Amyloid plaques in PSAPP mice bind less metal than plaques in human Alzheimer's disease

Abstract

Amyloid beta (Aβ) is the primary component of Alzheimer's disease (AD) plaques, a key pathological feature of the disease. Metal ions of zinc (Zn), copper (Cu), iron (Fe), and calcium (Ca) are elevated in human amyloid plaques and are thought to be involved in neurodegeneration. Transgenic mouse models of AD also exhibit amyloid plaques, but fail to exhibit the high degree of neurodegeneration observed in humans. In this study, we imaged the Zn, Cu, Fe, and Ca ion distribution in the PSAPP transgenic mouse model representing end-stage AD (N = 6) using synchrotron X-ray fluorescence (XRF) microprobe. In order to account for differences in density in the plaques, the relative protein content was imaged with synchrotron Fourier transform infrared microspectroscopy (FTIRM) on the same samples. FTIRM results revealed a 61% increase in protein content in the plaques compared to the surrounding tissue. After normalizing to protein density, we found that the PSAPP plaques contained only a 29% increase in Zn and there was actually less Cu, Fe, and Ca in the plaque compared to the surrounding tissue. Since metal binding to Aβ is thought to induce redox chemistry that is toxic to neurons, the reduced metal binding in PSAPP mice is consistent with the lack of neurodegeneration in these animals. These findings were in stark contrast to the high metal ion content observed in human AD plaques, further implicating the role of metal ions in human AD pathology.

Introduction

One of the key clinical features of Alzheimer's disease (AD) is the extracellular deposition of amyloid plaques primarily composed of a 39–43 amino acid protein called amyloid beta (Aβ). Aβ is derived from the proteolytic cleavage of a larger transmembrane glycoprotein, the amyloid precursor protein (APP), by β and γ secretases (Selkoe 2001). β-secretase first cleaves APP extracellularly, forming the N-terminus, and γ-secretase then cleaves APP in the intramembrane domain, creating the C-terminus. The two most frequently produced peptides are Aβ40, composed of 40 residues and found mostly in cerebrospinal fluid and vasculature, and Aβ42, composed of 42 residues and the main component of amyloid plaques (Roher et al., 1993). The formation of Aβ, even the deposition of diffuse plaques, is a normal cellular process, but in AD the peptide aggregates to form dense plaques in specific regions of the brain (Masters et al., 2006). But exactly how and why this misfolding occurs is still unknown.

Zinc (Zn), copper (Cu), iron (Fe), and calcium (Ca) ions are all present in the body under normal conditions, but their abundances are elevated in regions of the brain that are involved in AD (Bush, 2003, Lovell et al., 1998). The observation that human Aβ can bind to certain metal ions in vitro may indicate these ions play a role in increasing the protein's toxicity and the tendency to form plaques (Bush, 2003, Bush et al., 1994, Cuajungco and Faget, 2003, Maynard et al., 2005, Miura et al., 2000). Nuclear magnetic resonance (NMR) and X-ray absorption spectroscopy (XAS) studies of metal binding to human Aβ have revealed the presence of a low and high affinity binding site for both Cu and Zn via three N-terminal histidine imidazole rings and that the stoichiometry for Cu:Aβ is 1:1 and for Zn:Aβ ranging from 1:1 to 3:1 (Atwood et al., 2000, Clements et al., 1996, Streltsov, 2008).

The affinity for the Zn binding sites were measured as 100 nM and 5 μM, (Bush et al., 1994) and 10^− 10 M for Cu (Atwood et al., 2000). These affinities are well below physiological levels (∼ 100–150 μM (White et al., 2006)), supporting the possibility that Zn and Cu are likely to bind human Aβ in vivo, especially in AD where brain metal levels are known to be increased (Suh et al., 2000). Indeed, elevated levels of Zn, Cu, and Fe ions have been observed in amyloid plaques in human AD brain (Lovell et al., 1998, Miller et al., 2006, Suh et al., 2000) and studies involving Swedish mutant APP transgenic mice have shown that Zn is elevated in dense senile plaques (Lee et al., 1999). It has also recently been shown that Zn transporter proteins, which transport Zn ions into different intracellular compartments when intercellular Zn is elevated, are increased in AD and are abundantly expressed in human senile amyloid plaques (Smith et al., 2006, Zhang et al., 2008). Increased Fe was also found within the glial cells surrounding the plaques (Quintana et al., 2006). Excess Fe is typically sequestered by the iron storage protein ferritin and it was found that expression of ferritin is associated with degeneration of microglia in AD brain (Lopes et al., 2008). However, those previous studies primarily utilized histochemical methods, which can only detect free and loosely bound metal ions and cannot be accurately compared to the metal content in the surrounding healthy (non-plaque) tissue. In addition, previous studies that quantified metal content using proton and X-ray methods (Lovell et al., 1998, Miller et al., 2006) did not consider the elevated protein density in the plaque compared to the surrounding tissue. Thus, the observed increase in metal ions within the plaques may simply be attributed to an increase in protein density within the plaque and not an “accumulation” of excess metal ions or metalloproteins.

A number of mouse models have been developed to mimic one or more neuropathological features of AD, which has resulted in valuable progress in understanding AD progression. However, there is some evidence that they are not a complete representation of human AD (Schwab et al., 2004). While mice overexpressing mutant AD related genes exhibit many of the same neuropathological and behavioral features of human AD, including amyloid plaques, neurofibrillary tangles, motor impairments, and memory deficits (German and Eisch, 2004, Wirths et al., 2008), most transgenic models lack the widespread neuronal loss and severity of symptoms present in human AD pathology (Takeuchi et al., 2000). Additionally, the surrounding neurons appeared displaced by the plaques rather than damaged by them (Schwab et al., 2004). Exactly why this occurs is not known; however, mouse Aβ lacks a histidine at position 13. This residue has been shown to be critical in coordinating Cu and inducing aggregation in human Aβ, where wild-type mice show no aggregation (Atwood et al., 1998, Syme et al., 2004). Transgenic mice develop amyloid plaques that contain both endogenous murine Aβ and transgene-derived human Aβ (Pype et al., 2003, van Groen et al., 2006). Since both types are found in transgenic mouse plaques, this mixture may alter the metal binding characteristics of the peptide (Radde et al., 2008).

We have previously shown that end-stage human AD is characterized by the accumulation of Zn, Cu, Fe, and Ca that are co-localized with areas of high β-sheet protein and that the concentrations of metal inside the plaque are much higher than outside of the plaque (Miller et al., 2006). Here, those results are compared to analogous studies from the end stage of a PSAPP transgenic mouse model of AD, which overexpresses the presenilin (PS) 1 gene and the amyloid precursor protein (APP) gene. Synchrotron X-ray fluorescence (XRF) microprobe was used to image the metal content in the plaques and surrounding non-plaque tissue. In order to normalize to plaque protein density, synchrotron Fourier transform infrared microspectroscopy (FTIRM) was used on the same samples. Results revealed a dramatic decrease in metal binding in the PSAPP plaques compared to the human plaques. When normalized to protein density, human plaques showed elevated Zn, Cu, Fe, and Ca, whereas the PSAPP aggregates contained only elevated Zn. The differences in both neurodegeneration and metal accumulation between human AD and the PSAPP mouse model provide more evidence of the involvement of metal ions in AD.