Chan, H.W., Usiak, M., Selkoe, D.J., Perry, G. and Younkin, S.G., Soluble derivatives of the fl amyloid protein precursor of Alzheimer's disease are labeled by antisera to the fl amyloid protein, Biochem. Biophys. Res. Comrnun., 165 (1989) 182-188.
16 Probst, A., Anderton, B.H., Brion, J.-P. and Ulrich, J., Senile plaque neurites fail to demonstrate anti-paired helical filament and anti-microtubule-associated protein-tau immunoreactive proteins in the absence of neurofibrillary tangles in the neocortex, Acta Neuropathol., 77 (1989) 430-436. 17 Selkoe, D.J., Podlisny, M.B., Joachim, C.L., Vickers, E.A., Lee, G., Fritz, L.C. and Ostersdolf, T., /3-Amyloid precursor protein of Alzheimer disease occurs as 110- to 125-kilodalton membrane-associated proteins in neural and nonneural tissues, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7341-7345. 18 Shivers, B.D., Hilbich, C., Multhaup, G., Salbaum, M., Beyreuther, K. and Seeburg, P., Alzheimer's disease amyloidogenic glycoprotein: expression pattern in rat suggests a role in cell contact, EMBO J., 7 (1988) 1365-1370. 19 Shoji, M., Hirai, S. Yamaguchi, H., Harigaya, Y. and Kawarabayashi, T., Amyloid /3-protein precursor accumulates in dystrophic neurites of senile plaques in Alzheimer-type dementia, Brain Research, 512 (1990) 164-168. 20 Spillantini, M.G., Goedert, M., Jakes, R. and Klug, A., Topographical relationship between/3-amyloid and tau protein epitopes in tangle-bearing cells in Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 3252-3256. 21 Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 4506-4510. 22 Wischik, C.M., Novak, M., Bondareff, W., Harrington, C., Jakes, R., Harrington, K., Edwards, P. and Whitmore, J., Molecular dissection of Alzheimer pathology. In T. Miyatake, D.J. Selkoe and Y. lhara (Eds.), Molecular Biology and Genetics of Alzheimer's Disease, Elsevier, Amsterdam 1990, pp. 9-19. 23 Yamaguchi, H., Nakazato, Y., Hirai, S. and Shoji, M., Immunoelectron microscopic localization of amyloid /3 protein in diffuse plaques of the Alzheimer-type dementia, Brain Research, 508 (1990) 320-324. 24 Yamaguchi, H., Nakazato, Y., Hirai, S., Shoji, M. and Ihara, Y., Ultrastructure of the neuropil threads in the Alzheimer brain: their dendritic origin and accumulation in the senile plaques, Acta Neuropathol., 80 (1990) 368-374. 25 Yamaguchi, H., Nakazato, Y., Shoji, M., Okamoto, K., Ihara, Y., Morimatsu, M. and Hirai, S., Secondary deposition of/3 amyloid within extracellular neurofibrillary tangles in Alzheimer-type dementia, Am., J, Pathol., in press.
