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Aβ42, Presenilins, and Alzheimer’s Disease
Neurobiology of Aging, Vol. 19, No. 1S, pp. S11–S13, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00
PII:S0197-4580(98)00027-X
Ab42, Presenilins, and Alzheimer’s Disease T. IWATSUBO1 Department of Neuropathology and Neuroscience, Faculty of Pharmaceutical Sciences, University of Tokyo, 7–3-1 Hongo Bunkyoku, Tokyo 113, Japan IWATSUBO, T. Ab42, presenilins, and Alzheimer’s disease. NEUROBIOL AGING 19(1S) S11–S13, 1998.—The significance of amyloid b protein, especially those ending at the 42nd residue (Ab42), in the pathogenesis of familial Alzheimer’s disease (FAD) linked to the mutations of presenilins, was examined by transfection studies using cultured cells and immunohistochemical analysis of autopsied brains. The levels of Ab42 secreted from cells transfected with mutant presenilins linked to FAD, as well as the Ab42 burden in the cortices of patients with presenilin mutation were elevated. Thus, mutations in presenilin genes may enhance the production and deposition of Ab42 in the brains, thereby leading to Alzheimer’s disease. © 1998 Elsevier Science Inc. Alzheimer’s disease
Amyloid b protein
Ab42
Presenilin
THE deposition of amyloid b protein (Ab) as senile plaques (SP) and in the walls of cerebral blood vessels is one of the invariant features of the brains in patients with Alzheimer’s disease (AD). Ab is a 40 – 42 amino acid peptides that are proteolytically cleaved from a longer precursor protein termed b-amyloid precursor protein (bAPP). Ab deposition is thought to be closely related to the pathogenesis of AD because 1) it is exclusively seen in limited pathological conditions including AD, Down’s syndrome (DS), and aging; 2) accumulation of Ab as diffuse plaques (DPs) is the earliest neuropathological change thus far known in AD and DS; and, most importantly, 3) clinical manifestations of AD cosegregate with missense mutations located in the Ab flanking regions of bAPP in some pedigrees of early-onset familial AD (FAD). The discovery that Ab1– 40 is the major species in secreted Ab that is constitutively secreted into culture medium and cerebrospinal fluids implied that secreted Ab (1– 40) should be the major component of amyloid deposits in AD brains. However, the results derived from in vitro experiments that Ab ending at the 42nd residue (Ab42) polymerizes much faster than those ending at the 40th residue (Ab40) suggested that the C-terminal properties of Ab determine its aggregation potential, and, thus, important factors in b-amyloidogenesis (6). We examined cerebral cortices from patients with AD (3), DS (3,4), and non-demented aged individuals (ND) (2,5) by immunohistochemistry using monoclonal antibodies BA27 and BC05, that discriminate the C-terminus of Ab40 and Ab42, respectively, and found that Ab42 was invariably the predominant Ab species deposited as SP. Notably, DPs, one of the earliest form of Ab deposition, were exclusively positive for Ab42 (3,4), suggesting that Ab42 is the initially deposited Ab species in SPs. The missense mutations of bAPP at position 717 linked to FAD have been shown to increase the secretion of Ab42 in cells transfected with this gene (10,12). In accord with these findings, the ratio of Ab42-positive SP in the brains of patients with FAD with bAPP717 mutation (3), as well as the percentage of Ab42 in the blood plasma of patients with this mutation (7) were elevated.
