The Role of NAC in Amyloidogenesis in Alzheimer's Disease

Abstract

To the Editor-in-Chief:In 1993, Saitoh and colleagues identified the non-Aβ component of Alzheimer's disease (AD), amyloid (NAC), as an important element of amyloid-enriched fractions in AD brains.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar NAC is a 35-amino acid (aa. fragment derived from its 140-aa precursor, α-synuclein, which is now recognized to play a major role in Lewy body disease (LBD. pathogenesis.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar Furthermore, immunohistochemical studies with the anti-NAC-X1 antibody revealed that NAC is closely colocalized with Aβ in the AD plaques.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Moreover, biochemical studies showed that NAC is extremely hydrophobic and easily forms amyloid-like fibrils under physiological conditions.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar These results led us to the hypothesis that NAC might play an important role in Aβ aggregation and amyloidogenesis in AD.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google ScholarIn a recent issue of The American Journal of Pathology, Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar reported that NAC may not be associated with Aβ in the plaques. Using the antibody NAC42580 to label cortical and hippocampal sections of AD cases, the authors found no evidence of NAC immunoreactivity in the plaques. Furthermore, although the NAC42580 showed certain immunoreactivity in the urea extracts of the sodium dodecyl sulfate (SDS)-insoluble fractions in diffuse LBD and Parkinson's disease (PD) cases, there was no correlation between NAC and Aβ immunoreactivity in the same AD fractions. Based on this study and on a previous report,5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar the authors concluded that NAC may not be associated with Aβ in the plaques of AD.Several possibilities could be considered to explain the discrepancy between our results and those reported by Culvenor et al.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar First, differences in immunoreactivity could be due to differential properties of antibodies used in each study. NAC-X1 and NAC42580 were raised against epitopes derived from different portions of the NAC region: NAC1–9 for NAC-X11Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar and NAC15–31 for NAC42580.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar It is important to note that immunoblotting analysis showed that NAC-X1 preferentially recognizes aggregated forms of the NAC molecule over the monomeric ones.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Furthermore, in our experience, NAC-X1 does not immunoreact with soluble α-synuclein.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Thus, it can be predicted that our NAC-X1 antibody may be ideal to detect aggregated NAC in the plaques of AD. In contrast, immunoblotting analysis with the NAC42580 showed immunoreactivity with soluble α-synuclein in brain tissues.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar In fact, biochemical analysis of the NAC peptide by El-Agnaf et al6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar showed that the N-terminal region of NAC (NAC1–18. aggregates to form amyloid fibrils, while the C-terminal region (NAC19–35) remains soluble, suggesting that the N-terminal portion is essential for aggregation of NAC peptide.6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar Taken together, these observations might suggest that the NAC1–9 peptide could be naturally aggregated during inoculation in the rabbit, leading to the production of NAC-X1, which preferentially recognizes aggregated forms of NAC, whereas the C-terminal region of NAC may be less effective.Second, differences in antibody immunoreactivity might depend on the methods used for tissue preparation. In this regard, the NAC-X1 antibody immunolabeled plaques in paraformaldehyde-fixed vibratome sections, but not in formalin-fixed, paraffin-embedded tissue.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar, 3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar, 7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle Scholar, 8Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar In fact, both Bayer and Culvenor used archival formalin-fixed and/or paraffin-embedded tissues, rather than vibratome, for their immunohistochemical studies,4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar raising the possibility that fixation, solvents, and paraffin might destroy the NAC-X1 epitope. Further supporting the importance of tissue pretreatment and processing to detect the NAC epitope, we have recently shown that in vibratome sections pretreated with formic acid, the NAC-X1 antibody immunostained not only amyloid plaques and amyloid angiopathy, but also astroglial cells and granular neurons in LBD.7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle ScholarFinally, it is possible that the NAC-X1 antibody might be cross-reacting with or recognizing protein quaternary structure. In this regard, immunoblotting analysis confirmed that both monomeric and aggregated forms of Aβ were not immunoreactive with NAC-X1.