Brain Research. 537 (1990) 318-322 Elsevier
BRES 24451
Amyloid I~/A4 protein precursor is bound to neuroflbrillery ~ Alzh~mer-type dementia
in
Haruyasu Yamaguchi 1'2, Koji Ishiguro 2, Mikio Shoji 2, Tsuneo Yamazaki 2, Yoichi Nakazato 3, Yasuo Ihara 4 and Shunsaku Hirai: 1College of Medical Care and Technology; and Departments of 2Neurology and ~Pathology, School of Medicine; Gunma University, Maebashi, Gunma 371 (Japan) 4Tokyo MetropolitanInstitute of Gerontology, Tokyo (Japan) (Accepted 18 September 1990)
Key words: Amyloid fl/A4 protein precursor; Tau protein; Alzheimer-typedementia; Neurofibriilary tangle; Immunoelectron microscopy; Paired helical filament
Localization of amyloid 13/A4protein precursor (APP) in Alzheimer's neurofibrillary tangles (NFT) was examined immunohistochcmically. Antiserum directed to N-terminal of APP intensely labeled intracellular NFT and some neuropil threads. The NFr, extracted from ~heimer brains by detergent treatments, were also immunoreactivewith this antiserum. Antisera to other parts of APP labeled NFI" after the formic acid pretreatment. However, Western blot analysis of NFT demonstrated no immunoreactivebands with APP antiserum. These findingssuggest that APP is a minor component of the NFT. In the brains of Alzheimer-type dementia, major histopathological hallmarks are senile plaques and neurofibrillary tangles (NFT). The latter consist of both paired helical filaments (PHF) and straight filaments (SF) 24. Recent studies revealed that the major component of senile plaque amyloid is derived from the larger amyloid fl/A4 protein precursor (APP) 9. Some immunohistochemical studies, demonstrating positive labeling of NFT with fl/A4 protein antisera 1'6'12'2°, raise a question whether fl/A4 protein is an essential 'core' component of PHF. In the present study, we demonstrated that APP is a minor component of the NF-I', and that this can explain the positive labeling of intraneuronal NFT with some fl/A4 antisera. Labeling of the extracellular NFT with fl/A4 antiserum will be discussed elsewhere 25. First, we examined autopsy brains from 3 patients with Alzheimer-type dementia (65, 81, and 82 years old), and from 4 control patients (59, 67, 74, and 82 years old) with no neurological diseases. These brains were removed within 2 h after death. Frontal and temporal specimens were fixed in a 4% paraformaldehyde and 0.3% picric acid solution for 6-12 h and sectioned using cryostat. Some specimens were fixed in ethanol-polyethylene glycol mixture (Kryofix, Merck, E R . G . ) for 12-24 h and embedded in paraffin. Sections, 2-8 /~m thick, were pretreated with 2% Triton X-100 or 75% formic acid and then immunostained with antisera to synthetic peptides of
(1) APP18_38 (N-terminal of APP, W63N) 9A9, (2) APP577_ 596 (pre-fl/A4 protein region, Z31pre), (3) fl/A4 protein 1_ 28 (amyloidogenic internal portion of APP, APPs97_624, fl 28K) 23, (4) APP666_695 (C-terminal of APP, W61C)191 and (5) microtubule associated protein tau320__329(tau-C6) 1°, and ABC kits (Vector Lab, CA). Two APP antisera, W63N and W61C, have been reported to recognize 106-135 kDa APP of human brain extract by immunoblot analysis 19. The Z31pre also recognized the same APP bands in Western blot analysis (data not shown), while fl 28K did not. Some sections were counterstained with Congo red. Control sections were reacted with preabsorbed antisera 19. In the control brains, both W63N and W61C principally immunostained almost all neurons in every area examined in all 4 cases (Fig. la), although some glial cells in both gray and white matter were also labeled, agreeing with previous immunohistochemical studies using rat brains 3"18. However, Z31pre did not label any cellular structures. The immunoreactivity of NF'I', neuropil threads, and degenerating neurites of the senile plaque of the Alzheimer brains is summarized in Table I. Double staining with W63N and Congo red demonstrated that almost all intraneuronal NFT was positive with W63N (Fig. lb), while extracellular NFT was usually negative with W63N. Moreover, some of the tau-C6-positive neuropil threads
Correspondence: H. Yamaguchi, College of Medical Care and Technology,Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
319
Fig. 1. a: W63N labels almost all neurons; cryostat section, control brain, b: W63N intensely labels NFT and less intensely some neuropii threads, and weakly non-NFT-bearing neurons; cryostat section, AIzheimer brain, c-g: serial ethanol-fixed and paraffin-embedded sections of the Alzheimer brain immunostained after formic acid pretreatment, demonstrating positive NFT with W61C (c), W63N (d), Z31pre (e), and tau-C6 (f). Z31pre showed diffuse background staining, fl 28K (g) labels the amyloid component of senile plaque and amyloid angiopathy, but not NFT. Arrow, arrowhead and asterisk indicate the same NFT, amyloidotic vessel and senile plaque, respectively, h: Sarkosyl-NFT, immunoreactive with W63N. Bar = 20/zm.
