Alzheimer disease periventricular white matter lesions exhibit specific proteomic profile alterations

Abstract

The white matter (WM) represents approximately half the cerebrum volume and is profoundly affected in Alzheimer's disease (AD). However, both the WM responses to AD as well as potential influences of this compartment to dementia pathogenesis remain comparatively neglected. Neuroimaging studies have revealed WM alterations are commonly associated with AD and renewed interest in examining the pathologic basis and importance of these changes. In AD subjects, immunohistochemistry and electron microscopy revealed changes in astrocyte morphology and myelin loss as well as up to 30% axonal loss in areas of WM rarefaction when measured against non-demented control (NDC) tissue. Comparative proteomic analyses were performed on pooled samples of periventricular WM (PVWM) obtained from AD (n=4) and NDC (n=5) subjects with both groups having a mean age of death of 86 years. All subjects had an apolipoprotein E ε3/3 genotype with the exception of one NDC subject who was ε2/3. Urea-detergent homogenates were analyzed using two different separation techniques: 2-dimensional isoelectric focusing/reverse-phase chromatography and 2-dimensional difference gel electrophoresis (2D-DIGE). Proteins with different expression levels between the 2 diagnostic groups were identified using MALDI-Tof/Tof mass spectrometry. In addition, Western blots were used to quantify proteins of interest in individual AD and NDC cases. Our proteomic studies revealed that when WM protein pools were loaded at equal amounts of total protein for comparative analyses, there were quantitative differences between the 2 groups. Molecules related to cytoskeleton maintenance, calcium metabolism and cellular survival such as glial fibrillary acidic protein, vimentin, tropomyosin, collapsin response mediator protein-2, calmodulin, S100-P, annexin A1, α-internexin, α- and β-synuclein, α-B-crystalline, fascin-1, ubiquitin carboxyl-terminal esterase and thymosine were altered between AD and NDC pools. Our experiments suggest that WM activities become globally impaired during the course of AD with significant morphological, biochemical and functional consequential implications for gray matter function and cognitive deficits. These observations may endorse the hypothesis that WM dysfunction is not only a consequence of AD pathology, but that it may precipitate and/or potentiate AD dementia.

Figure 1

Coronal sections from the frontal lobe at the level of the genu of the corpus callosum and immunohistological sections of deep WM stained with anti-GFAP/Alexa Fluor 488. A) A representative section of a NDC individual (case #4) showing an intense and homogeneous distribution of the H&E stain throughout the WM. B) An example of extensive WM rarefaction in AD (case #13). In this section, the apparent pallor of the H&E stain reveals extensive WM loss comprising the periventricular and deep WM. The boxed areas in both cases depict the areas of PVWM tissue used in the proteomic analysis and the arrows designate the superior angle of the anterior horn of the lateral ventricle. C) Immunohistological section of the deep WM of NDC case #4 astrocytes. The morphology of the astrocyte shows a stellar body with fine ramified fibrous processes with smooth surfaces that extend over long distances. D) Section of the deep WM from AD case #13. Most of the astrocyte bodies resemble the protoplasmic type with shorter processes of variable caliber. There is an extensive distribution of granular positive GFAP that may correspond to dilated astrocytic cell processes probably filling areas of extensive axonal demise (see Figure 2). Magnification = 200X. Scale bar in (C) also applies to (D). CC, corpus callosum; CG, cingulate gyrus; SFG, superior frontal gyrus; MFG, middle frontal gyrus; IFG, inferior frontal gyrus.

Figure 2

Electron micrographs of frontal WM. A) Electron micrograph section of a 90 year old NDC female, with a total WMR score of 0, showing a homogeneous distribution of axons with different degrees of myelination. B) An 87 year old AD female with a total WMR score of 10, demonstrating a severe loss of axons and myelin surrounded by dilated astrocytic processes (designated by asterisks) with a ‘watery appearance’ that fill the gaps left by axonal downfall that may explain the granular positive GFAP impression shown in Figure 1D. Axonal counting revealed a difference of 30% loss in the AD case relative to the NDC. Magnification = 2000X. Scale bar in (A) applies to (B). The tissues were fixed in the immediate postmortem in 2% paraformaldehyde, 1.25% glutaraldehyde, 0.075% calcium chloride in 0.1 M Na cacodylate buffer pH 7.4. The electron micrographs are a courtesy of Dr. Bernd Bohrmann, Hoffman-La Roche, Basel Switzerland.

Figure 3

2D-DIGE analysis of the NDC pool (A) and AD pool (B) of PVWM pellet proteins. C) Zoomed in area of the NDC gel showing identified spots in more detail. The numbers in (C) correspond to Table 2 spot numbers with mass spectrometry protein identifications. Isoelectric points (pI) are given across the top of each gel and molecular weigh in kiloDaltons (kDa) along the left side. Note that pI and molecular weight are approximate.

Figure 4

After fractionation based on pH, AD (red) and NDC (green) PVWM soluble protein pools were separated on a reverse phase column. After filtering the data, 5 fractions were selected for mass spectrometry protein identification. A) Fraction I mass spectrometry results are shown in Table 3. B) Fractions II and III mass spectrometry results are shown in Table 5. C) Fraction IV mass spectrometry results are shown in Table 5 and Fraction V shown in Table 6.

Figure 5

1D SDS-PAGE Western blots of individual AD and NDC subjects. Antibody details are given in supplementary Table I. Case numbers provided at the top of each blot correspond to those in Table 1: Cases 1-5 = NDC and cases 10-13 = AD. All blots were stripped and re-probed with actin and are provided below each blot. Statistical analysis was performed with an unpaired, 2-tailed, t-test.