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Therefore, it is unlikely that NAC-X1 cross-reacts with Aβ.In summary, we would like to argue that it is necessary to continue exploring the hypothesis that NAC plays an important role in plaque formation and AD. The essential difficulty in obtaining conclusive evidence may be due to the lack of information as to the mechanisms by which NAC is generated from its precursor, α-synuclein.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar In this regard, the results of this study by Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar are potentially interesting, because their immunoblotting analysis of brain homogenates showed that in addition to full length α-synuclein at 18kd, NAC42850 recognized faint bands at 12 and 6 kd, supporting the possibility that α-synuclein undergoes degradation. We expect that further characterization of these short fragments might disclose a yet unknown mechanism of α-synuclein proteolysis, leading to a better understanding of the role of α-synuclein and NAC in the pathogenesis of AD.Author's Reply:The central questions we have tried to address are whether intact α-synuclein (αSN) or its proteolytic product, the non-Aβ component of Alzheimer's disease (AD) amyloid, NAC, αSN(61–95), ever occurs as an extracellular aggregate, and, if so, whether there is any special relationship with Aβ deposition. Earlier studies by Saitoh, Masliah, and colleagues1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Scopus (1200) Google Scholar, 2Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Google Scholar, 3Masliah E Iwai A Mallory M Ueda K Saitoh T Altered presynaptic protein NACP is associated with plaque formation and neurodegeneration in Alzheimer's disease.Am J Pathol. 1996; 148: 201-210PubMed Google Scholar, 4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar using antibodies to NAC(1–9) indicated that it may be an integral but minor component of Aβ amyloid plaques. Our antibodies to NAC(15–31)5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar, 6Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (61) Google Scholar and NAC(11–23) (unpublished) fail to reveal an association of NAC with the extracellular Aβ amyloid deposits in AD. There is however, intracellular NAC immunoreactivity in abnormal neuritic structures in close association with Aβ amyloid plaques restricted to the CA1 region of the hippocampus in cases of AD combined with cortical Lewy bodies.The response of Hashimoto and colleagues suggests our data reflect a lack of reactivity with aggregated NAC; however, all our antibodies react with aggregated αSN as found in Lewy bodies. The suggestion that differences may be due to tissue preparation protocols is also unlikely, because our antibodies were reactive with Lewy bodies after formalin fixation, paraffin sectioning, and formic acid treatment. Hashimoto and colleagues themselves report no difference of labeling of vibratome or paraffin sections after formic acid pretreatment of tissue.4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar We therefore suspect that the reactivity of the NAC(1–9) antibody with AD plaques may be due to cross-reactivity with Aβ, since residues 6–8 of NAC share identity with the C-terminal region of Aβ(36–38), as identified earlier by Han et al.7Han H Weinreb PH Lansbury PT The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by β-amyloid: is NAC a common trigger or target in neurodegenerative disease?.Chem Biol. 1995; 2: 163-169Abstract Full Text PDF PubMed Scopus (280) Google ScholarOur Western blot analysis of SDS-insoluble extracts of brain tissue from cases of dementia with Lewy bodies indicates that aggregates of αSN contain full-length as well as truncated species, including a putative NAC fragment of about 6 kd. In pure AD cases, we are unable to demonstrate accumulation of SDS-insoluble αSN or a NAC fragment in the same fractions that contain the SDS-insoluble Aβ. peptide.5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar In a further study of four AD cases, SDS-insoluble αSN or the NAC fragment was not detectable (Campbell et al, manuscript submitted). Our combined immunocytochemical and biochemical data therefore indicate that αSN and its proteolytic products accumulate largely in intracellular compartments, independent of extracellular Aβ amyloid deposits. To the Editor-in-Chief:In 1993, Saitoh and colleagues identified the non-Aβ component of Alzheimer's disease (AD), amyloid (NAC), as an important element of amyloid-enriched fractions in AD brains.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar NAC is a 35-amino acid (aa. fragment derived from its 140-aa precursor, α-synuclein, which is now recognized to play a major role in Lewy body disease (LBD. pathogenesis.