Evaluation of Copper Chelation Therapy in a Transgenic Rat Model of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of the amyloid ß (Aß) protein in blood vessels and leads to hemorrhages, strokes, and dementia in elderly individuals. Recent reports have shown elevated copper levels colocalized with vascular amyloid in human CAA and Alzheimer's disease patients, which have been suggested to contribute to cytotoxicity through the formation of reactive oxygen species. Here, we treated a transgenic rat model of CAA (rTg-DI) with the copper-specific chelator, tetrathiomolybdate (TTM), via intraperitoneal (IP) administration for 6 months to determine if it could lower copper content in vascular amyloid deposits and modify CAA pathology. Results showed that TTM treatment led to elevated Aß load in the hippocampus of the rTg-DI rats and increased microbleeds in the wild type (WT) animals. X-ray fluorescence microscopy was performed to image the distribution of copper and revealed a surprising increase in copper colocalized with Aß aggregates in TTM-treated rTg-DI rats. Unexpectedly, we also found an increase in the copper content in unaffected vessels of both rTg-DI and WT animals. These results show that IP administration of TTM was ineffective in removing copper from vascular Aß aggregates in vivo and increased the development of disease pathology in CAA.

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.

Copper accumulation and the effect of chelation treatment on cerebral amyloid angiopathy compared to parenchymal amyloid plaques.

Accumulation of fibrillar amyloid ß-protein (Aß) in parenchymal plaques and in blood vessels of the brain, the latter condition known as cerebral amyloid angiopathy (CAA), are hallmark pathologies of Alzheimer's disease (AD) and related disorders. Cerebral amyloid deposits have been reported to accumulate various metals, most notably copper and zinc. Here we show that, in human AD, copper is preferentially accumulated in amyloid-containing brain blood vessels compared to parenchymal amyloid plaques. In light of this observation, we evaluated the effects of reducing copper levels in Tg2576 mice, a transgenic model of AD amyloid pathologies. The copper chelator, tetrathiomolybdate (TTM), was administered to twelve month old Tg2576 mice for a period of five months. Copper chelation treatment significantly reduced both CAA and parenchymal plaque load in Tg2576 mice. Further, copper chelation reduced parenchymal plaque copper content but had no effect on CAA copper levels in this model. These findings indicate that copper is associated with both CAA deposits and parenchymal amyloid plaques in humans, but less in Tg2576 mice. TTM only reduces copper levels in plaques in Tg2576 mice. Reducing copper levels in the brain may beneficially lower amyloid pathologies associated with AD.

Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution.

We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM). This approach enables visualization of LBT-tagged proteins while simultaneously measuring the elemental distribution in cells at a spatial resolution necessary for visualizing cell membranes and eukaryotic subcellular organelles.

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam.

X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area. Here, XRF nanotomography was used to image the elemental distribution in individual E. coli bacterial cells using a sub-15 nm beam at the Hard X-ray Nanoprobe beamline (HXN, 3-ID) at NSLS-II. These measurements were simultaneously combined with ptychography to image structural components of the cells. The cells were embedded in small (3-20 µm) sodium chloride crystals, which provided a non-aqueous matrix to retain the three-dimensional structure of the E. coli while collecting data at room temperature. Results showed a generally uniform distribution of calcium in the cells, but an inhomogeneous zinc distribution, most notably with concentrated regions of zinc at the polar ends of the cells. This work demonstrates that simultaneous two-dimensional ptychography and XRF nanotomography can be performed with a sub-15 nm beam size on unfrozen biological cells to co-localize elemental distribution and nanostructure simultaneously.

Imaging Nutrient Distribution in the Rhizosphere Using FTIR Imaging.

Symbiotic associations in the rhizosphere between plants and microorganisms lead to efficient changes in the distribution of nutrients that promote growth and development for each organism involved. Understanding these nutrient fluxes provides insight into the molecular dynamics involved in nutrient transport from one organism to the other. To study such a nutrient flow, a new application of Fourier transform infrared imaging (FTIRI) was developed that entailed growing Populus tremulodes seedlings on a thin, nutrient-enriched Phytagel matrix that allows pixel to pixel measurement of the distribution of nutrients, in particular, nitrate, in the rhizosphere. The FTIR spectra collected from ammonium nitrate in the matrix indicated the greatest changes in the spectra at 1340 cm-1 due to the asymmetric stretching vibrations of nitrate. For quantification of the nitrate concentration in the rhizosphere of experimental plants, a calibration curve was generated that gave the nitrate concentration at each pixel in the chemical image. These images of the poplar rhizosphere showed evidence for symbiotic sharing of nutrients between the plant and the fungi, Laccaria bicolor, where the nitrate concentration was five times higher near mycorrhizal roots than further out into the rhizosphere. This suggested that nitrates are acquired and transported from the media toward the plant root by the fungi. Similarly, the sucrose used in the growth media as a carbon source was depleted around the fungi, suggesting its uptake and consumption by the system. This study is the first of its kind to visualize and quantify the nutrient availability associated with mycorrhizal interactions, indicating that FTIRI has the ability to monitor nutrient changes with other microorganisms in the rhizosphere as a key step for understanding nutrient flow processes in more diverse biological systems.