An electrostatic cluster guides Aß40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aß peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer's disease (AD). We investigate the structure of human-derived Aß40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in in vitro Aß40 fibril structures. One population has an ordered N-terminal fold comprised of two ß-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations can guide fibril formation.

Impact of Aß40 and Aß42 Fibrils on the Transcriptome of Primary Astrocytes and Microglia.

Fibrillar amyloid ß-protein (Aß) deposits in the brain, which are primarily composed of Aß40 or Aß42 peptides, are key pathological features of Alzheimer's disease (AD) and related disorders. Although the underlying mechanisms are still not clear, the Aß fibrils can trigger a number of cellular responses, including activation of astrocytes and microglia. In addition, fibril structures of the Aß40 and Aß42 peptides are known to be polymorphic, which poses a challenge for attributing the contribution of different Aß sequences and structures to brain pathology. Here, we systematically treated primary astrocytes and microglia with single, well-characterized polymorphs of Aß40 or Aß42 fibrils, and performed bulk RNA sequencing to assess cell-specific changes in gene expression. A greater number of genes were up-regulated by Aß42 fibril-treated glial cells (251 and 2133 genes in astrocyte and microglia, respectively) compared with the Aß40 fibril-treated glial cells (191 and 251 genes in astrocytes and microglia, respectively). Immunolabeling studies in an AD rat model with parenchymal fibrillar Aß42 plaques confirmed the expression of PAI-1, MMP9, MMP12, CCL2, and C1r in plaque-associated microglia, and iNOS, GBP2, and C3D in plaque-associated astrocytes, validating markers from the RNA sequence data. In order to better understand these Aß fibril-induced gene changes, we analyzed gene expression patterns using the Ingenuity pathway analysis program. These analyses further highlighted that Aß42 fibril treatment up-regulated cellular activation pathways and immune response pathways in glial cells, including IL1ß and TNFα in astrocytes, and microglial activation and TGFß1 in microglia. Further analysis revealed that a number of disease-associated microglial (DAM) genes were surprisingly suppressed in Aß40 fibril treated microglia. Together, the present findings indicate that Aß42 fibrils generally show similar, but stronger, stimulating activity of glial cells compared with Aß40 fibril treatment.

Insights into Cerebral Amyloid Angiopathy Type 1 and Type 2 from Comparisons of the Fibrillar Assembly and Stability of the Aß40-Iowa and Aß40-Dutch Peptides.

Two distinct diseases are associated with the deposition of fibrillar amyloid-ß (Aß) peptides in the human brain in an age-dependent fashion. Alzheimer's disease is primarily associated with parenchymal plaque deposition of Aß42, while cerebral amyloid angiopathy (CAA) is associated with amyloid formation of predominantly Aß40 in the cerebral vasculature. In addition, familial mutations at positions 22 and 23 of the Aß sequence can enhance vascular deposition in the two major subtypes of CAA. The E22Q (Dutch) mutation is associated with CAA type 2, while the D23N (Iowa) mutation is associated with CAA type 1. Here we investigate differences in the formation and structure of fibrils of these mutant Aß peptides in vitro to gain insights into their biochemical and physiological differences in the brain. Using Fourier transform infrared and nuclear magnetic resonance spectroscopy, we measure the relative propensities of Aß40-Dutch and Aß40-Iowa to form antiparallel structure and compare these propensities to those of the wild-type Aß40 and Aß42 isoforms. We find that both Aß40-Dutch and Aß40-Iowa have strong propensities to form antiparallel ß-hairpins in the first step of the fibrillization process. However, there is a marked difference in the ability of these peptides to form elongated antiparallel structures. Importantly, we find marked differences in the stability of the protofibril or fibril states formed by the four Aß peptides. We discuss these differences with respect to the mechanisms of Aß fibril formation in CAA.

Human cerebral vascular amyloid contains both antiparallel and parallel in-register Aß40 fibrils.

The accumulation of fibrillar amyloid-ß (Aß) peptides alongside or within the cerebral vasculature is the hallmark of cerebral amyloid angiopathy (CAA). This condition commonly co-occurs with Alzheimer's disease (AD) and leads to cerebral microbleeds, intracranial hemorrhages, and stroke. CAA also occurs sporadically in an age-dependent fashion and can be accelerated by the presence of familial Aß mutant peptides. Recent studies using Fourier transform infrared (FTIR) spectroscopy of vascular Aß fibrils derived from rodents containing the double E22Q/D23N mutations indicated the presence of a novel antiparallel ß-sheet structure. To address whether this structure is associated solely with the familial mutations or is a common feature of CAA, we propagated Aß fibrils from human brain vascular tissue of patients diagnosed with nonfamilial CAA. Aß fibrils were isolated from cerebral blood vessels using laser capture microdissection in which specific amyloid deposits were removed from thin slices of the brain tissue. Transmission electron microscopy revealed that these deposits were organized into a tight meshwork of fibrils, which FTIR measurements showed could serve as seeds to propagate the growth of Aß40 fibrils for structural studies. Solid-state NMR measurements of the fibrils propagated from vascular amyloid showed they contained a mixture of parallel, in-register, and antiparallel ß-sheet structures. The presence of fibrils with antiparallel structure derived from vascular amyloid is distinct from the typical parallel, in-register ß-sheet structure that appears in fibrils derived from parenchymal amyloid in AD. These observations reveal that different microenvironments influence the structures of Aß fibrils in the human brain.

Copper stabilizes antiparallel ß-sheet fibrils of the amyloid ß40 (Aß40)-Iowa variant.

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long ß-amyloid peptide (Aß40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aß42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel ß-sheet, whereas previous studies by other researchers have reported parallel ß-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.