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar Furthermore, immunohistochemical studies with the anti-NAC-X1 antibody revealed that NAC is closely colocalized with Aβ in the AD plaques.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Moreover, biochemical studies showed that NAC is extremely hydrophobic and easily forms amyloid-like fibrils under physiological conditions.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar These results led us to the hypothesis that NAC might play an important role in Aβ aggregation and amyloidogenesis in AD.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google ScholarIn a recent issue of The American Journal of Pathology, Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar reported that NAC may not be associated with Aβ in the plaques. Using the antibody NAC42580 to label cortical and hippocampal sections of AD cases, the authors found no evidence of NAC immunoreactivity in the plaques. Furthermore, although the NAC42580 showed certain immunoreactivity in the urea extracts of the sodium dodecyl sulfate (SDS)-insoluble fractions in diffuse LBD and Parkinson's disease (PD) cases, there was no correlation between NAC and Aβ immunoreactivity in the same AD fractions. Based on this study and on a previous report,5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar the authors concluded that NAC may not be associated with Aβ in the plaques of AD.Several possibilities could be considered to explain the discrepancy between our results and those reported by Culvenor et al.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar First, differences in immunoreactivity could be due to differential properties of antibodies used in each study. NAC-X1 and NAC42580 were raised against epitopes derived from different portions of the NAC region: NAC1–9 for NAC-X11Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar and NAC15–31 for NAC42580.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar It is important to note that immunoblotting analysis showed that NAC-X1 preferentially recognizes aggregated forms of the NAC molecule over the monomeric ones.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Furthermore, in our experience, NAC-X1 does not immunoreact with soluble α-synuclein.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Thus, it can be predicted that our NAC-X1 antibody may be ideal to detect aggregated NAC in the plaques of AD. In contrast, immunoblotting analysis with the NAC42580 showed immunoreactivity with soluble α-synuclein in brain tissues.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar In fact, biochemical analysis of the NAC peptide by El-Agnaf et al6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar showed that the N-terminal region of NAC (NAC1–18. aggregates to form amyloid fibrils, while the C-terminal region (NAC19–35) remains soluble, suggesting that the N-terminal portion is essential for aggregation of NAC peptide.6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar Taken together, these observations might suggest that the NAC1–9 peptide could be naturally aggregated during inoculation in the rabbit, leading to the production of NAC-X1, which preferentially recognizes aggregated forms of NAC, whereas the C-terminal region of NAC may be less effective.Second, differences in antibody immunoreactivity might depend on the methods used for tissue preparation. In this regard, the NAC-X1 antibody immunolabeled plaques in paraformaldehyde-fixed vibratome sections, but not in formalin-fixed, paraffin-embedded tissue.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar, 3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar, 7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle Scholar, 8Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar In fact, both Bayer and Culvenor used archival formalin-fixed and/or paraffin-embedded tissues, rather than vibratome, for their immunohistochemical studies,4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar raising the possibility that fixation, solvents, and paraffin might destroy the NAC-X1 epitope. Further supporting the importance of tissue pretreatment and processing to detect the NAC epitope, we have recently shown that in vibratome sections pretreated with formic acid, the NAC-X1 antibody immunostained not only amyloid plaques and amyloid angiopathy, but also astroglial cells and granular neurons in LBD.7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle ScholarFinally, it is possible that the NAC-X1 antibody might be cross-reacting with or recognizing protein quaternary structure. In this regard, immunoblotting analysis confirmed that both monomeric and aggregated forms of Aβ were not immunoreactive with NAC-X1.