Recently, it has been shown that plasma levels of Ab42 are elevated in patients with early-onset FAD carrying mutations in presenilin 1 (PS1) on chromosome 14, as well as in presenilin 2 (PS2) on chromosome 1 (11). In this study, we tested the hypothesis that missense mutations in PS genes cause FAD by enhancing the production and deposition of Ab42 by 1) transfection experiments of cDNAs encoding human PS2 of wild-type and with a mutation linked to FAD; and 2) morphometric analysis of the autopsied brain tissues from patients carrying mutant PS1 and PS2 genes. MATERIALS AND METHODS
COS-1 cells were transfected with cDNAs encoding wild-type or N141I mutant PS2 together with human APP cDNAs. Mouse neuroblastoma neuro2a (N2a) cells were transfected with wildtype or N141I mutant PS3 cDNAs, and cells stably expressing these cDNAs were cloned. The levels of Ab40 and Ab42 secreted from these cells were quantitated by two-site ELISAs as described (13). Sections from formalin-fixed, paraffin embedded frontal cortices from eight cases of FAD with five different PS1 mutations (8) and six cases with Volga German (N141I) PS2 mutation (9) were immunostained with BC05 and BA27, and the density or percentage area covered by immunopositive deposits were calculated by computer-assisted morphometry as described (3,4). RESULTS AND DISCUSSION
The mean level 6 S.E. of Abx-42 secreted from COS cells transiently transfected with N141I mutant PS2 and bAPP cDNAs was 53.2 6 6.7 pM (n 5 3), which was 1.8-fold compared with that from cells with wild-type PS2 (29.6 6 1.7 pM). The mean level 6 S.E. of Ab42 secreted from three independent N2a cell lines stably expressing N141I mutant PS2 was 262.9 6 14.9 pM (n 5 4 per each clone), which were ;8 times higher than those in cells with wild-type PS2 (33.6 6 3.6 pM). The density and percentage area of Ab42-positive SP were
1 Address correspondence to: Takeshi Iwatsubo, Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7–3-1 Hongo Bunkyoku, Tokyo 113-0033, Japan; E-mail: [email protected].
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FIG. 1. Morphometric analysis of Ab deposition in the brains of patients with familial Alzheimer’s disease. Mean density (mm2) and percentage area of plaques covered by Ab42- (sum of white and black columns) or Ab40-positive (black column) plaques 6 S.E. are shown. The white part represents those of Ab42-positive, Ab40-negative plaques. PS1: familial Alzheimer’s disease (FAD) with mutations in PS1 genes. PS2: FAD (Volga German families) with N141I PS2 mutation. APP717: FAD with bAPP mutation at position 717. Elderly DS: Down’s syndrome over the age of 50. Sporadic AD: AD without family history.
both markedly increased in PS1 mutants (223.9 6 124.9/mm2, 14.0 6 5.4%), these being significantly higher than those in sporadic AD (sAD; p , 0.01 by ANOVA). In contrast, those values in N141I PS2 mutants (133.5 6 66.2/mm2, 7.9 6 3.1%) were similar to those in sAD, although Ab42 was again the predominant species deposited in these brains (Fig. 1). These present data strongly suggest that mutations in PS genes cause FAD by fostering the production and deposition of Ab42. Thus, the increase in the production and deposition of Ab42 is a common feature of all three types of early-onset FAD caused by mutations in distinct genes (i.e., bAPP, PS1, and PS2). The mechanism by which mutant PS leads to the increased secretion of Ab42 is unknown at present; mutant PS may interact with bAPP and change its conformation in a way to be more susceptible to Ab42 cleavage by some “chaperone-like” effect, or, alternatively, it may directly affect the activity of g-secretase(s). Mutant PS may change the protein trafficking in the intracellular vesicular system in which Ab is produced. In this context, recent discovery that
Ab42 is produced in endoplasmic reticulum (1) dovetails with the localization of PS to endoplasmic reticulum, suggesting the direct or indirect interaction of these two molecules in this compartment. Thus, there emerges a scheme in which a number of pathogenic genes and their downstream pathways converge into the production and deposition of Ab, especially Ab42 species, leading to the development of AD. However, the possibility of some additional pathogenetic mechamisms that are related to, or independent from, the production and deposition of Ab42 should also be explored. ACKNOWLEDGEMENTS
The author thanks Drs. Kei Maruyama, Taisuke Tomita, Takaomi C. Saido, David M.A. Mann and all other collaborators for their significant contributions to this study. Especially, the molecular biological studies of presenilins were conducted under the direction of K.M. at the Laboratory of Neurochemistry, National Institute for Physiological Sciences, Japan, as well as in the University of Tokyo.
