American Journal of Pathology, Vol. 141, No. 2, Augut 1992 Copyight C Amamn Association of Pathologists
Alzheimer's Disease 3-amyloid Precursor Protein Expression in the Nucleus Basalis of Meynert
Greer M. Murphy, Jr.,* Barry D. Greenberg, William G. Ellis,11 Lysia S. Forno,t Shahriar M. Salamat,11 Patricia A. Gonzalez-DeWhitt,t David E. Lowery, Jared R. Tinklenberg,* and Lawrence F. Engt From the Departments of Psychiaby and Behavioral Sciences and Pathology,t Stanford University School of Medicine, and the Veterans Affairs Medical Center, Palo Alto, Califomia, The Upjohn Co.,* Kalamazoo, Michigan, and the Department of Pathology,1I School of Medicine, University of California, Davis, Calfornia
the cerebral cortex as well as to a variety of subcortical structures in the primate brain.23 A cholinergic deficit in the cerebral cortex may contribute to cognitive decline in AD,4 possibly due to degenerative changes in the nbM. The ,B-amyloid precursor protein (IAPP) is believed to give rise to the 3-amyloid peptide (PAP), which accumulates in plaques and vessels in AD.-7 Transcription of ,APP mRNA in the nbM has been reported to be abnormal in AD,8'9 but the protein itself has not been examined in this region. It has been reported that in hippocampus and cerebral cortex, ,APP is found in intracellular neurofibrillary tangles (NFT) and the adjacent cytoplasm,10 11 sometimes along with components of the paired helical filament (PHF) such as tau. This study examined neurons in the nbM in AD for expression of ,BAPP to more fully understand the relationship between IAPP and NFTs in this critically important cell group.
The nucleus basalis ofMeynert (nbM) was examined using immunocytochemistry for P-amyloid precursor protein (IAPP) expression in Alzbeimer's disease (AD). In mild AD cases, light labeling of the cell body and proximal processes was observee4 and small intracellular structures were labeled rarely. In the more severe cases, intense cytoplasmic PAPP labeling was seen, often along with small APP-positive structures. Double-labeling experiments demonstrated that in the more severe cases these small structures were also decorated by a neurofibrillary tangle (NFT) antiserum. Other neurons in the severe cases showed incorporation of iAPP into large inclusions which were also labeled with the NFT antiserumn However, some large inclusions in the severe cases were labeled by the NFT antiserum but contained no PAPP. Extraneuronal NFTs did not show PAPP labeling and did not react with an antibody to the n-amyloid peptide. These results suggest that increased expression of PAPP coincides with intracellular NA formation in the nbM, but that the formation of extraneuronal NFTs results in a loss of IAPP immunoreactivity. (AmJPathol 1992, 141:357-361)
Material included nine cases with clinical and neuropathologic diagnoses of Alzheimer's disease, and one 38-year-old case with Down's Syndrome (DS) (Table 1). For ,APP immunostaining, blocks from the basal forebrain and superior temporal lobe were immersed immediately at autopsy in methacam (60% methanol, 30% chloroform, 10% glacial acetic acid) at 40C for 24 to 48 hours, then embedded in paraffin; 5-,Lm sections were cut at the level of the anterior commissure so as to include the giant neurons of the nbM scattered throughout the substantia innominata. The sections corresponded to Figure 1 OG and 1 OH of Hedreen et al.12 The remainder of the brain was fixed in formalin and a comprehensive gross and microscopic neuropathologic workup was performed. Cases were classified as mild, moderate, or severe AD using a semiquantitative scale based on the
The nucleus basalis of Meynert (nbM) is a focus of neuropathologic change in Alzheimer's disease (AD).1 Normally, this cell group provides cholinergic innervation to
Supported by the Dana Foundation, DVA, NIMH, and the State of Califomia. Accepted for publication January 31, 1992. Address reprint requests to Dr. G. M. Murphy, Jr., Psychiatry Service (1 16A3), Palo Alto Veterans Affairs Medical Center, Palo Alto, CA 94304.
