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 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.