- Email: [email protected]
Thiorphan-induced neprilysin inhibition raises amyloid β levels in rabbit cortex and cerebrospinal fluid
Neuroscience Letters 350 (2003) 178–180 www.elsevier.com/locate/neulet
Thiorphan-induced neprilysin inhibition raises amyloid b levels in rabbit cortex and cerebrospinal fluid Amanda J. Newella, Lucia I. Suea, Sarah Scotta, Paula K. Rauschkolbb, Douglas G. Walkera, Pamela E. Potterb, Thomas G. Beacha,* a
Sun Health Research Institute, 10515 West Santa Fe Drive, Sun City, AZ 85351, USA b Midwestern University, Glendale, AZ, USA
Received 14 May 2003; received in revised form 18 July 2003; accepted 21 July 2003
Abstract Studies on the pathogenesis of Alzheimer’s disease (AD) suggest overproduction of amyloid b (Ab) may not be the only pathogenic route to AD. Decreased degradation of Ab is another possible disease mechanism. Neprilysin is a neutral endopeptidase that has been proposed to be the major enzyme responsible for Ab degradation. Studies have reported correlations between Ab deposition and neprilysin activity in the human brain. This study shows that intracerebroventricular infusion of thiorphan, a neprilysin inhibitor, raises cortical and cerebrospinal fluid (CSF) Ab concentrations in rabbits. Rabbits treated with thiorphan for 5 days had levels of CSF and cortical Ab40 that were 147 and 142% of the control group, respectively. Results for Ab42 showed a similar trend. The results indicate that age-related decreases of neprilysin could lead to increased brain concentrations of Ab, plaque formation, and AD. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Amyloid b; Thiorphan; Neprilysin; Cerebral cortex; Rabbits; Animal model
Research over the last decade has established that brain accumulation and histologic deposition of a single peptide known as amyloid b (Ab) is a critical event in Alzheimer’s disease (AD). Many investigators believe that Ab overproduction and deposition leads to all of the other relevant pathologic changes in the disease, including neurofibrillary tangle formation, loss of synapses, neuronal death, and dementia [3]. Preventing this accumulation may prevent AD. Overproduction of Ab may not be the only route to AD. Attention has focused recently on the possibility that decreased degradation of Ab could also lead to Ab deposition [9]. Turnover of Ab in the brain is rapid and it has been estimated that very small decreases in its removal would lead to deposition [9]. Although several enzymes are likely to participate in the proteolysis of Ab, it appears that one, termed neprilysin, may be the most important. Neprilysin is a multifunctional neutral endopeptidase that was first investigated in relation to its ability to degrade enkephalin and substance P [6,11,12]. In a series of recent *
Corresponding author. Tel.: þ1-623-876-5643; fax: þ 1-623-876-5461. E-mail address: [email protected] (T.G. Beach).
experiments, Saido and colleagues have shown that neprilysin is the major degrading pathway for Ab in the brain [5,6,11,12]. Inhibition or genetic absence of neprilysin activity leads to increased brain Ab concentrations [5,6]. In humans, cortical neprilysin protein and message are inversely related to the density and location of Ab deposits in the non-demented elderly and in subjects with AD [13, 14], suggesting that loss of neprilysin activity may be an age-related change that leads to Ab deposition. Prolonged neprilysin inhibition in rodents by brain parenchymal infusion of thiorphan produced local Ab deposition [6], although this was not replicated by another group using intracerebroventricular infusion [4]. We therefore decided to repeat these experiments, with some minor variations. We have used rabbits as our experimental animal, rather than rodents, since rodent Ab differs from the human peptide by having three different amino acids, and there is some evidence that this difference leads to decreased amyloidogenic processing of b-amyloid precursor protein (bAPP) and/or decreased Ab aggregation [2,7,8]. It is possible, therefore, that the rodent Ab sequence may make rodents resistant to Ab overproduction and/or deposition. Since the rabbit amino acid sequence in and around the Ab
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00902-9
A.J. Newell et al. / Neuroscience Letters 350 (2003) 178–180