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Table 1. Case Material Case Sex M
2 3 4 5 6 7 8 9 10
M
F F F M M
F M M
AD
=
Age
Diagnosis
64 75 77 78 80 80 83 86 87 38
AD, severe AD, severe AD, moderate AD, severe AD, mild AD, severe AD, moderate AD, severe AD, severe DS with mild AD
Alzheimer's disease; DS
=
Down's syndrome.
number of plaques and NFTs in silver-stained sections from a variety of brain regions. In practice, this evaluation gives results similar to the system of Khachaturian.13 All cases were free of other neuropathologic changes, except for a single Lewy body (LB) in the locus ceruleus in case 4 and a single small cortical infarct in cases 3 and 6. For antibody production, 3APP-6957 was generated using a baculovirus expression system. A termination codon was engineered at two positions C-terminal to the PAP domain, functionally deleting the C-terminal 56 amino acids. Expressed ,BAPP was purified from conditioned media to apparent homogeneity through a series of chromatographic steps. The protein was shown to extend from the natural IAPP N-terminus. These details and C-terminal sequence analysis are reported elsewhere.14 Polyclonal antisera to this purified protein were raised in rabbits by standard methods. Use of these antisera was previously reported.10 15'16 To identify NFTs, an antiserum to SDS-isolated PHF was used.17 We also used a monoclonal antibody raised to a synthetic peptide corresponding to residues 1-28 of the ,BAP (California Biotechnology, Mountain View, CA). The epitope for this reagent has been mapped to residues 1-10 of the synthetic PAP immunogen.
,BAPP immunocytochemistry was performed using the peroxidase-antiperoxidase (PAP) method,18 or the avidin-biotin (AB) method (Vectastain ABC Kit, Vector Labs, Burlingame, CA). NFT and PAP immunocytochemistry were performed with the AB method. For both the PAP and the AB reactions, sections were pretreated with 1% H202 in methanol for 30 minutes, and diaminobenzidine (DAB) was used as the chromogen. A pre-immune serum for the 3APP antibody was used to check for nonspecific reactivity. As previously reported by Cras et al,10 formic acid pretreatment did not enhance the reactivity of the ,BAPP reagent in methacarn-fixed tissue. Likewise, formic acid did not improve the performance of the PAP antibody in tissue treated with methacarn. For double labeling, the ,APP reaction was performed first, using either the PAP or the AB methods, and DAB was used as
the chromogen. After treatment in diluted aqueous HCI (99 ml of H20 and 1 ml of 30% HCI) for 1 hour, secondary labeling for NFT was done using the AB method. The NFT reaction was visualized with 4-chloro-1 -naphthol.
Results ,BAPP expression in the nbM was found to vary considerably among the AD cases. In the mild AD case, and in the DS case, nbM neurons showed a uniform granular labeling of the soma with the PAPP antiserum. This ,APP labeling was considerably stronger than the background signal obtained with the pre-immune antiserum. Labeling often extended into proximal nerve cell processes (Figure 1). Rarely neurons in the mild AD case showed an aggregation of 3APP-positive material into small intracellular structures (Figure 1). These structures were not observed in the DS case. In the sections from the mild AD case and the DS case that were reacted with the NFT antiserum or with an antiserum to mixed tau/MAP-2 epitopes,19 no intracellular structures were detected. In the moderate and severe AD cases, neurons usually showed more intense cytoplasmic ,BAPP immunoreactivity, often in a punctate pattern in the perinuclear region (Figure 2). Small 3APP-containing structures, which were also labeled with the NFT antiserum, were seen in many cells in moderate and severe cases (Figures 2, 3). Other neurons in severe AD cases showed heavy labeling of large intracellular inclusions with the ,APP antiserum (Figure 4). Double labeling revealed that these large inclusions reacted with both the 3APP and the NFT antisera (Figures 3, 5). Double labeling also showed that some neurons in the moderate and severe cases with intense perinuclear ,APP had no NFT formation (Figures
3, 5). Occasionally, neurons in the moderate and severe cases displayed intracellular inclusions that were not strongly labeled for IAPP (Figure 6), although they were labeled by the NFT antiserum. Often there was an aggregation of 3APP-positive material around the edges of these inclusions. None of the inclusions in the nbM were eosinophilic. Extracellular NFTs in the nbM were not labeled with the ,APP antisera, although they were strongly reactive with the NFT reagent (Figures 3, 5). Neither intracellular nor extracellular NFTs were labeled with the PAP antibody, even though strong labeling of neuritic plaque amyloid in the adjacent amygdala was observed (data not shown).