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Therefore, it is unlikely that NAC-X1 cross-reacts with Aβ.In summary, we would like to argue that it is necessary to continue exploring the hypothesis that NAC plays an important role in plaque formation and AD. The essential difficulty in obtaining conclusive evidence may be due to the lack of information as to the mechanisms by which NAC is generated from its precursor, α-synuclein.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar In this regard, the results of this study by Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar are potentially interesting, because their immunoblotting analysis of brain homogenates showed that in addition to full length α-synuclein at 18kd, NAC42850 recognized faint bands at 12 and 6 kd, supporting the possibility that α-synuclein undergoes degradation. We expect that further characterization of these short fragments might disclose a yet unknown mechanism of α-synuclein proteolysis, leading to a better understanding of the role of α-synuclein and NAC in the pathogenesis of AD. In 1993, Saitoh and colleagues identified the non-Aβ component of Alzheimer's disease (AD), amyloid (NAC), as an important element of amyloid-enriched fractions in AD brains.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar NAC is a 35-amino acid (aa. fragment derived from its 140-aa precursor, α-synuclein, which is now recognized to play a major role in Lewy body disease (LBD. pathogenesis.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar Furthermore, immunohistochemical studies with the anti-NAC-X1 antibody revealed that NAC is closely colocalized with Aβ in the AD plaques.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Moreover, biochemical studies showed that NAC is extremely hydrophobic and easily forms amyloid-like fibrils under physiological conditions.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar These results led us to the hypothesis that NAC might play an important role in Aβ aggregation and amyloidogenesis in AD.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar In a recent issue of The American Journal of Pathology, Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar reported that NAC may not be associated with Aβ in the plaques. Using the antibody NAC42580 to label cortical and hippocampal sections of AD cases, the authors found no evidence of NAC immunoreactivity in the plaques. Furthermore, although the NAC42580 showed certain immunoreactivity in the urea extracts of the sodium dodecyl sulfate (SDS)-insoluble fractions in diffuse LBD and Parkinson's disease (PD) cases, there was no correlation between NAC and Aβ immunoreactivity in the same AD fractions. Based on this study and on a previous report,5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar the authors concluded that NAC may not be associated with Aβ in the plaques of AD. Several possibilities could be considered to explain the discrepancy between our results and those reported by Culvenor et al.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar First, differences in immunoreactivity could be due to differential properties of antibodies used in each study. NAC-X1 and NAC42580 were raised against epitopes derived from different portions of the NAC region: NAC1–9 for NAC-X11Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar and NAC15–31 for NAC42580.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar It is important to note that immunoblotting analysis showed that NAC-X1 preferentially recognizes aggregated forms of the NAC molecule over the monomeric ones.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Furthermore, in our experience, NAC-X1 does not immunoreact with soluble α-synuclein.3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar Thus, it can be predicted that our NAC-X1 antibody may be ideal to detect aggregated NAC in the plaques of AD. In contrast, immunoblotting analysis with the NAC42580 showed immunoreactivity with soluble α-synuclein in brain tissues.4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar In fact, biochemical analysis of the NAC peptide by El-Agnaf et al6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar showed that the N-terminal region of NAC (NAC1–18. aggregates to form amyloid fibrils, while the C-terminal region (NAC19–35) remains soluble, suggesting that the N-terminal portion is essential for aggregation of NAC peptide.6El-Agnaf OM Bodles Am Guthrie DJS Harriott P Irvine B The N-terminal region of non-Aβ component of Alzheimer's disease amyloid is responsible for its tendency to assume β-sheet, and aggregates to form fibrils.Eur J Biochem. 1998; 258: 157-163Crossref PubMed Scopus (74) Google Scholar Taken together, these observations might suggest that the NAC1–9 peptide could be naturally aggregated during inoculation in the rabbit, leading to the production of NAC-X1, which preferentially recognizes aggregated forms of NAC, whereas the C-terminal region of NAC may be less effective. Second, differences in antibody immunoreactivity might depend on the methods used for tissue preparation. In this regard, the NAC-X1 antibody immunolabeled plaques in paraformaldehyde-fixed vibratome sections, but not in formalin-fixed, paraffin-embedded tissue.