Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity.
The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.
Bodles
A M
AM
Centre for Peptide and Protein Engineering, School of Biology and Biochemistry, The Queens University of Belfast, Medical Biology Centre, Belfast, UK.
Guthrie
D J
DJ
Greer
B
B
Irvine
G B
GB
D000097:Acetonitriles; D000544:Alzheimer Disease; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000633:toxicity*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects*; D002942:Circular Dichroism; D005089:Exocytosis; D006801:Humans; D020894:Microfibrils / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology; D016716:PC12 Cells; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity*; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology; D011487:Protein Conformation; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051843:Synucleins; D051844:alpha-Synuclein
D000097:Acetonitriles; D000682:Amyloid; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C497604:SNCA protein, human; C497606:Snca protein, rat; D051843:Synucleins; D051844:alpha-Synuclein; C032159:acetonitrile