Discussion These results show that variation exists among AD cases in the expression of ,APP in nbM neurons. Comparison
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Figure 1. Top left: Neurons in the nbM, case 5. There is a uniform cytoplosmic labeling in the cell soma and proxoimal cell processes with the PAPP antiserum. A small intranearonal structure (arrowhead) is pIAPP-positive. Figure 2. Top right: Punctate cytoplasmic labelling (arrowhead) and intraneuronal inclusion (arrow), APP antiserum, case 8. An inclusion that is not strongly labeled for IAPP is also present (open arrow). Figure 3. Middle left: Double labeling of PAPP (brown) and NFT (purple), case 8. Neuron at arrowhead shows small areas of NFT formation against a background of ,BAPP. Another neuron (arrow) shows heavy PAPP expression but no NFT. An extraneuronal NFT (open arrow) is not labeled by ,BAPP antiserum. Figure 4. Middle right: Intraneuronal NFT in nbM strongly labeled with P.APP antiserum, case 1. Figure 5. Bottom left: Double labeling with APP (brown), and NFT(purple), case 8. Two neurons (arrowbeads) show simultaneous strong labeling for f.APP and NFT Extraneuronal NFTs are not labeled with 3APP antiserum (open arrows). Figure 6. Bottom right: Neurofibrillary tangle unlabeled by 3APP antiserum (arrowhead), case 8. Note accumulation of fIAPP around perfpbery of inclusion.
of mild, moderate, and severe AD cases suggests there may be a progression of ,APP expression. Mild AD and DS cases show uniform, low levels of ,APP labeling in nbM neurons. In moderate and severe AD cases, PAPP immunoreactivity in nbM cells is more intense, especially in the perinuclear region. Increased neuronal IAPP im-
munolabeling in AD has also been reported in the pyramidal layer of Ammon's horn, perhaps contained in lysosomes.2021 Perinuclear accumulation of iAPP, similar to that observed in the nbM in the present study, has also been reported in muscle cells22 and in several cell
lines.23
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The enhanced 3APP labeling of nbM cells observed in moderate and severe AD cases in the present study could be due to transcriptional abnormalities translated to the protein level, because there is evidence of increased 3APP mRNA in the nbM in AD. Increases in total IAPP mRNA,8 as well as the PAPP-695 splicing variant9 have been reported with in situ hybridization. Alternatively, the normal metabolism of ,APP may be impaired in AD, resulting in its intracellular accumulation. A number of studies have reported that dystrophic neurites in plaques show strong ,APP immunoreactivity.24 If some of these neurites are axon terminals of nbM neurons, BAPP in them could originate in the cell body, as IAPP is transported anterogradely along axons.25 IAPP and ,BAP have been colocalized in plaques, which may indicate that processing of ,APP to ,BAP then occurs in situ.16'26 Comparisons of 3APP expression in nbM neurons and in neurites located in nbM projection fields will be needed to test the hypothesis of anterograde transport of IAPP in AD brain. In addition to increased cytoplasmic immunoreactivity for ,APP, many nbM neurons in the moderate and severe AD cases showed small areas of NFT formation. If these cells are representative of an early or intermediate stage of neuronal degeneration, increased neuronal ,BAPP immunoreactivity may coincide with NFT formation. Other neurons in the moderate and severe cases showed large ,APP-positive intraneuronal NFTs. This suggests that as neuronal degeneration proceeds, ,BAPP is incorporated into or becomes associated with many large NFTs. This could be due to impaired clearance of ,BAPP from dying cells. A few PAPP-positive inclusions were detected in the mild AD case. Other sections from this case labeled with NFT, and tau/MAP-2 antisera did not show inclusions in the nbM. This could indicate that ,BAPP accumulates in the nbM before NFT and tau/MAP-2 epitopes. Alternatively, because of the low frequency of RAPP-positive inclusions in the mild AD case, the sections reacted with the NFT and tau/MAP-2 antisera may have been negative due to chance alone. Additional mild AD cases will be needed to clarify this issue. Although most intraneuronal inclusions in the nbM in the moderate and severe cases contained ,APP, some did not. If the PAPP-negative inclusions were LB, strong eosinophilia and a lack of NFT labeling would be expected.27 The absence of LB elsewhere in the brain in our sample makes it unlikely that these inclusions were LB. ,APP does not persist in a form recognizable to our antiserum in extraneuronal tangles. Yamaguchi et a11" have likewise reported an absence of ,BAPP in extraneuronal tangles in the frontal and temporal cortices. ,APP associated with NFTs may therefore be sensitive to pro-
cesses involved in the conversion of intracellular NFTs to extracellular tombstone tangles. Further, in the extracellular space, ,BAPP in tombstone tangles may be vulnerable to proteolytic degradation. Trypsin pretreatment has been shown to reduce the ,APP immunoreactivity of NFTs extracted from AD brain.11 Labeling of extracellular hippocampal and cortical NFTs with PAP antibodies has been reported in AD and other neuropathologic conditions,28-30 although in many studies this has not been observed. In this study, the ,BAP monoclonal antibody did not label extracellular tangles in the nbM. However, it did label a few extracellular tangles in the pyramidal layer of Ammon's horn in the same cases (unpublished observation). This may mean that there are differences among anatomic regions in the reactivity of NFTs to PAP antibodies, perhaps because NFT epitopes vary from region to region. Other antigenic properties of NFTs have also been reported to depend on anatomic location.31 In summary, ,APP expression by neurons in the nbM varies among AD cases, but appears to increase with progression of the disease. Neurons often simultaneously showed increased expression of 3APP and the formation of NFTs. Some intraneuronal NFTs in the nbM were labeled by ,BAPP and NFT antisera, but extracellular tangles reacted with NFT antiserum alone. PAP was not observed in intracellular or extracellular tangles in the nbM. These results indicate that for many neurons in the nbM, cell death is preceded by an overexpression and/or accumulation of ,BAPP, but ,BAPP is lost in the extracellular
tangle.