REFERENCES 1. Cook, D. G.; Forman, M.; Sung, J. C.; Leight, S.; Kolson, D. L.; Iwatsubo, T.; Lee, V. M.-Y.; Doms, R. W. Alzheimer Ab(1– 42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nature Med. 3:1021–1023; 1997. 2. Fukumoto, H.; Asami-Odaka, A.; Suzuki, N.; Shimada, H.; Ihara, Y.; Iwatsubo, T. Amyloid b protein deposition in normal aging has the same characteristics as that in Alzheimer’s disease. Predominance of
Ab42(43) and association of Ab40 with cored plaques. Am. J. Pathol. 148:259 –265; 1996. 3. Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Visualization of Ab42(43) and Ab40 in senile plaques with end-specific Ab monoclonals: evidence that an initially deposited species is Ab42(43). Neuron 13:45–53; 1994. 4. Iwatsubo, T.; Mann, D. M. A.; Odaka, A.; Suzuki, N.; Ihara, Y.
Ab42 AND PRESENILIN IN AD
5.
6.
7.
8.
9.
Amyloid b protein (Ab) deposition. Ab42(43) precedes Ab40 in Down syndrome. Ann. Neurol. 37:294 –299; 1995. Iwatsubo, T.; Saido, T. C.; Mann, D. M. A.; Lee, V. M.-Y.; Trojanowski, J. Q. Full-length amyloid-b(1– 42(43)) as well as aminoterminally modified and truncated amyloid-b42(43) deposit in diffuse plaques. Am. J. Pathol. 149:1823–1830; 1996. Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. Jr. The carboxy terminus of the b amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer’s disease. Biochemistry 32:4693– 46971; 1993. Kosaka, T.; Imagawa, M.; Seki, K.; Arai, H.; Sasaki, H.; Tsuji, S.; Asami-Odaka, A.; Fukushima, T.; Imai, K.; Iwatsubo, T. The bAPP717 Alzheimer mutation increases the percentage of plasma amyloid b protein ending at Ab42(43). Neurology 48:741–745; 1997. Mann, D. M. A.; Iwatsubo, T.; Cairns, N. J.; Lantos, P. L.; Nochlin, D.; Sumi, S. M.; Bird, T. D.; Poorkaj, P.; Hardy, J.; Hutton, M.; Prihar, G.; Crook, R.; Rossor, M. N.; Haltia, M. Amyloid b protein (Ab) deposition in chromosome 14-linked Alzheimer’s disease: Predominance of Ab42(43). Ann. Neurol. 40:149 –156; 1996. Mann, D. M. A.; Iwatsubo, T.; Nochlin, D.; Sumi, M.; Levy-Lahad, E.; Bird, T. D. Amyloid (Ab) deposition in chromosome 1-linked Alzheimer’s disease.—The Volga German families. Ann. Neurol. 41:52– 57; 1997.
S13 10. Maruyama, K.; Tomita, T.; Shinozaki, K.; Kume, H.; Asada, H.; Saido, T. C.; Ishiura, S.; Iwatsubo, T.; Obata, K. Familial Alzheimer’s disease-linked mutations at Val717 of amyloid precursor protein are specific for the increased secretion of Ab42(43). Biochem. Biophys. Res. Comm. 227:730 –735; 1996. 11. Scheuner, D.; Eckman, C.; Jensen, M.; Song, X.; Citron, M.; Suzuki, N.; Bird, T. D.; Hardy, J.; Hutton, M.; Kukull, W.; Larson, E.; Levy-Lahad, E.; Viitanen, M.; Peskind, E.; Poorkaj, P.; Schellenberg, G.; Tanzi, R.; Wasco, W.; Lannfelt, L.; Selkoe, D.; Younkin, S. Secreted amyloid b-protein similar to that in the senile plaques of Alzheimer’s disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer’s disease. Nature Med. 2:864 – 870; 1996. 12. Suzuki, N.; Cheung, T. T.; Cai X.-D.; Odaka, A.; Otvos, L. Jr.; Eckman, C.; Golde, T. E.; Younkin, S. G. An increased percentage of long amyloid b-protein is secreted by familial amyloid b-protein precursor (bAPP717) mutants. Science 264:1336 –1340; 1994. 13. Tomita, T.; Maruyama, K.; Saido, T. C.; Kume, H.; Shinozaki, K.; Tokuhiro, S.; Capell, A.; Walter, J.; Gru¨nberg, J.; Haass, C.; Iwatsubo, T.; Obata, K. The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid b protein ending at the 42nd (or 43rd) residue. Proc. Natl. Acad. Sci. USA 94:2025–2030; 1997.