region of bAPP (complete peptides encoded by exons 15, 16 and 17) is identical to that of humans [1], this possibility is avoided. We infused thiorphan unilaterally into the lateral ventricle with indwelling intracerebroventricular cannulae (Plastics One, Inc, Roanoke, VA) connected to subcutaneous osmotic pumps (model 2ML1, Durect Corp., Cupertino, CA). Thiorphan or vehicle (25% absolute ethanol, 25% distilled water and 50% DMSO) were infused at a rate of 10 ml/h (1 mg/kg per day). Rabbits were euthanized after 5 days of treatment, the brains dissected out fresh and processed for detection of Ab by immunohistochemical, ELISA and immunoblot methods. Immunohistochemical localization of Ab was performed using paraformaldehyde-fixed tissue and standard immunoperoxidase methods utilizing the antibody 10D5 (amino acids 1 – 16 of Ab; Elan Pharmaceuticals, South San Francisco, CA). Cerebrospinal fluid (CSF) and fresh-frozen cerebral cortex was assayed using a commercially-available Ab ELISA kit (Biosource, Camarillo, CA). Frozen cortical tissue was extracted in four volumes of 5.5 M guanidine hydrochloride [10] in 50 mM Tris buffer at pH 8.0, following which the homogenate was left to stand at room temperature overnight. Homogenates were then diluted 1:10 in the Biosource diluent and centrifuged at 14,000 rpm for 20 min at 48C with the supernatant used for ELISA. Cerebrospinal fluid for ELISA analysis was diluted with Biosource diluent at 1:20 for Ab40 and 1:4 for Ab42. Immunoblot analysis of CSF Ab was performed to verify the ELISA results. Undiluted CSF samples were run on 4 – 12% NuPage Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. The membranes were boiled in phosphate-buffered saline buffer for 10 min to enhance antibody binding and were then incubated with the 6E10 antibody (Signet Laboratories, Dedham, MA), which recognizes amino acids 1 –16 of Ab, thereby labeling both Ab40 and Ab42. Bound antibody was detected with peroxidase-labeled anti-mouse IgG followed by incubation with chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). The location and intensity of bands was assessed using a ChemiImager 4000 blot imaging system (Alpha Innotech, San Leandro, CA) and by exposure of Xray film. The ELISA results showed that thiorphan treatment increased both cortical and CSF concentrations of Ab. Cerebrospinal fluid levels of Ab40 and 42 were increased to 147% (19.27 versus 13.08 ng/ml) and 120% (2.48 versus 2.06 ng/ml), respectively, of control animals (Fig. 1A). The result with Ab40 was significant (P ¼ 0:003, unpaired, twotailed t-test). Thiorphan treatment raised cortical concentrations of Ab40 to 142% (1.25 versus 0.88 pmol/g) of control animals (P ¼ 0:03, unpaired, two-tailed t-test) while the concentrations of cortical Ab42 were not significantly altered (Fig. 1B). The immunoblot results (Fig. 2) showed that CSF Ab (combined Ab40 and 42) was significantly elevated by thiorphan treatment (129% of control values,
179
Fig. 1. Graphs depicting the effect of thiorphan on CSF (A); and cortical Ab (B). Thiorphan administration resulted in significant increases in mean CSF (to 147% of control, P ¼ 0:003, unpaired t-test) and cortical Ab40 (to 142% of control, P ¼ 0:03, unpaired t-test). SD ¼ standard deviation.
P , 0:05, Mann – Whitney U-test). Immunohistochemical staining with the 10D5 antibody did not reveal evidence of Ab deposition or intraneuronal immunoreactivity in thiorphan-treated animals. As previously reported by others, intracerebral thiorphan infusion raises CNS Ab levels [4 – 6]. In contrast to previous reports, however, we did not see immunohistochemical evidence of increased Ab levels, either in the form of plaque-like structures [6] or intraneuronal staining [4]. It may be that longer infusion durations are required to produce histologically-detectable Ab as the aforementioned studies used durations of 30 and 37 days while we used a duration of 5 days. Our result may also help resolve a confounding issue brought up by a previous investigation [4], in which it was suggested that the effect of thiorphan infusion on Ab may be due to the use of ascorbic acid as a preservative in the infusing fluid. As we did not use ascorbic acid in our infusion solution, the observed increases in Ab could not have been due to this. We found that thiorphan had a greater effect on Ab40 than on Ab42. In contrast, others have reported the reverse to be true, in both in vitro [6,11] and in vivo [5,6] experiments. The reason for this discrepancy is unclear, but may be due to the different extraction methods, treatment duration or animal species used. In conclusion, these results demonstrate that in vivo inhibition of endogenous neprilysin activity via intracerebroventricular infusion of thiorphan results in increased concentrations of Ab in the CSF and cerebral cortex. This supports the theory that loss of brain neprilysin activity
Fig. 2. Image of immunoblot analysis of Ab from CSF from control and thiorphan-treated animals. The mean intensity of the 4 kD band in thiorphan-treated animals was significantly increased (129% of control, P , 0:05, Mann –Whitney U-test) compared to control animals. See text for additional discussion.