Acknowledgments The authors thank Dr. Dennis Selkoe for the PHF-NFT and the tau/MAP-2 antisera; Dr. Barbara Cordell for valuable advice and the PAP antiserum; Erin Murphy, Karen Stultz, and Raj Shrivastava for assistance in the immunostaining; Leslie Fiedler for manuscript editing; and Yuen-Ling Lee for helpful advice.
References 1. Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, DeLong MR: Alzheimer's disease and senile dementia: Loss of neurons in the basal forebrain. Science 1982, 215:12371239 2. Kitt CA, Mitchell SJ, DeLong MR, Wainer BH, Price DL: Fiber pathways of basal forebrain cholinergic neurons in monkeys. Brain Res 1987, 406:192-206 3. Mesulam M-M, Mufson EJ, Levey Al, Wainer BH: Cholinergic innervation of cortex by the basal forebrain: Cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata),
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and hypothalamus in the rhesus monkey. J Comp Neurol 1983, 214:170-197 Perry EK, Tomlinson BE, Blessed G, Bergmann K, Gibson PH, Perry RH: Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. Br Med J 1978, 2:1457-1459 Glenner GG, Wong CM: Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984,120:885-890 Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K: Amyloid plaque core protein in Alzheimer's disease and Down's syndrome. Proc Natl Acad Sci USA 1985, 82:4245-4249 Kang J, Lemaire H-G, Unterbeck A, Salbaum JM, Masters C, Grzeschik K-H, Multhaup G, Beyreuther K, Muller-Hill B: The precursor of Alzheimer's disease amyloid A4 protein resembles a cell surface receptor. Nature 1987, 325:733736 Cohen ML, Golde TE, Usiak MF, Younkin LH, Younkin SG: In situ hybridization of nucleus basalis neurons shows increased ,B-amyloid mRNA in Alzheimer's disease. Proc Natl Acad Sci USA 1988, 85:1227-1231 Palmert MR, Golde TE, Cohen ML, Kovacs DM, Tanzi RE, Gusella JF, Usiak MF, Younkin LH, Younkin SG: Amyloid protein precursor messenger RNAs: Differential expression in Alzheimer's disease. Science 1988, 241:1080-1084 Cras P, Kawai M, Siedlak S, Mulvihill P, Gambetti P, Lowery D, Gonzalez-DeWhitt P, Greenberg BD, Perry G: Neuronal and microglial involvement in 3-amyloid protein deposition in Alzheimer's disease. Am J Pathol 1990, 137:241-246 Yamaguchi H, Ishiguro K, Shoji M, Yamazaki T, Nakazato Y, Ihara Y, Hirai S: Amyloid ,3/A4 protein precursor is bound to neurofibrillary tangles in Alzheimer-type dementia. Brain Res 1990, 537:318-322 Hedreen JC, Struble RG, Whitehouse PJ, Price DL: Topography of the magnocellular basal forebrain system in human brain. J Neuropathol Exp Neurol 1984, 43:1-21 Khachaturian ZS: Diagnosis of Alzheimer's disease. Arch Neurol 1985, 42:1097-1105 Lowery DE, Pasternak J, Gonzalez-DeWhitt PA, ZurcherNeely H, Tomich C-SC, Altman RA, Fairbanks MB, Heinrikson RL, Younkin SG, Greenberg BD: Alzheimer amyloid precursor protein produced by recombinant baculovirus expression: Proteolytic processing and protease inhibitory properties. J Biol Chem 1991, 266:19842-19850 Arai H, Lee VM-Y, Otvos L, Greenberg BD, Lowery DE, Sharma SK, Schmidt ML, Trojanowski JQ: Defined neurofilament, y, and ,B-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques. Proc Natl Acad Sci USA 1990, 87:2249-2253 Murphy GM, Murphy E, Greenberg BD, Cordell B, Eng LF, Ellis WG, Forno LS, Salamat SM, Gonzalez-DeWhitt PA, Lowery DE, Tinklenberg JR: Alzheimer's Disease: ,3-amyloid precursor protein expression in plaques varies among cytoarchitectonic areas of the medial temporal lobe. Neurosci Lett 1991, 131:100-104 Ihara Y, Abraham C, Selkoe DJ: Antibodies to paired helical