PII:S0197-4580(98)00027-X
Ab42, Presenilins, and Alzheimer’s Disease T. IWATSUBO1 Department of Neuropathology and Neuroscience, Faculty of Pharmaceutical Sciences, University of Tokyo, 7–3-1 Hongo Bunkyoku, Tokyo 113, Japan IWATSUBO, T. Ab42, presenilins, and Alzheimer’s disease. NEUROBIOL AGING 19(1S) S11–S13, 1998.—The significance of amyloid b protein, especially those ending at the 42nd residue (Ab42), in the pathogenesis of familial Alzheimer’s disease (FAD) linked to the mutations of presenilins, was examined by transfection studies using cultured cells and immunohistochemical analysis of autopsied brains. The levels of Ab42 secreted from cells transfected with mutant presenilins linked to FAD, as well as the Ab42 burden in the cortices of patients with presenilin mutation were elevated. Thus, mutations in presenilin genes may enhance the production and deposition of Ab42 in the brains, thereby leading to Alzheimer’s disease. © 1998 Elsevier Science Inc. Alzheimer’s disease
Amyloid b protein
Ab42
Presenilin
THE deposition of amyloid b protein (Ab) as senile plaques (SP) and in the walls of cerebral blood vessels is one of the invariant features of the brains in patients with Alzheimer’s disease (AD). Ab is a 40 – 42 amino acid peptides that are proteolytically cleaved from a longer precursor protein termed b-amyloid precursor protein (bAPP). Ab deposition is thought to be closely related to the pathogenesis of AD because 1) it is exclusively seen in limited pathological conditions including AD, Down’s syndrome (DS), and aging; 2) accumulation of Ab as diffuse plaques (DPs) is the earliest neuropathological change thus far known in AD and DS; and, most importantly, 3) clinical manifestations of AD cosegregate with missense mutations located in the Ab flanking regions of bAPP in some pedigrees of early-onset familial AD (FAD). The discovery that Ab1– 40 is the major species in secreted Ab that is constitutively secreted into culture medium and cerebrospinal fluids implied that secreted Ab (1– 40) should be the major component of amyloid deposits in AD brains. However, the results derived from in vitro experiments that Ab ending at the 42nd residue (Ab42) polymerizes much faster than those ending at the 40th residue (Ab40) suggested that the C-terminal properties of Ab determine its aggregation potential, and, thus, important factors in b-amyloidogenesis (6). We examined cerebral cortices from patients with AD (3), DS (3,4), and non-demented aged individuals (ND) (2,5) by immunohistochemistry using monoclonal antibodies BA27 and BC05, that discriminate the C-terminus of Ab40 and Ab42, respectively, and found that Ab42 was invariably the predominant Ab species deposited as SP. Notably, DPs, one of the earliest form of Ab deposition, were exclusively positive for Ab42 (3,4), suggesting that Ab42 is the initially deposited Ab species in SPs. The missense mutations of bAPP at position 717 linked to FAD have been shown to increase the secretion of Ab42 in cells transfected with this gene (10,12). In accord with these findings, the ratio of Ab42-positive SP in the brains of patients with FAD with bAPP717 mutation (3), as well as the percentage of Ab42 in the blood plasma of patients with this mutation (7) were elevated.