320 TABLE I lmmunolabeling of neurofibrillary tangles (NFT), neuropil threads and senile plaque neurites with antisera to amyloid fl/A4 protein precursor (A PP) and tau -
negative, + occasionally positive, + often positive, + + mostly positive. APP18_3s* (W63N)
Without formic acid Intraneuronal NFT Extracellular NFT Neuropil threads Senile plaque neurites
+ +
With formic acid*** Intraneuronal NFI' Neuropil threads Senile plaque neurites
++ + ++
APP577_596(Z31pre) APP597_624(f128K)
++
APP~,r,6_695(W61C)
Tau32o_329**(Tau-C6)
_+ ++
++ + ++ ++
+ + ++
++ ++ ++
-
++ + ++
* Kang's sequence 9, ** Lee's sequence 1°, *** deformity and decreased Congophilia. (or curly fibers) were positive with W63N (Fig. lb). The cytoplasm of both N F T bearing and non-bearing neurons was weakly labeled. The W61C labeled NFT-bearing neurons, although the NFT itself was often negative. The formic acid pretreatment enhanced the labeling of NFT both with W63N (Fig. ld) and W61C (Fig. lc). The Z31pre labeled N F T only after the formic acid pretreatment (Fig. le). The diffuse background staining of Z31pre was denser in the plaque areas of the formic acid treated sections. It was not easy to distinguish extracellular NFT after the formic acid pretreatment, because this treatment deformed the tissue structure and diminished the Congophilia of NFT. The fl 28K (Fig. lg) labeled senile plaque amyloid but was consistently negative with intraneuronal NFT, even after the formic acid. The tau-C6 showed neuropil threads and both intraneuronal and extraceUular NFT (Fig. If). Second, we examined the ultrastructural localization of W63N in 2 Alzheimer brains. Vibratome sections of
paraformaldehyde-fixed tissues were immunostained with either W63N or normal rabbit serum and then embedded in epoxy resin 23. Ultrathin sections, observed without heavy metal staining, showed intense immunoreaction to both intracellular N F T (Fig. 2a) and neuropil threads (Fig. 2b,c). Immunoreaction products were located on P H F and SF at a high magnification (Fig. 2c). The circular profiles of P H F and SF represent poor penetration of the antiserum. The cytoplasm of these neurons showed diffuse and weak labeling. Third, we examined the immunoreactivity of detergent-treated N F T in order to clarify whether A P P simply adheres to P H F and SF or is incorporated into them. NFT were extracted from 3 Alzheimer brains by homogenization and centrifugation in Tris-buffered saline (TBSNFF) 1°. In addition, the T B S - N F T fraction was treated with either 1% Sarkosyl for 1 h at 4 °C (Sarkosyl-NFT), 2% Triton X-100 for 30 rain at room temperature (Triton-NFT), 2% trypsin for 1 h at 37 °C (Trypsin-NFT),
O Fig. 2. Immunoelectron micrograph using W63N, demonstrating positive NFT (a) and neuropil threads (b,c). Reaction products localized on pathological filaments (c). Bars = 200 nm.
