Association among amyloid plaque, lipid, and creatine in hippocampus of TgCRND8 mouse model for Alzheimer disease

Abstract

Amyloid peptide (Aβ) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30-50 μm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.

FIGURE 1.

Morphology of mouse brain tissue and characteristic spectra from FTIR-FPA. A, unstained hippocampus from a 14-month-old transgenic mouse mounted on gold covered slide. Locations of the dentate gyrus (DG) and cornu ammonis (CA) neurons are indicated. B, IR map of the hippocampal tissue illustrated processed for the intensity of the CH₂ symmetric stretch. Regions with low intensity correspond to neuron, medium intensity to gray matter, and high intensity to white matter. Arrow in FTIR map indicates highest lipid content in the alveus (white matter bundle leaving the hippocampus). Box outlines area for depth profile experiment. Scale bar, 100 μm. C, IR spectra of characteristic tissue types. Plaque core shows a distinct amide I doublet and low absorption in the CH₂ region. White matter has a high lipid bilayer concentration, causing intense CH₂ absorption. Creatine deposits generate two distinct peaks around 1400 cm⁻¹ and 1300 cm⁻¹. Arrows indicate peak maxima of the band envelopes used for the various analyses.

FIGURE 2.

Increase of creatine in hippocampus of TgCRND8 mouse with age. FTIR-FPA maps of hippocampal tissue from littermate control and TgCRND8 mice, sacrificed at 5, 8, 11, 14, and 17 months are shown. Age of littermate pair in months is shown in the upper left corner of first processed image in each row. Each map has been processed for the band areas of lipid carbonyl and of creatine. Relative amounts are portrayed as gray scale using one set of limits for total lipid and one for total creatine. Creatine deposits are concentrated over the pyramidal cell layer of the hippocampus formation. Scale bar, 100 μm.

FIGURE 3.

A, depth profile of hippocampal tissue through three consecutive sections of 14-month-old TgCRND8 mouse brain. FTIR-FPA maps were processed for band area of CH₂ symmetric stretch to show tissue morphology (gray scale below). Creatine locations from map processed on a creatine band area at 1400 cm⁻¹ are overlaid (color gradient below). Scale bar, 100 μm. B, sFTIR analysis of dense core plaque from 17-month-old TgCRND8 mouse. a, light microscope image. b–d, sFTIR map was processed for: β-sheet in amide I region (b), creatine band at 1400 cm⁻¹ (c), and lipid membrane from band area of CH₂ symmetric stretch (d). Scale bar, 50 μm.

FIGURE 4.

sFTIR maps of a dense core plaque from 14-month TgCRND8 mouse (see box, Fig. 1B) depth profiled through nine consecutive sections. Panel A, row I shows tissue as seen under visible light microscope. Red dots denote pixel centers from mapping software. Rows II–IV show sFTIR maps processed for lipid, plaque, and creatine, respectively. Row V shows superposition of high lipid (l), creatine (Cr), and dense core plaque (Aβ), created by layering pixels containing highest levels of each component. Scale bar, 20 μm. B, sFTIR spectra in region of the amide I band, through sections 1–9, Row I, numbered black squares. C, sFTIR spectra in region of amide I band, taken at lettered squares in section 7. Vertical scale shows log(1/R) in 0.5 units; spectra are offset for clarity. Gray bars indicate spectral region characteristic for β-sheet conformation.