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November 29, 2006

Co-Localization of -Amyloid Deposits and Metal Accumulation in Alzheimer’s Disease

Q. Wang¹, T.P. Telivala¹, R.J. Smith¹, A. Lanzirotti², J. Miklossy³, and L.M. Miller^1
1National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY; ²Consortium for Advanced Radiation Sources, University of Chicago, Chicago, IL; ³Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, BC

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease. It is characterized by the misfolding and plaque-like accumulation of a naturally occurring protein, amyloid beta (A) in the brain. This misfolding process has been associated with the binding of metal ions such as Fe, Cu, and Zn in vitro. In this work, the secondary structure of the amyloid plaques in human AD brain tissue was imaged in situ using synchrotron Fourier transform infrared microspectroscopy (FTIRM). The results were correlated spatially with the metal ion distribution in the identical tissue, as determined using synchrotron x-ray fluorescence (XRF) microprobe. Results revealed “hot spots” of accumulated Zn and Cu ions that were co-localized with the elevated regions of -sheet protein, suggesting that metal ions may play a role in amyloid plaque formation in human Alzheimer’s disease.

From left to right: Randy Smith, Adele Qi Wang, Antonio Lanzirotti and Lisa Miller.

Alzheimer’s disease (AD) is a progressive brain disorder that gradually destroys a person’s memory, ability to learn, reason, make judgments, communicate, and carry out daily activities. The brain in AD is characterized by the presence of amyloid plaques, which consist of small deposits of a peptide called amyloid (A). In vitro evidence suggests that metal ions such as Cu, Zn, Fe and Mn may play a role in the misfolding of A in AD. However, the functions of these metal ions and A misfolding in the disease process are not well understood. The overall aim of this research is to obtain an in situ structural and mechanistic picture of how metal ions in the brain are involved in A formation and plaque aggregation in AD.

In this work, thin cryosections (~ 10 m) of brain tissue from patients with neuropathologically confirmed AD were studied. The locations of the amyloid plaques were visualized by green fluorescence using Thioflavin S staining and epifluorescence microscopy (Figure 1B). FTIRM carried out at NSLS beamline U10B showed that the amyloid plaques had elevated -sheet content, as demonstrated by a strong Amide I absorbance at 1625 cm^-1, which was different from the FTIR spectrum of A in vitro (Figure 2A). The correlation image generated based on peak height ratio of 1625 / 1657 cm^-1 (Figure 1C) revealed that regions of elevated -sheet content in the AD tissue corresponded well with amyloid deposits as identified by Thioflavin staining.

Using XRF microprobe at NSLS beamline X26A and APS beamline 13-ID, we found that the background content of Ca, Fe, Cu, and Zn in AD vs. control tissue were similar; however the metal distribution in AD tissue was not uniform. Specifically, “hot spots” of accumulated Ca, Fe, Cu, and Zn ions were observed. The SXRF images of Zn and Cu can be seen in Figure 1D and 1E, respectively. A strong spatial correlation (r² = 0.97) was found between the locations of the Cu and Zn ions. The elevated Zn and Cu in the “hot spot” is also evident in representative XRF spectra (Figure 2B).

Figure 1. (A) Bright field and (B) Epifluorescence image of human AD tissue stained with Thioflavin S. (C) Single channel color FTIR correlation image of -sheet protein (green). (D) Single channel color SXRF microprobe image of Zn (red). (E) Single channel color SXRF microprobe image of Cu (blue). (F) The RGB correlation image.

In order to correlate the misfolded amyloid protein and metal distribution in the tissue, a RGB image was generated with Zn content in red channel, -sheet protein content in the green channel, and Cu content in the blue channel. Results revealed the co-localization of Cu, Zn and -sheet protein in the amyloid plaques in AD human tissue (Figure 1F). Neither plaques nor accumulated metal hot spots were observed in control brain tissue.

Figure 2. (A) Infrared spectra of Thioflavin-positive area (red) and Thioflavin-negative area (black) of AD tissue. For comparison, the FTIR spectrum of purified A peptide in vitro is shown (green). (B) SXRF microprobe spectra from Thioflavin-positive area (red) and Thioflavin-negative area (black).

In summary, these results provide increasing evidence that the formation of amyloid plaques from the A peptide is associated with metal ions in the brain. Here we show for the first time a direct strong spatial correlation between AD plaques and metal ions in the brain, emphasizing the role of metal ions in AD etiology. In the future, exploring earlier stages of disease and in-situ probing metal-protein binding will be of interest for understanding the disease pathogenesis and mechanism.

BEAMLINES
U10B, X26A

FUNDING
National Institutes of Health

PUBLICATION
L.M. Miller, Q. Wang, T.P. Telivala, R.J. Smith, A. Lanzirotti and J. Miklossy, “Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with -amyloid deposits in Alzheimer’s disease,” Journal of Structural Biology, 155(1), 30-3 (2006).

FOR MORE INFORMATION
Lisa M. Miller
Brookhaven National Laboratory
National Synchrotron Light Source
Upton, NY
Email: lmiller@bnl.gov

Adele Qi Wang
Brookhaven National Laboratory
National Synchrotron Light Source
Upton, NY
Email: qwang@bnl.gov