321 or a combination of 2% sodium dodecyl sulfate and 0.05% mercaptoethanol solution for 10 min at 95 °C (SDS-NFT), and then washed. Aliquots of these 5 kinds of NFT samples were dropped on slides, air dried, and then immunostained. Even after the removal of soluble components from NFT by the Sarkosyl, Triton, or SDS treatment, W63N labeled NFT, as well as tau-C6 did (Fig. lh), while Z31pre, fl 28K, and W61C did not. The trypsin-NFT was negative with all APP antisera but was weakly positive with tau-C6. Fourth, we tried to demonstrate W63N-immunoreactive bands of NFT in the Western blot analysis. Sarkosylinsoluble fractions from the Alzheimer brains 14 were solubilized in a routine SDS/mercaptoethanol buffer by heating, then electrophoresed, blotted on nitrocellulose membranes, and immunostained for W63N or Z31pre. However, we could not detect any immunoreactive bands or smear patterns (data not shown), yet antiserum to PHF showed smear patterns for the same specimen 7. Here, we will summarize our results: (1) antiserum to N-terminal of APP labeled the PHF and SF of both NFT and neuropil threads. (2) This antiserum recognized the detergent-insoluble component of NFT. (3) The formic acid pretreatment exposed other APP epitopes of NFT. (4) Smear pattern was not recognized in the Western blot analysis. From these results, we conclude that small amounts of APP are bound to PHF and SF of both NFT and neuropil threads. Using APP antisera, we have already shown positive labeling of senile plaque neurites together with negative labeling of N F T 19. In this study, we used the same antisera but got a positive staining of NFT. This is because (1) paraformaldehyde-cryostat or ethanol-paraffin sections showed better immunoreactivity than the previously used paraformaldehyde-paraffin sections, and (2) pretreatment of the sections with either Triton X-100 or formic acid greatly enhanced the labeling of APP. We used 4 kinds of rabbit polyclonal antisera to the APP sequence. Three (W63N, Z31pre and W61C) recognized APP in the immunoblot analysis, and therefore labeled NFT. This agreed with Cras's report 4 demonstrating an occasional labeling of NFT and neuropil threads with antibodies to recombinantly expressed APP. In contrast, one antiserum (fl 28K) did not recognize APP and did not label NFT, like other
polyclonal antiserum to fl/A4 protein 17. It is likely that epitopes of amyloidogenic fl protein region are hidden in the large APP molecule because of its hydrophobicity. However, polyclonal and monoclonal antibodies directed to the synthetic peptide of fl/A4 protein1_11 or 1-12 have been shown to label NET 12'20'22, while these antibodies did not label amyloid deposits. We suppose that these antibodies recognize APP. Moreover, the sequence of fl/A4 protein1_12 is contained in the secreted form of APE and antisera to this part of the fl protein recognize the secreted form of APP 15. Recent direct chemical analysis of NFT has revealed that major components of NFT are the carboxyl third of tau 1°'21 and ubiquitin 13, but not APP 1°. Fragments of MAP5 and whole molecules of tau are also incorporated in the outer coat of PHF 5"7. These microtubule associated proteins are processed, fragmentated, and then aggregated to form P H E It is likely that APP is also processed and fragmentated in the P H E This would explain the variable patterns of NFT labeling with our 3 kinds of antisera that recognize APP. A ligation study of the peripheral nerve has revealed that APP is transported by fast axonal flow, which depends on the microtubular system 11. Because microtubular transportation system is disturbed in the NFTbearing neurons, APP may accumulate in their cytoplasm and processes, and subsequently, it may be fragmentated and incorporated into PHF and SF. In the Alzheimer brains, PHF and SF appears in 3 kinds of structures: NFT, senile plaque neurites, and neuropil threads 24. Some previous immunohistochemical studies have shown the presence of APP in senile plaque neurites 2'4's'19. In the Alzheimer brains, the senile plaque neurites were immunoreactive with both APP and tau antisera 19, while in the brains of non-demented subjects, they were negative with APP 2 and tau 16 antisera. In other words, senile plaque neurites, which lack PHF and SF, were negative with APP antiserum. These results, taken with ours, led us to stress co-localization of APP with PHF and SE Finally, we emphasize that APP is a minor, not a main, component of NFT. It may be bound to PHF and SF when they are formed in the cytoplasm (NFT), dendrites (neuropil threads), and degenerating neurites of senile plaques.
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Lowery, D., Gonzalez-DeWhitt, P., Greenberg, B. and Perry, G., Neuronal and microglial involvement in fl-amyloid protein deposition in Alzheimer disease, Am. J. Pathol., 137 (1990) 241-246. 5 Hasegawa, M., Arai, T. and Ihara, Y., Immunochemical
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