Atherosclerotic lesions and mitochondria DNA deletions in brain microvessels: implication in the pathogenesis of Alzheimer's disease

Abstract

The pathogenesis that is primarily responsible for Alzheimer's disease (AD) and cerebrovascular accidents (CVA) appears to involve chronic hypoperfusion. We studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD biopsy samples and two transgenic mouse models of AD, yeast artificial chromosome (YAC) and C57B6/SJL Tg (+), which overexpress human amyloid beta precursor protein (AbetaPP). In situ hybridization using probes for normal and 5 kb deleted human and mouse mitochondrial DNA (mtDNA) was performed along with immunocytochemistry using antibodies against the Abeta peptide processed from AbetaPP, 8-hydroxy-2'-guanosine (8OHG), and cytochrome c oxidase (COX). More amyloid deposition, oxidative stress markers as well as mitochondrial DNA deletions and structural abnormalities were present in the vascular walls of the human AD samples and the AbetaPP-YAC and C57B6/SJL Tg (+) transgenic mice compared to age-matched controls. Ultrastructural damage in perivascular cells highly correlated with endothelial lesions in all samples. Therefore, pharmacological interventions, directed at correcting the chronic hypoperfusion state, may change the natural course of the development of dementing neurodegeneration.

Keywords: Alzheimer’s disease; atherosclerosis; brain hypoperfusion; electron microscopy; transgenic animals; vascular and mitochondrial lesions.

Figure 1

Ultrastructural characteristics of brain microvessels from human Alzheimer disease brain biopsies. A: Microvessels with minimal changes did not show any particular pathology in the ultrastructure of the vascular endothelium; however, perivascular cells show the presence of vacuolar degenerative structures in the matrix (indicated by single arrow). B: Vascular endothelium and perivascular cells of damaged microvessels display ultrastructural lesions in their cytoplasmic organelles especially mitochondria. Completely damaged matrices appeared to be permanent features of all of these cells (indicated by single arrow). Original magnification ×20,000 (A and B). C: Vascular endothelium with non-reversible damage have completely damaged mitochondria and destructive changes in the membranous structures. The basal membrane (BM) is very thick and occupies a large area of the vascular wall. Original magnification ×12,000.

Figure 2

Features of amyloid deposition in a neuronal cell body and the extracellular matrix in AβPP-YAC aged transgenic mouse brain (PAP pre-embedding immunocytochemistry). Amyloid depositions are associated with the formation of PHF-like structures (double asterisk in figure E). Original magnification A–D ×20,000. E–F ×25,000.

Figure 3

Features of APP immunostaining in frontal cortical microvessels in aged AβPP-YAC mice determined by PAP pre-embedding immunocytochemistry. A, B and C are without counter staining. D is with counter staining. Asterisks indicate amyloid deposition. Original magnification ×12,000.

Figure 4

Electron microscopic subcellular features of brain cortical microvessels from 24-month old AβPP-YAC mice. A: Vascular endothelial cells display lipid vacuoles in their cytoplasmic matrices (single arrow). Perivascular cells have “giant”- sized lipid/amyloid granules in their cytoplasmic matrices (indicated by asterisk). Original magnification: ×12,000. B: Microvessels with amyloid “angiopathy” are characterized by the accumulation of large-sized amyloid/lipid granules in the matrices of perivascular cells (indicated by asterisk). Vacuolar structures with lipid filled amyloid deposition (single arrow) are also present in the matrices of perivascular cells. Original magnification: ×12,000.

Figure 5

Age-matched control, non-transgenic mice did not show any particular changes in their neuronal ultrastructure. Lipofuscin was present in some neurons (indicate by asterisk in figure A). Original magnification of A and B: ×5,000, and ×20,000, respectively.

Figure 6

Ultrastructural characteristics of the neuronal damage in AβPP-YAC transgenic mouse hippocampus. Mitochondrial lesions were associated with lipofuscin formation in the neuronal cell body (asterisk) and mitochondria appeared to be a major substrate for lipofuscin formation (single arrows). Original magnification: A, E, and F ×5,000. B, C, and D ×20,000 respectively.

Figure 7

Ultrastructural characteristics of cortical neurons in C57B6/SJL transgenic mouse (Tg +) brain. The main characteristic of neuronal damage appeared to be the transformation of completely damaged mitochondria (mitochondria without any residues of mitochondrial cristae; indicated by single arrows) into the lipofuscin granules, characterised by their cluster type localization in the neuronal cell body. Original magnification: A and B ×5,000; C and D ×20,000, respectively.

Figure 8

Electron microscopic determination of mitochondrial DNA signals visualized by using wild type and chimeric 5 kb deleted mtDNA probes in a human postmortem Alzheimer’s disease (AD) brain (A–B) and 24-month old AβPP-YAC transgenic mouse brain (C–D). A and B: AD brain microvessels endothelium and perivascular cells show clusters of wild type mtDNA containing positive signals visualized by indirect colloidal gold techniques (indicated by the dark dots). Original magnification: A and B ×10,000 and ×25,000, respectively. C–D: AβPP-YAC transgenic mice show the presence of clusters of chimeric 5 kb deleted mitochondria DNA positive signals throughout the matrices of vascular and perivascular cells (single arrows). Original magnification: C and D ×30,000 and ×20,000, respectively. Abbreviations: BM, basal membrane; EC, endothelial cell; ER, erythrocytes; N, cell nucleus; VL, vessel lumen; PAP, peroxidase-anti-peroxidase; PHF, paired helical filaments.