Iowa variant of familial Alzheimer's disease: accumulation of posttranslationally modified AbetaD23N in parenchymal and cerebrovascular amyloid deposits

Abstract

Mutations within the amyloid-beta (Abeta) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer's disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AbetaD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Abeta molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Abeta species-the Iowa brain Abeta peptidome-contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Abeta cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia.

Figure 1

Immunohistochemical analysis of parenchymal and vascular lesions in the Iowa pedigree. Vascular anti-Aβ (4G8) immunoreactivity (A); preamyloid lesions (arrows, B); vasocentric plaques (double arrow, C); Congo red positive, birefringent vascular deposits (D and E); and thioflavin S fluorescent vascular deposits (F). Scale bar represents 200 μm in A and 100 μm in B–F.

Figure 2

WB and MS analysis of PBS-extracted Aβ. Aβ species retrieved from fractions extracted from 100 mg of microvessel-depleted frontal cortex (A); 10 mg of microvessels and leptomeningeal vessels recovered from 100 mg of frontal cortex (B). C: Theoretical and experimental m/z values. WB: ∗ indicates residual Ig light and heavy chains from the IP. MS: ** illustrates nonspecific peaks also present in negative controls.

Figure 3

WB and MS analysis of SDS-extracted Aβ. Aβ species retrieved from 100 mg of microvessel-depleted frontal cortex (A); 10 mg of microvessels and leptomeningeal vessels recovered from 100 of mg frontal cortex (B). C: Theoretical and experimental m/z values. WB: * indicates residual Ig chains. MS: ** indicates nonspecific peaks appearing in the negative controls.

Figure 4

WB and MS analysis of fibrillar Aβ deposits. A: Parenchymal FA extracts of frontal cortex obtained from either 0.25 or 2.5 mg of tissue for WB and MS, respectively. B: FA extracts from leptomeningeal and microvessel fractions obtained from either 0.025- or 0.25-mg vessels for WB and MS, respectively; C: Theoretical and experimental m/z values. MS: ** indicates nonspecific peaks appearing in the negative controls.

Figure 5

Biochemical heterogeneity of amyloid lesions. A: Location of Iowa mutation on the Aβ1-40 sequence and the T1, T2, T3, and T4 tryptic peptides; B: RP-HPLC of tryptic digests of wild-type (D23) and variant (N23) synthetic homologues, as well as of Iowa FA-extracts; C: MALDI-TOF MS of T3a and T3b in reflectron mode. Monoisotopic mass of protonated T3 from N23 is 1324.69 Da and from D23 is 1325.67; D: Detection of isoAsp residues. Inset: Schematic representation of enzymatic reaction for isoAsp identification. RP-HPLC profiles indicate the presence of S-adenosyl-homocysteine in tryptic fragments T1, T2, and T3 but not in T4.

Figure 6

Effect of Asp acid isomerization and D23N mutation on Aβ fibrillogenesis assessed by thioflavin-T binding. Fluorescence evaluation (excitation/emission wavelengths 435/490 nm, respectively) of Thioflavin T binding assay of the samples collected at the different time points during the 3-days duration of the experiments was performed as described in Materials and Methods. The data are representative of three independent experiments open circles, AβD23N isoAsp1,7 (solid line); closed circles, AβD23N Asp1,7 (dash line); closed squares, Aβ40 isoAsp7, 23 (solid line); open squares, Aβ40 isoAsp1,7, 23 (dash line); triangles, wild-type Aβ40 (solid line).