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filaments in Alzheimer's disease do not recognize normal brain proteins. Nature 1983, 304:727-730 Sternberger LA: Immunocytochemistry, 3rd edition. New York, Wiley, 1986, pp 90-209 Joachim CL, Morris BM, Kosik KS, Selkoe DJ: Tau antisera recognize neurofibrillary tangles in a range of neurodegenerative disorders. Ann Neurol 1987, 22:514-520 Benowitz LI, Rodriguez W, Paskevich P, Mufson EJ, Schenk D, Neve RL: The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exp Neurol 1989, 106:237-250 Cole G, Masliah E, Huynh TV, DeTeresa R, Terry RD, Okuda C, Saitoh T: An antiserum against amyloid (3-protein precursor detects a unique peptide in Alzheimer's brain. Neurosci Lett 1989, 100:340-346 Zimmerman K, Herget T, Salbaum JM, Schubert W, Hilbich C, Cramer M, Masters CL, Multhaup G, Kang J, Lemaire H-G, Beyreuther K, Starzinski-Powitz A: Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle. EMBO J 1988, 7:367-372 Shelton ER, Cohn R, Fish L, Obernolte R, Tahilramani R, Nestor JJ, Chan HW: Characterization of ,B-amyloid precursor proteins with or without the protease-inhibitor domain using anti-peptide antibodies. J Neurochem 1990, 55:6069 Cole GM, Masliah E, Shelton ER, Chan HW, Terry RD, Saitoh T: Accumulation of amyloid precursor fragment in Alzheimer plaques. Neurobiol Aging 1991, 12:85-91 Koo EH, Sisodia SS, Archer DR, Martin U, Weidemann A, Beyreuther K, Fischer P, Masters CL, Price DL: Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport. Proc Natl Acad Sci USA 1990, 87: 1561-1565 Martin U, Sisodia SS, Koo EH, Cork LC, Dellovade TL, Weidemann A, Beyreuther K, Masters C, Price DL: Amyloid precursor protein in aged nonhuman primates. Proc Natl Acad Sci USA 1991, 88:1461-1465 Tiller-Borcich JK, Forno LS: Parkinson's disease and dementia with neuronal inclusions in the cerebral cortex: Lewy bodies or Pick bodies. J Neuropathol Exp Neurol 1988, 47: 526-535 Hyman BT, Van Hoesen GW, Beyreuther K, Masters CL: A4 amyloid protein immunoreactivity is present in Alzheimer's disease neurofibrillary tangles. Neurosci Lett 1989, 101: 352-355 Allsop D, Haga S, Bruton C, Ishii T, Roberts GW: Neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid f-protein of Alzheimer's disease. Am J Pathol 1990,136:255-260 Yamaguchi H, Nakazato Y, Shoji M, Okamato K, Ihara Y, Morimatsu M, Hirai S: Secondary deposition of beta amyloid within extracellular neurofibrillary tangles in Alzheimer's-type dementia. Am J Pathol 1991, 138:699-705 Tabaton M, Perry G, Autilio-Gambetti L, Manetto V, Gambetti P: Influence of neuronal location on antigenic properties of neurofibrillary tangles. Ann Neurol 1988, 23:604-610
Alzheimer's Disease 3-amyloid Precursor Protein Expression in the Nucleus Basalis of Meynert
Greer M. Murphy, Jr.,* Barry D. Greenberg, William G. Ellis,11 Lysia S. Forno,t Shahriar M. Salamat,11 Patricia A. Gonzalez-DeWhitt,t David E. Lowery, Jared R. Tinklenberg,* and Lawrence F. Engt From the Departments of Psychiaby and Behavioral Sciences and Pathology,t Stanford University School of Medicine, and the Veterans Affairs Medical Center, Palo Alto, Califomia, The Upjohn Co.,* Kalamazoo, Michigan, and the Department of Pathology,1I School of Medicine, University of California, Davis, Calfornia
the cerebral cortex as well as to a variety of subcortical structures in the primate brain.23 A cholinergic deficit in the cerebral cortex may contribute to cognitive decline in AD,4 possibly due to degenerative changes in the nbM. The ,B-amyloid precursor protein (IAPP) is believed to give rise to the 3-amyloid peptide (PAP), which accumulates in plaques and vessels in AD.-7 Transcription of ,APP mRNA in the nbM has been reported to be abnormal in AD,8'9 but the protein itself has not been examined in this region. It has been reported that in hippocampus and cerebral cortex, ,APP is found in intracellular neurofibrillary tangles (NFT) and the adjacent cytoplasm,10 11 sometimes along with components of the paired helical filament (PHF) such as tau. This study examined neurons in the nbM in AD for expression of ,BAPP to more fully understand the relationship between IAPP and NFTs in this critically important cell group.