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar, 3Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (191) Google Scholar, 7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle Scholar, 8Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar In fact, both Bayer and Culvenor used archival formalin-fixed and/or paraffin-embedded tissues, rather than vibratome, for their immunohistochemical studies,4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (85) Google Scholar raising the possibility that fixation, solvents, and paraffin might destroy the NAC-X1 epitope. Further supporting the importance of tissue pretreatment and processing to detect the NAC epitope, we have recently shown that in vibratome sections pretreated with formic acid, the NAC-X1 antibody immunostained not only amyloid plaques and amyloid angiopathy, but also astroglial cells and granular neurons in LBD.7Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen L, Masliah E: C-terminal α-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol 1999, in pressGoogle Scholar Finally, it is possible that the NAC-X1 antibody might be cross-reacting with or recognizing protein quaternary structure. In this regard, immunoblotting analysis confirmed that both monomeric and aggregated forms of Aβ were not immunoreactive with NAC-X1.1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Google Scholar Therefore, it is unlikely that NAC-X1 cross-reacts with Aβ. In summary, we would like to argue that it is necessary to continue exploring the hypothesis that NAC plays an important role in plaque formation and AD. The essential difficulty in obtaining conclusive evidence may be due to the lack of information as to the mechanisms by which NAC is generated from its precursor, α-synuclein.2Hashimoto M Masliah E Alpha-synuclein in Lewy body disease and Alzheimer's disease.Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (214) Google Scholar In this regard, the results of this study by Culvenor et al4Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar are potentially interesting, because their immunoblotting analysis of brain homogenates showed that in addition to full length α-synuclein at 18kd, NAC42850 recognized faint bands at 12 and 6 kd, supporting the possibility that α-synuclein undergoes degradation. We expect that further characterization of these short fragments might disclose a yet unknown mechanism of α-synuclein proteolysis, leading to a better understanding of the role of α-synuclein and NAC in the pathogenesis of AD. Author's Reply:The central questions we have tried to address are whether intact α-synuclein (αSN) or its proteolytic product, the non-Aβ component of Alzheimer's disease (AD) amyloid, NAC, αSN(61–95), ever occurs as an extracellular aggregate, and, if so, whether there is any special relationship with Aβ deposition. Earlier studies by Saitoh, Masliah, and colleagues1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Scopus (1200) Google Scholar, 2Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Google Scholar, 3Masliah E Iwai A Mallory M Ueda K Saitoh T Altered presynaptic protein NACP is associated with plaque formation and neurodegeneration in Alzheimer's disease.Am J Pathol. 1996; 148: 201-210PubMed Google Scholar, 4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar using antibodies to NAC(1–9) indicated that it may be an integral but minor component of Aβ amyloid plaques. Our antibodies to NAC(15–31)5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar, 6Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (61) Google Scholar and NAC(11–23) (unpublished) fail to reveal an association of NAC with the extracellular Aβ amyloid deposits in AD. There is however, intracellular NAC immunoreactivity in abnormal neuritic structures in close association with Aβ amyloid plaques restricted to the CA1 region of the hippocampus in cases of AD combined with cortical Lewy bodies.The response of Hashimoto and colleagues suggests our data reflect a lack of reactivity with aggregated NAC; however, all our antibodies react with aggregated αSN as found in Lewy bodies. The suggestion that differences may be due to tissue preparation protocols is also unlikely, because our antibodies were reactive with Lewy bodies after formalin fixation, paraffin sectioning, and formic acid treatment. Hashimoto and colleagues themselves report no difference of labeling of vibratome or paraffin sections after formic acid pretreatment of tissue.4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar We therefore suspect that the reactivity of the NAC(1–9) antibody with AD plaques may be due to cross-reactivity with Aβ, since residues 6–8 of NAC share identity with the C-terminal region of Aβ(36–38), as identified earlier by Han et al.7Han H Weinreb PH Lansbury PT The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by β-amyloid: is NAC a common trigger or target in