Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms beta-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157-163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 degrees C the full-length peptide and fragments NAC(3-18) and NAC(1-18) scrambled sequence [NAC(1-18 s)] were toxic, whereas fragments NAC(1-13) and NAC(6-14) were not. CD indicates that NAC(3-18) and NAC(1-18 s) exhibit beta-sheet secondary structure in aqueous solution, whereas NAC(1-13) and NAC(6-14) do not. NAC(3-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1-18 s) it does not come out of solution to a significant degree. Fragments NAC(1-13) and NAC(6-14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.

[1]  M. Shearman,et al.  The Intracellular Component of Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction Is Specifically Inhibited by β‐Amyloid Peptides , 1995 .

[2]  G. Glenner,et al.  Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein , 1984 .

[3]  D. Selkoe,et al.  Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. , 1999, The Journal of biological chemistry.

[4]  Robert L. Nussbaum,et al.  Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease , 1997 .

[5]  Peter T. Lansbury,et al.  Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease , 1998, Nature Medicine.

[6]  L. Thal,et al.  Expression Pattern of Synucleins (Non‐Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α‐Synuclein) During Murine Brain Development , 1998 .

[7]  E. Masliah,et al.  Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  P. S. St George-Hyslop,et al.  Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. , 1987, Science.

[9]  M. L. Schmidt,et al.  α-Synuclein in Lewy bodies , 1997, Nature.

[10]  P. Lansbury,et al.  The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease? , 1995, Chemistry & biology.

[11]  H. Wiśniewski,et al.  Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Michel Goedert,et al.  Familial Parkinson's disease: The awakening of α-synuclein , 1997, Nature.

[13]  E. Masliah,et al.  Non-A beta component of Alzheimer's disease amyloid (NAC) is amyloidogenic. , 1995, Biochemistry.

[14]  P. Højrup,et al.  Binding of Abeta to alpha- and beta-synucleins: identification of segments in alpha-synuclein/NAC precursor that bind Abeta and NAC. , 1997, The Biochemical journal.

[15]  Hitoshi Takahashi,et al.  NACP, a presynaptic protein, immunoreactivity in Lewy bodies in Parkinson's disease , 1997, Neuroscience Letters.

[16]  R. Jakes,et al.  Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α‐synuclein protein implicated in Parkinson's disease , 1998, FEBS letters.

[17]  J. Mikkelsen,et al.  Isoform-specific binding of human apolipoprotein E to the non-amyloid beta component of Alzheimer's disease amyloid. , 1997, Brain research. Molecular brain research.

[18]  Peter T. Lansbury,et al.  Assembly of Aβ Amyloid Protofibrils: An in Vitro Model for a Possible Early Event in Alzheimer's Disease† , 1999 .

[19]  P. Lansbury.  Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Neill,et al.  Aggregates from mutant and wild‐type α‐synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β‐sheet and amyloid‐like filaments , 1998, FEBS letters.

[21]  D. Brems,et al.  Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. , 1994, Molecular pharmacology.

[22]  B. Yankner,et al.  Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[23]  E. Masliah,et al.  Altered presynaptic protein NACP is associated with plaque formation and neurodegeneration in Alzheimer's disease. , 1996, The American journal of pathology.

[24]  K. Grzeschik,et al.  The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor , 1987, Nature.

[25]  P. Frey,et al.  Conformations of synthetic beta peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells. , 1996, Biochimica et biophysica acta.

[26]  M. Lerman,et al.  Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease. , 1987, Science.

[27]  H. Mori,et al.  Tissue-dependent alternative splicing of mRNA for NACP, the precursor of non-A beta component of Alzheimer's disease amyloid. , 1994, Biochemical and biophysical research communications.

[28]  W. Honer,et al.  Abnormal accumulation of NACP/alpha-synuclein in neurodegenerative disorders. , 1998, The American journal of pathology.

[29]  L. Iversen,et al.  Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[30]  G. Irvine,et al.  The N‐terminal region of non‐Aβ component of Alzheimer's Disease amyloid is responsible for its tendency to assume β‐sheet and aggregate to form fibrils , 1998 .