Recently, it has been shown that plasma levels of Ab42 are elevated in patients with early-onset FAD carrying mutations in presenilin 1 (PS1) on chromosome 14, as well as in presenilin 2 (PS2) on chromosome 1 (11). In this study, we tested the hypothesis that missense mutations in PS genes cause FAD by enhancing the production and deposition of Ab42 by 1) transfection experiments of cDNAs encoding human PS2 of wild-type and with a mutation linked to FAD; and 2) morphometric analysis of the autopsied brain tissues from patients carrying mutant PS1 and PS2 genes. MATERIALS AND METHODS
COS-1 cells were transfected with cDNAs encoding wild-type or N141I mutant PS2 together with human APP cDNAs. Mouse neuroblastoma neuro2a (N2a) cells were transfected with wildtype or N141I mutant PS3 cDNAs, and cells stably expressing these cDNAs were cloned. The levels of Ab40 and Ab42 secreted from these cells were quantitated by two-site ELISAs as described (13). Sections from formalin-fixed, paraffin embedded frontal cortices from eight cases of FAD with five different PS1 mutations (8) and six cases with Volga German (N141I) PS2 mutation (9) were immunostained with BC05 and BA27, and the density or percentage area covered by immunopositive deposits were calculated by computer-assisted morphometry as described (3,4). RESULTS AND DISCUSSION
The mean level 6 S.E. of Abx-42 secreted from COS cells transiently transfected with N141I mutant PS2 and bAPP cDNAs was 53.2 6 6.7 pM (n 5 3), which was 1.8-fold compared with that from cells with wild-type PS2 (29.6 6 1.7 pM). The mean level 6 S.E. of Ab42 secreted from three independent N2a cell lines stably expressing N141I mutant PS2 was 262.9 6 14.9 pM (n 5 4 per each clone), which were ;8 times higher than those in cells with wild-type PS2 (33.6 6 3.6 pM). The density and percentage area of Ab42-positive SP were
1 Address correspondence to: Takeshi Iwatsubo, Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7–3-1 Hongo Bunkyoku, Tokyo 113-0033, Japan; E-mail: [email protected].
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IWATSUBO
FIG. 1. Morphometric analysis of Ab deposition in the brains of patients with familial Alzheimer’s disease. Mean density (mm2) and percentage area of plaques covered by Ab42- (sum of white and black columns) or Ab40-positive (black column) plaques 6 S.E. are shown. The white part represents those of Ab42-positive, Ab40-negative plaques. PS1: familial Alzheimer’s disease (FAD) with mutations in PS1 genes. PS2: FAD (Volga German families) with N141I PS2 mutation. APP717: FAD with bAPP mutation at position 717. Elderly DS: Down’s syndrome over the age of 50. Sporadic AD: AD without family history.
both markedly increased in PS1 mutants (223.9 6 124.9/mm2, 14.0 6 5.4%), these being significantly higher than those in sporadic AD (sAD; p , 0.01 by ANOVA). In contrast, those values in N141I PS2 mutants (133.5 6 66.2/mm2, 7.9 6 3.1%) were similar to those in sAD, although Ab42 was again the predominant species deposited in these brains (Fig. 1). These present data strongly suggest that mutations in PS genes cause FAD by fostering the production and deposition of Ab42. Thus, the increase in the production and deposition of Ab42 is a common feature of all three types of early-onset FAD caused by mutations in distinct genes (i.e., bAPP, PS1, and PS2). The mechanism by which mutant PS leads to the increased secretion of Ab42 is unknown at present; mutant PS may interact with bAPP and change its conformation in a way to be more susceptible to Ab42 cleavage by some “chaperone-like” effect, or, alternatively, it may directly affect the activity of g-secretase(s). Mutant PS may change the protein trafficking in the intracellular vesicular system in which Ab is produced. In this context, recent discovery that
Ab42 is produced in endoplasmic reticulum (1) dovetails with the localization of PS to endoplasmic reticulum, suggesting the direct or indirect interaction of these two molecules in this compartment. Thus, there emerges a scheme in which a number of pathogenic genes and their downstream pathways converge into the production and deposition of Ab, especially Ab42 species, leading to the development of AD. However, the possibility of some additional pathogenetic mechamisms that are related to, or independent from, the production and deposition of Ab42 should also be explored. ACKNOWLEDGEMENTS
The author thanks Drs. Kei Maruyama, Taisuke Tomita, Takaomi C. Saido, David M.A. Mann and all other collaborators for their significant contributions to this study. Especially, the molecular biological studies of presenilins were conducted under the direction of K.M. at the Laboratory of Neurochemistry, National Institute for Physiological Sciences, Japan, as well as in the University of Tokyo.