180
A.J. Newell et al. / Neuroscience Letters 350 (2003) 178–180
could be a pathogenic mechanism leading to age-related Ab deposition and AD.
[7]
Acknowledgements [8]
This work was supported by a grant from the National Institute on Aging (RO1NS38674) to T.G. Beach.
References [1] J.S. Davidson, R.L. West, P. Kotikalapudi, L.E. Maroun, Sequence and methylation in the beta/A4 region of the rabbit amyloid precursor protein gene, Biochem. Biophys. Res. Commun. 188 (1992) 905–911. [2] B. DeStrooper, M. Simons, G. Multhaup, F. Van Leuven, K. Beyreuther, C.G. Dotti, Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence, EMBO J. 14 (1995) 4932–4938. [3] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science 297 (2002) 353–356. [4] B. Hauss-Wegrzyniak, G.L. Wenk, Beta-amyloid deposition in the brains of rats chronically infused with thiorphan or lipopolysaccharide: the role of ascorbic acid in the vehicle, Neurosci. Lett. 322 (2002) 75–78. [5] N. Iwata, S. Tsubuki, Y. Takaki, K. Shirotani, B. Lu, N.P. Gerard, C. Gerard, E. Hama, H.J. Lee, T.C. Saido, Metabolic regulation of brain Abeta by neprilysin, Science 292 (2001) 1550–1552. [6] N. Iwata, S. Tsubuki, Y. Takaki, K. Watanabe, M. Sekiguchi, E. Hosoki, M. Kawashima-Morishima, H.J. Lee, E. Hama, Y. SekineAizawa, T.C. Saido, Identification of the major Abeta1–42-degrading catabolic pathway in brain parenchyma: suppression leads to
[9]
[10]
[11]
[12]
[13]
[14]
biochemical and pathological deposition, Nat. Med. 6 (2000) 143 –150. L. Otvos Jr, G.I. Szendrei, V.M. Lee, H.H. Mantsch, Human and rodent Alzheimer beta-amyloid peptides acquire distinct conformations in membrane-mimicking solvents, Eur. J. Biochem. 211 (1993) 249 –257. A.G. Reaume, D.S. Howland, S.P. Trusko, M.J. Savage, D.M. Lang, B.D. Greenberg, R. Siman, R.W. Scott, Enhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer’s disease mutations and a ‘humanized’ Abeta sequence, J. Biol. Chem. 271 (1996) 23380–23388. T.C. Saido, Alzheimer’s disease as proteolytic disorders: anabolism and catabolism of beta-amyloid, Neurobiol. Aging 19 (1998) S69–S75. M.J. Savage, S.P. Trusko, D.S. Howland, L.R. Pinsker, S. Mistretta, A.G. Reaume, B.D. Greenberg, R. Siman, R.W. Scott, Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester, J. Neurosci. 18 (1998) 1743–1752. K. Shirotani, S. Tsubuki, N. Iwata, Y. Takaki, W. Harigaya, K. Maruyama, S. Kiryu-Seo, H. Kiyama, H. Iwata, T. Tomita, T. Iwatsubo, T.C. Saido, Neprilysin degrades both amyloid beta peptides 1–40 and 1– 42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases, J. Biol. Chem. 276 (2001) 21895–21901. Y. Takaki, N. Iwata, S. Tsubuki, S. Taniguchi, S. Toyoshima, B. Lu, N.P. Gerard, C. Gerard, H.J. Lee, K. Shirotani, T.C. Saido, Biochemical identification of the neutral endopeptidase family member responsible for the catabolism of amyloid beta peptide in the brain, J. Biochem. (Tokyo) 128 (2000) 897 –902. K. Yasojima, H. Akiyama, E.G. McGeer, P.L. McGeer, Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide, Neurosci. Lett. 297 (2001) 97–100. K. Yasojima, E.G. McGeer, P.L. McGeer, Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer’s disease and normal brain, Brain Res. 919 (2001) 115 –121.