neurodegenerative disease?.Chem Biol. 1995; 2: 163-169Abstract Full Text PDF PubMed Scopus (280) Google ScholarOur Western blot analysis of SDS-insoluble extracts of brain tissue from cases of dementia with Lewy bodies indicates that aggregates of αSN contain full-length as well as truncated species, including a putative NAC fragment of about 6 kd. In pure AD cases, we are unable to demonstrate accumulation of SDS-insoluble αSN or a NAC fragment in the same fractions that contain the SDS-insoluble Aβ. peptide.5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar In a further study of four AD cases, SDS-insoluble αSN or the NAC fragment was not detectable (Campbell et al, manuscript submitted). Our combined immunocytochemical and biochemical data therefore indicate that αSN and its proteolytic products accumulate largely in intracellular compartments, independent of extracellular Aβ amyloid deposits. The central questions we have tried to address are whether intact α-synuclein (αSN) or its proteolytic product, the non-Aβ component of Alzheimer's disease (AD) amyloid, NAC, αSN(61–95), ever occurs as an extracellular aggregate, and, if so, whether there is any special relationship with Aβ deposition. Earlier studies by Saitoh, Masliah, and colleagues1Ueda K Fukushima H Masliah E Xia Y Iwai A Otero D Kondo J Ihara Y Saitoh T Molecular cloning of a novel component of amyloid in Alzheimer's disease.Proc Natl Acad Sci USA. 1993; 90: 11282-11286Crossref PubMed Scopus (1200) Google Scholar, 2Iwai A Yoshimoto M Masliah E Saitoh T Non-Aβ component of Alzheimer's disease amyloid (NAC) is amyloidogenic.Biochemistry. 1995; 34: 10139-10145Crossref PubMed Google Scholar, 3Masliah E Iwai A Mallory M Ueda K Saitoh T Altered presynaptic protein NACP is associated with plaque formation and neurodegeneration in Alzheimer's disease.Am J Pathol. 1996; 148: 201-210PubMed Google Scholar, 4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar using antibodies to NAC(1–9) indicated that it may be an integral but minor component of Aβ amyloid plaques. Our antibodies to NAC(15–31)5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar, 6Bayer TA Jakala P Hartmann T Havas L McLean C Culvenor JG Li Q-X Masters CL Falkai P Beyreuther K α-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease β-amyloid plaque cores.Neurosci Lett. 1999; 266: 213-216Crossref PubMed Scopus (61) Google Scholar and NAC(11–23) (unpublished) fail to reveal an association of NAC with the extracellular Aβ amyloid deposits in AD. There is however, intracellular NAC immunoreactivity in abnormal neuritic structures in close association with Aβ amyloid plaques restricted to the CA1 region of the hippocampus in cases of AD combined with cortical Lewy bodies. The response of Hashimoto and colleagues suggests our data reflect a lack of reactivity with aggregated NAC; however, all our antibodies react with aggregated αSN as found in Lewy bodies. The suggestion that differences may be due to tissue preparation protocols is also unlikely, because our antibodies were reactive with Lewy bodies after formalin fixation, paraffin sectioning, and formic acid treatment. Hashimoto and colleagues themselves report no difference of labeling of vibratome or paraffin sections after formic acid pretreatment of tissue.4Takeda A Hashimoto M Mallory M Sundsmo M Hansen L Sisk A Masliah E Human NACP/α-synuclein distribution in Lewy body disease.Lab Invest. 1998; 78: 1169-1177PubMed Google Scholar We therefore suspect that the reactivity of the NAC(1–9) antibody with AD plaques may be due to cross-reactivity with Aβ, since residues 6–8 of NAC share identity with the C-terminal region of Aβ(36–38), as identified earlier by Han et al.7Han H Weinreb PH Lansbury PT The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by β-amyloid: is NAC a common trigger or target in neurodegenerative disease?.Chem Biol. 1995; 2: 163-169Abstract Full Text PDF PubMed Scopus (280) Google Scholar Our Western blot analysis of SDS-insoluble extracts of brain tissue from cases of dementia with Lewy bodies indicates that aggregates of αSN contain full-length as well as truncated species, including a putative NAC fragment of about 6 kd. In pure AD cases, we are unable to demonstrate accumulation of SDS-insoluble αSN or a NAC fragment in the same fractions that contain the SDS-insoluble Aβ. peptide.5Culvenor JG McLean CA Cutt S Campbell BCV Maher F Jakala P Hartmann T Beyreuther K Masters CL Li Q-X Non-Aβ component of Alzheimer's disease amyloid (NAC) revisited: NAC and α-synuclein are not associated with Aβ amyloid.Am J Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Google Scholar In a further study of four AD cases, SDS-insoluble αSN or the NAC fragment was not detectable (Campbell et al, manuscript submitted). Our combined immunocytochemical and biochemical data therefore indicate that αSN and its proteolytic products accumulate largely in intracellular compartments, independent of extracellular Aβ amyloid deposits.