The nucleus basalis ofMeynert (nbM) was examined using immunocytochemistry for P-amyloid precursor protein (IAPP) expression in Alzbeimer's disease (AD). In mild AD cases, light labeling of the cell body and proximal processes was observee4 and small intracellular structures were labeled rarely. In the more severe cases, intense cytoplasmic PAPP labeling was seen, often along with small APP-positive structures. Double-labeling experiments demonstrated that in the more severe cases these small structures were also decorated by a neurofibrillary tangle (NFT) antiserum. Other neurons in the severe cases showed incorporation of iAPP into large inclusions which were also labeled with the NFT antiserumn However, some large inclusions in the severe cases were labeled by the NFT antiserum but contained no PAPP. Extraneuronal NFTs did not show PAPP labeling and did not react with an antibody to the n-amyloid peptide. These results suggest that increased expression of PAPP coincides with intracellular NA formation in the nbM, but that the formation of extraneuronal NFTs results in a loss of IAPP immunoreactivity. (AmJPathol 1992, 141:357-361)
Material included nine cases with clinical and neuropathologic diagnoses of Alzheimer's disease, and one 38-year-old case with Down's Syndrome (DS) (Table 1). For ,APP immunostaining, blocks from the basal forebrain and superior temporal lobe were immersed immediately at autopsy in methacam (60% methanol, 30% chloroform, 10% glacial acetic acid) at 40C for 24 to 48 hours, then embedded in paraffin; 5-,Lm sections were cut at the level of the anterior commissure so as to include the giant neurons of the nbM scattered throughout the substantia innominata. The sections corresponded to Figure 1 OG and 1 OH of Hedreen et al.12 The remainder of the brain was fixed in formalin and a comprehensive gross and microscopic neuropathologic workup was performed. Cases were classified as mild, moderate, or severe AD using a semiquantitative scale based on the
The nucleus basalis of Meynert (nbM) is a focus of neuropathologic change in Alzheimer's disease (AD).1 Normally, this cell group provides cholinergic innervation to
Supported by the Dana Foundation, DVA, NIMH, and the State of Califomia. Accepted for publication January 31, 1992. Address reprint requests to Dr. G. M. Murphy, Jr., Psychiatry Service (1 16A3), Palo Alto Veterans Affairs Medical Center, Palo Alto, CA 94304.
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Table 1. Case Material Case Sex M
2 3 4 5 6 7 8 9 10
M
F F F M M
F M M
AD
=
Age
Diagnosis
64 75 77 78 80 80 83 86 87 38
AD, severe AD, severe AD, moderate AD, severe AD, mild AD, severe AD, moderate AD, severe AD, severe DS with mild AD
Alzheimer's disease; DS
=
Down's syndrome.
number of plaques and NFTs in silver-stained sections from a variety of brain regions. In practice, this evaluation gives results similar to the system of Khachaturian.13 All cases were free of other neuropathologic changes, except for a single Lewy body (LB) in the locus ceruleus in case 4 and a single small cortical infarct in cases 3 and 6. For antibody production, 3APP-6957 was generated using a baculovirus expression system. A termination codon was engineered at two positions C-terminal to the PAP domain, functionally deleting the C-terminal 56 amino acids. Expressed ,BAPP was purified from conditioned media to apparent homogeneity through a series of chromatographic steps. The protein was shown to extend from the natural IAPP N-terminus. These details and C-terminal sequence analysis are reported elsewhere.14 Polyclonal antisera to this purified protein were raised in rabbits by standard methods. Use of these antisera was previously reported.10 15'16 To identify NFTs, an antiserum to SDS-isolated PHF was used.17 We also used a monoclonal antibody raised to a synthetic peptide corresponding to residues 1-28 of the ,BAP (California Biotechnology, Mountain View, CA). The epitope for this reagent has been mapped to residues 1-10 of the synthetic PAP immunogen.