Thiorphan-induced neprilysin inhibition raises amyloid b levels in rabbit cortex and cerebrospinal fluid Amanda J. Newella, Lucia I. Suea, Sarah Scotta, Paula K. Rauschkolbb, Douglas G. Walkera, Pamela E. Potterb, Thomas G. Beacha,* a
Sun Health Research Institute, 10515 West Santa Fe Drive, Sun City, AZ 85351, USA b Midwestern University, Glendale, AZ, USA
Received 14 May 2003; received in revised form 18 July 2003; accepted 21 July 2003
Abstract Studies on the pathogenesis of Alzheimer’s disease (AD) suggest overproduction of amyloid b (Ab) may not be the only pathogenic route to AD. Decreased degradation of Ab is another possible disease mechanism. Neprilysin is a neutral endopeptidase that has been proposed to be the major enzyme responsible for Ab degradation. Studies have reported correlations between Ab deposition and neprilysin activity in the human brain. This study shows that intracerebroventricular infusion of thiorphan, a neprilysin inhibitor, raises cortical and cerebrospinal fluid (CSF) Ab concentrations in rabbits. Rabbits treated with thiorphan for 5 days had levels of CSF and cortical Ab40 that were 147 and 142% of the control group, respectively. Results for Ab42 showed a similar trend. The results indicate that age-related decreases of neprilysin could lead to increased brain concentrations of Ab, plaque formation, and AD. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Amyloid b; Thiorphan; Neprilysin; Cerebral cortex; Rabbits; Animal model
Research over the last decade has established that brain accumulation and histologic deposition of a single peptide known as amyloid b (Ab) is a critical event in Alzheimer’s disease (AD). Many investigators believe that Ab overproduction and deposition leads to all of the other relevant pathologic changes in the disease, including neurofibrillary tangle formation, loss of synapses, neuronal death, and dementia [3]. Preventing this accumulation may prevent AD. Overproduction of Ab may not be the only route to AD. Attention has focused recently on the possibility that decreased degradation of Ab could also lead to Ab deposition [9]. Turnover of Ab in the brain is rapid and it has been estimated that very small decreases in its removal would lead to deposition [9]. Although several enzymes are likely to participate in the proteolysis of Ab, it appears that one, termed neprilysin, may be the most important. Neprilysin is a multifunctional neutral endopeptidase that was first investigated in relation to its ability to degrade enkephalin and substance P [6,11,12]. In a series of recent *
Corresponding author. Tel.: þ1-623-876-5643; fax: þ 1-623-876-5461. E-mail address: [email protected] (T.G. Beach).
experiments, Saido and colleagues have shown that neprilysin is the major degrading pathway for Ab in the brain [5,6,11,12]. Inhibition or genetic absence of neprilysin activity leads to increased brain Ab concentrations [5,6]. In humans, cortical neprilysin protein and message are inversely related to the density and location of Ab deposits in the non-demented elderly and in subjects with AD [13, 14], suggesting that loss of neprilysin activity may be an age-related change that leads to Ab deposition. Prolonged neprilysin inhibition in rodents by brain parenchymal infusion of thiorphan produced local Ab deposition [6], although this was not replicated by another group using intracerebroventricular infusion [4]. We therefore decided to repeat these experiments, with some minor variations. We have used rabbits as our experimental animal, rather than rodents, since rodent Ab differs from the human peptide by having three different amino acids, and there is some evidence that this difference leads to decreased amyloidogenic processing of b-amyloid precursor protein (bAPP) and/or decreased Ab aggregation [2,7,8]. It is possible, therefore, that the rodent Ab sequence may make rodents resistant to Ab overproduction and/or deposition. Since the rabbit amino acid sequence in and around the Ab
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00902-9
A.J. Newell et al. / Neuroscience Letters 350 (2003) 178–180