REFERENCES 1. Cook, D. G.; Forman, M.; Sung, J. C.; Leight, S.; Kolson, D. L.; Iwatsubo, T.; Lee, V. M.-Y.; Doms, R. W. Alzheimer Ab(1– 42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nature Med. 3:1021–1023; 1997. 2. Fukumoto, H.; Asami-Odaka, A.; Suzuki, N.; Shimada, H.; Ihara, Y.; Iwatsubo, T. Amyloid b protein deposition in normal aging has the same characteristics as that in Alzheimer’s disease. Predominance of
Ab42(43) and association of Ab40 with cored plaques. Am. J. Pathol. 148:259 –265; 1996. 3. Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Visualization of Ab42(43) and Ab40 in senile plaques with end-specific Ab monoclonals: evidence that an initially deposited species is Ab42(43). Neuron 13:45–53; 1994. 4. Iwatsubo, T.; Mann, D. M. A.; Odaka, A.; Suzuki, N.; Ihara, Y.
Ab42 AND PRESENILIN IN AD
5.
6.
7.
8.
9.
Amyloid b protein (Ab) deposition. Ab42(43) precedes Ab40 in Down syndrome. Ann. Neurol. 37:294 –299; 1995. Iwatsubo, T.; Saido, T. C.; Mann, D. M. A.; Lee, V. M.-Y.; Trojanowski, J. Q. Full-length amyloid-b(1– 42(43)) as well as aminoterminally modified and truncated amyloid-b42(43) deposit in diffuse plaques. Am. J. Pathol. 149:1823–1830; 1996. Jarrett, J. T.; Berger, E. P.; Lansbury, P. T. Jr. The carboxy terminus of the b amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer’s disease. Biochemistry 32:4693– 46971; 1993. Kosaka, T.; Imagawa, M.; Seki, K.; Arai, H.; Sasaki, H.; Tsuji, S.; Asami-Odaka, A.; Fukushima, T.; Imai, K.; Iwatsubo, T. The bAPP717 Alzheimer mutation increases the percentage of plasma amyloid b protein ending at Ab42(43). Neurology 48:741–745; 1997. Mann, D. M. A.; Iwatsubo, T.; Cairns, N. J.; Lantos, P. L.; Nochlin, D.; Sumi, S. M.; Bird, T. D.; Poorkaj, P.; Hardy, J.; Hutton, M.; Prihar, G.; Crook, R.; Rossor, M. N.; Haltia, M. Amyloid b protein (Ab) deposition in chromosome 14-linked Alzheimer’s disease: Predominance of Ab42(43). Ann. Neurol. 40:149 –156; 1996. Mann, D. M. A.; Iwatsubo, T.; Nochlin, D.; Sumi, M.; Levy-Lahad, E.; Bird, T. D. Amyloid (Ab) deposition in chromosome 1-linked Alzheimer’s disease.—The Volga German families. Ann. Neurol. 41:52– 57; 1997.
S13 10. Maruyama, K.; Tomita, T.; Shinozaki, K.; Kume, H.; Asada, H.; Saido, T. C.; Ishiura, S.; Iwatsubo, T.; Obata, K. Familial Alzheimer’s disease-linked mutations at Val717 of amyloid precursor protein are specific for the increased secretion of Ab42(43). Biochem. Biophys. Res. Comm. 227:730 –735; 1996. 11. Scheuner, D.; Eckman, C.; Jensen, M.; Song, X.; Citron, M.; Suzuki, N.; Bird, T. D.; Hardy, J.; Hutton, M.; Kukull, W.; Larson, E.; Levy-Lahad, E.; Viitanen, M.; Peskind, E.; Poorkaj, P.; Schellenberg, G.; Tanzi, R.; Wasco, W.; Lannfelt, L.; Selkoe, D.; Younkin, S. Secreted amyloid b-protein similar to that in the senile plaques of Alzheimer’s disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer’s disease. Nature Med. 2:864 – 870; 1996. 12. Suzuki, N.; Cheung, T. T.; Cai X.-D.; Odaka, A.; Otvos, L. Jr.; Eckman, C.; Golde, T. E.; Younkin, S. G. An increased percentage of long amyloid b-protein is secreted by familial amyloid b-protein precursor (bAPP717) mutants. Science 264:1336 –1340; 1994. 13. Tomita, T.; Maruyama, K.; Saido, T. C.; Kume, H.; Shinozaki, K.; Tokuhiro, S.; Capell, A.; Walter, J.; Gru¨nberg, J.; Haass, C.; Iwatsubo, T.; Obata, K. The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid b protein ending at the 42nd (or 43rd) residue. Proc. Natl. Acad. Sci. USA 94:2025–2030; 1997.