,BAPP immunocytochemistry was performed using the peroxidase-antiperoxidase (PAP) method,18 or the avidin-biotin (AB) method (Vectastain ABC Kit, Vector Labs, Burlingame, CA). NFT and PAP immunocytochemistry were performed with the AB method. For both the PAP and the AB reactions, sections were pretreated with 1% H202 in methanol for 30 minutes, and diaminobenzidine (DAB) was used as the chromogen. A pre-immune serum for the 3APP antibody was used to check for nonspecific reactivity. As previously reported by Cras et al,10 formic acid pretreatment did not enhance the reactivity of the ,BAPP reagent in methacarn-fixed tissue. Likewise, formic acid did not improve the performance of the PAP antibody in tissue treated with methacarn. For double labeling, the ,APP reaction was performed first, using either the PAP or the AB methods, and DAB was used as
the chromogen. After treatment in diluted aqueous HCI (99 ml of H20 and 1 ml of 30% HCI) for 1 hour, secondary labeling for NFT was done using the AB method. The NFT reaction was visualized with 4-chloro-1 -naphthol.
Results ,BAPP expression in the nbM was found to vary considerably among the AD cases. In the mild AD case, and in the DS case, nbM neurons showed a uniform granular labeling of the soma with the PAPP antiserum. This ,APP labeling was considerably stronger than the background signal obtained with the pre-immune antiserum. Labeling often extended into proximal nerve cell processes (Figure 1). Rarely neurons in the mild AD case showed an aggregation of 3APP-positive material into small intracellular structures (Figure 1). These structures were not observed in the DS case. In the sections from the mild AD case and the DS case that were reacted with the NFT antiserum or with an antiserum to mixed tau/MAP-2 epitopes,19 no intracellular structures were detected. In the moderate and severe AD cases, neurons usually showed more intense cytoplasmic ,BAPP immunoreactivity, often in a punctate pattern in the perinuclear region (Figure 2). Small 3APP-containing structures, which were also labeled with the NFT antiserum, were seen in many cells in moderate and severe cases (Figures 2, 3). Other neurons in severe AD cases showed heavy labeling of large intracellular inclusions with the ,APP antiserum (Figure 4). Double labeling revealed that these large inclusions reacted with both the 3APP and the NFT antisera (Figures 3, 5). Double labeling also showed that some neurons in the moderate and severe cases with intense perinuclear ,APP had no NFT formation (Figures
3, 5). Occasionally, neurons in the moderate and severe cases displayed intracellular inclusions that were not strongly labeled for IAPP (Figure 6), although they were labeled by the NFT antiserum. Often there was an aggregation of 3APP-positive material around the edges of these inclusions. None of the inclusions in the nbM were eosinophilic. Extracellular NFTs in the nbM were not labeled with the ,APP antisera, although they were strongly reactive with the NFT reagent (Figures 3, 5). Neither intracellular nor extracellular NFTs were labeled with the PAP antibody, even though strong labeling of neuritic plaque amyloid in the adjacent amygdala was observed (data not shown).
Discussion These results show that variation exists among AD cases in the expression of ,APP in nbM neurons. Comparison
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Figure 1. Top left: Neurons in the nbM, case 5. There is a uniform cytoplosmic labeling in the cell soma and proxoimal cell processes with the PAPP antiserum. A small intranearonal structure (arrowhead) is pIAPP-positive. Figure 2. Top right: Punctate cytoplasmic labelling (arrowhead) and intraneuronal inclusion (arrow), APP antiserum, case 8. An inclusion that is not strongly labeled for IAPP is also present (open arrow). Figure 3. Middle left: Double labeling of PAPP (brown) and NFT (purple), case 8. Neuron at arrowhead shows small areas of NFT formation against a background of ,BAPP. Another neuron (arrow) shows heavy PAPP expression but no NFT. An extraneuronal NFT (open arrow) is not labeled by ,BAPP antiserum. Figure 4. Middle right: Intraneuronal NFT in nbM strongly labeled with P.APP antiserum, case 1. Figure 5. Bottom left: Double labeling with APP (brown), and NFT(purple), case 8. Two neurons (arrowbeads) show simultaneous strong labeling for f.APP and NFT Extraneuronal NFTs are not labeled with 3APP antiserum (open arrows). Figure 6. Bottom right: Neurofibrillary tangle unlabeled by 3APP antiserum (arrowhead), case 8. Note accumulation of fIAPP around perfpbery of inclusion.