region of bAPP (complete peptides encoded by exons 15, 16 and 17) is identical to that of humans [1], this possibility is avoided. We infused thiorphan unilaterally into the lateral ventricle with indwelling intracerebroventricular cannulae (Plastics One, Inc, Roanoke, VA) connected to subcutaneous osmotic pumps (model 2ML1, Durect Corp., Cupertino, CA). Thiorphan or vehicle (25% absolute ethanol, 25% distilled water and 50% DMSO) were infused at a rate of 10 ml/h (1 mg/kg per day). Rabbits were euthanized after 5 days of treatment, the brains dissected out fresh and processed for detection of Ab by immunohistochemical, ELISA and immunoblot methods. Immunohistochemical localization of Ab was performed using paraformaldehyde-fixed tissue and standard immunoperoxidase methods utilizing the antibody 10D5 (amino acids 1 – 16 of Ab; Elan Pharmaceuticals, South San Francisco, CA). Cerebrospinal fluid (CSF) and fresh-frozen cerebral cortex was assayed using a commercially-available Ab ELISA kit (Biosource, Camarillo, CA). Frozen cortical tissue was extracted in four volumes of 5.5 M guanidine hydrochloride [10] in 50 mM Tris buffer at pH 8.0, following which the homogenate was left to stand at room temperature overnight. Homogenates were then diluted 1:10 in the Biosource diluent and centrifuged at 14,000 rpm for 20 min at 48C with the supernatant used for ELISA. Cerebrospinal fluid for ELISA analysis was diluted with Biosource diluent at 1:20 for Ab40 and 1:4 for Ab42. Immunoblot analysis of CSF Ab was performed to verify the ELISA results. Undiluted CSF samples were run on 4 – 12% NuPage Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. The membranes were boiled in phosphate-buffered saline buffer for 10 min to enhance antibody binding and were then incubated with the 6E10 antibody (Signet Laboratories, Dedham, MA), which recognizes amino acids 1 –16 of Ab, thereby labeling both Ab40 and Ab42. Bound antibody was detected with peroxidase-labeled anti-mouse IgG followed by incubation with chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). The location and intensity of bands was assessed using a ChemiImager 4000 blot imaging system (Alpha Innotech, San Leandro, CA) and by exposure of Xray film. The ELISA results showed that thiorphan treatment increased both cortical and CSF concentrations of Ab. Cerebrospinal fluid levels of Ab40 and 42 were increased to 147% (19.27 versus 13.08 ng/ml) and 120% (2.48 versus 2.06 ng/ml), respectively, of control animals (Fig. 1A). The result with Ab40 was significant (P ¼ 0:003, unpaired, twotailed t-test). Thiorphan treatment raised cortical concentrations of Ab40 to 142% (1.25 versus 0.88 pmol/g) of control animals (P ¼ 0:03, unpaired, two-tailed t-test) while the concentrations of cortical Ab42 were not significantly altered (Fig. 1B). The immunoblot results (Fig. 2) showed that CSF Ab (combined Ab40 and 42) was significantly elevated by thiorphan treatment (129% of control values,
179
Fig. 1. Graphs depicting the effect of thiorphan on CSF (A); and cortical Ab (B). Thiorphan administration resulted in significant increases in mean CSF (to 147% of control, P ¼ 0:003, unpaired t-test) and cortical Ab40 (to 142% of control, P ¼ 0:03, unpaired t-test). SD ¼ standard deviation.
P , 0:05, Mann – Whitney U-test). Immunohistochemical staining with the 10D5 antibody did not reveal evidence of Ab deposition or intraneuronal immunoreactivity in thiorphan-treated animals. As previously reported by others, intracerebral thiorphan infusion raises CNS Ab levels [4 – 6]. In contrast to previous reports, however, we did not see immunohistochemical evidence of increased Ab levels, either in the form of plaque-like structures [6] or intraneuronal staining [4]. It may be that longer infusion durations are required to produce histologically-detectable Ab as the aforementioned studies used durations of 30 and 37 days while we used a duration of 5 days. Our result may also help resolve a confounding issue brought up by a previous investigation [4], in which it was suggested that the effect of thiorphan infusion on Ab may be due to the use of ascorbic acid as a preservative in the infusing fluid. As we did not use ascorbic acid in our infusion solution, the observed increases in Ab could not have been due to this. We found that thiorphan had a greater effect on Ab40 than on Ab42. In contrast, others have reported the reverse to be true, in both in vitro [6,11] and in vivo [5,6] experiments. The reason for this discrepancy is unclear, but may be due to the different extraction methods, treatment duration or animal species used. In conclusion, these results demonstrate that in vivo inhibition of endogenous neprilysin activity via intracerebroventricular infusion of thiorphan results in increased concentrations of Ab in the CSF and cerebral cortex. This supports the theory that loss of brain neprilysin activity
Fig. 2. Image of immunoblot analysis of Ab from CSF from control and thiorphan-treated animals. The mean intensity of the 4 kD band in thiorphan-treated animals was significantly increased (129% of control, P , 0:05, Mann –Whitney U-test) compared to control animals. See text for additional discussion.
