title: Alzheimer's disease amyloid β-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils.
abstract: Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aβ proteins and their aggregation states or fibrillar structure.
authors.affiliation: Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA. joseph@mayo.edu
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Poduslo
authors.forename: Kyle G
authors.initials: KG
authors.lastname: Howell
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Olson
authors.forename: Marina
authors.initials: M
authors.lastname: Ramirez-Alvarado
authors.forename: Karunya K
authors.initials: KK
authors.lastname: Kandimalla
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005454:Fluorescence Polarization; D006639:Histidine / Q000235:genetics; D006801:Humans; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D006639:Histidine; D004121:Dimethyl Sulfoxide