of mild, moderate, and severe AD cases suggests there may be a progression of ,APP expression. Mild AD and DS cases show uniform, low levels of ,APP labeling in nbM neurons. In moderate and severe AD cases, PAPP immunoreactivity in nbM cells is more intense, especially in the perinuclear region. Increased neuronal IAPP im-
munolabeling in AD has also been reported in the pyramidal layer of Ammon's horn, perhaps contained in lysosomes.2021 Perinuclear accumulation of iAPP, similar to that observed in the nbM in the present study, has also been reported in muscle cells22 and in several cell
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The enhanced 3APP labeling of nbM cells observed in moderate and severe AD cases in the present study could be due to transcriptional abnormalities translated to the protein level, because there is evidence of increased 3APP mRNA in the nbM in AD. Increases in total IAPP mRNA,8 as well as the PAPP-695 splicing variant9 have been reported with in situ hybridization. Alternatively, the normal metabolism of ,APP may be impaired in AD, resulting in its intracellular accumulation. A number of studies have reported that dystrophic neurites in plaques show strong ,APP immunoreactivity.24 If some of these neurites are axon terminals of nbM neurons, BAPP in them could originate in the cell body, as IAPP is transported anterogradely along axons.25 IAPP and ,BAP have been colocalized in plaques, which may indicate that processing of ,APP to ,BAP then occurs in situ.16'26 Comparisons of 3APP expression in nbM neurons and in neurites located in nbM projection fields will be needed to test the hypothesis of anterograde transport of IAPP in AD brain. In addition to increased cytoplasmic immunoreactivity for ,APP, many nbM neurons in the moderate and severe AD cases showed small areas of NFT formation. If these cells are representative of an early or intermediate stage of neuronal degeneration, increased neuronal ,BAPP immunoreactivity may coincide with NFT formation. Other neurons in the moderate and severe cases showed large ,APP-positive intraneuronal NFTs. This suggests that as neuronal degeneration proceeds, ,BAPP is incorporated into or becomes associated with many large NFTs. This could be due to impaired clearance of ,BAPP from dying cells. A few PAPP-positive inclusions were detected in the mild AD case. Other sections from this case labeled with NFT, and tau/MAP-2 antisera did not show inclusions in the nbM. This could indicate that ,BAPP accumulates in the nbM before NFT and tau/MAP-2 epitopes. Alternatively, because of the low frequency of RAPP-positive inclusions in the mild AD case, the sections reacted with the NFT and tau/MAP-2 antisera may have been negative due to chance alone. Additional mild AD cases will be needed to clarify this issue. Although most intraneuronal inclusions in the nbM in the moderate and severe cases contained ,APP, some did not. If the PAPP-negative inclusions were LB, strong eosinophilia and a lack of NFT labeling would be expected.27 The absence of LB elsewhere in the brain in our sample makes it unlikely that these inclusions were LB. ,APP does not persist in a form recognizable to our antiserum in extraneuronal tangles. Yamaguchi et a11" have likewise reported an absence of ,BAPP in extraneuronal tangles in the frontal and temporal cortices. ,APP associated with NFTs may therefore be sensitive to pro-
cesses involved in the conversion of intracellular NFTs to extracellular tombstone tangles. Further, in the extracellular space, ,BAPP in tombstone tangles may be vulnerable to proteolytic degradation. Trypsin pretreatment has been shown to reduce the ,APP immunoreactivity of NFTs extracted from AD brain.11 Labeling of extracellular hippocampal and cortical NFTs with PAP antibodies has been reported in AD and other neuropathologic conditions,28-30 although in many studies this has not been observed. In this study, the ,BAP monoclonal antibody did not label extracellular tangles in the nbM. However, it did label a few extracellular tangles in the pyramidal layer of Ammon's horn in the same cases (unpublished observation). This may mean that there are differences among anatomic regions in the reactivity of NFTs to PAP antibodies, perhaps because NFT epitopes vary from region to region. Other antigenic properties of NFTs have also been reported to depend on anatomic location.31 In summary, ,APP expression by neurons in the nbM varies among AD cases, but appears to increase with progression of the disease. Neurons often simultaneously showed increased expression of 3APP and the formation of NFTs. Some intraneuronal NFTs in the nbM were labeled by ,BAPP and NFT antisera, but extracellular tangles reacted with NFT antiserum alone. PAP was not observed in intracellular or extracellular tangles in the nbM. These results indicate that for many neurons in the nbM, cell death is preceded by an overexpression and/or accumulation of ,BAPP, but ,BAPP is lost in the extracellular
tangle.
Acknowledgments The authors thank Dr. Dennis Selkoe for the PHF-NFT and the tau/MAP-2 antisera; Dr. Barbara Cordell for valuable advice and the PAP antiserum; Erin Murphy, Karen Stultz, and Raj Shrivastava for assistance in the immunostaining; Leslie Fiedler for manuscript editing; and Yuen-Ling Lee for helpful advice.
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