180
A.J. Newell et al. / Neuroscience Letters 350 (2003) 178–180
could be a pathogenic mechanism leading to age-related Ab deposition and AD.
[7]
Acknowledgements [8]
This work was supported by a grant from the National Institute on Aging (RO1NS38674) to T.G. Beach.
References [1] J.S. Davidson, R.L. West, P. Kotikalapudi, L.E. Maroun, Sequence and methylation in the beta/A4 region of the rabbit amyloid precursor protein gene, Biochem. Biophys. Res. Commun. 188 (1992) 905–911. [2] B. DeStrooper, M. Simons, G. Multhaup, F. Van Leuven, K. Beyreuther, C.G. Dotti, Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence, EMBO J. 14 (1995) 4932–4938. [3] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science 297 (2002) 353–356. [4] B. Hauss-Wegrzyniak, G.L. Wenk, Beta-amyloid deposition in the brains of rats chronically infused with thiorphan or lipopolysaccharide: the role of ascorbic acid in the vehicle, Neurosci. Lett. 322 (2002) 75–78. [5] N. Iwata, S. Tsubuki, Y. Takaki, K. Shirotani, B. Lu, N.P. Gerard, C. Gerard, E. Hama, H.J. Lee, T.C. Saido, Metabolic regulation of brain Abeta by neprilysin, Science 292 (2001) 1550–1552. [6] N. Iwata, S. Tsubuki, Y. Takaki, K. Watanabe, M. Sekiguchi, E. Hosoki, M. Kawashima-Morishima, H.J. Lee, E. Hama, Y. SekineAizawa, T.C. Saido, Identification of the major Abeta1–42-degrading catabolic pathway in brain parenchyma: suppression leads to
[9]
[10]
[11]
[12]
[13]
[14]
biochemical and pathological deposition, Nat. Med. 6 (2000) 143 –150. L. Otvos Jr, G.I. Szendrei, V.M. Lee, H.H. Mantsch, Human and rodent Alzheimer beta-amyloid peptides acquire distinct conformations in membrane-mimicking solvents, Eur. J. Biochem. 211 (1993) 249 –257. A.G. Reaume, D.S. Howland, S.P. Trusko, M.J. Savage, D.M. Lang, B.D. Greenberg, R. Siman, R.W. Scott, Enhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer’s disease mutations and a ‘humanized’ Abeta sequence, J. Biol. Chem. 271 (1996) 23380–23388. T.C. Saido, Alzheimer’s disease as proteolytic disorders: anabolism and catabolism of beta-amyloid, Neurobiol. Aging 19 (1998) S69–S75. M.J. Savage, S.P. Trusko, D.S. Howland, L.R. Pinsker, S. Mistretta, A.G. Reaume, B.D. Greenberg, R. Siman, R.W. Scott, Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester, J. Neurosci. 18 (1998) 1743–1752. K. Shirotani, S. Tsubuki, N. Iwata, Y. Takaki, W. Harigaya, K. Maruyama, S. Kiryu-Seo, H. Kiyama, H. Iwata, T. Tomita, T. Iwatsubo, T.C. Saido, Neprilysin degrades both amyloid beta peptides 1–40 and 1– 42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases, J. Biol. Chem. 276 (2001) 21895–21901. Y. Takaki, N. Iwata, S. Tsubuki, S. Taniguchi, S. Toyoshima, B. Lu, N.P. Gerard, C. Gerard, H.J. Lee, K. Shirotani, T.C. Saido, Biochemical identification of the neutral endopeptidase family member responsible for the catabolism of amyloid beta peptide in the brain, J. Biochem. (Tokyo) 128 (2000) 897 –902. K. Yasojima, H. Akiyama, E.G. McGeer, P.L. McGeer, Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide, Neurosci. Lett. 297 (2001) 97–100. K. Yasojima, E.G. McGeer, P.L. McGeer, Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer’s disease and normal brain, Brain Res. 919 (2001) 115 –121.