Glial vascular degeneration in CADASIL

Abstract

CADASIL is a genetic vascular dementia caused by mutations in the Notch 3 gene on Chromosome 19. However, little is known about the mechanisms of vascular degeneration. We characterized upstream components of Notch signaling pathways that may be disrupted in CADASIL, by measuring expression of insulin, IGF-1, and IGF-2 receptors, Notch 1, Notch 3, and aspartyl-(asparaginyl)-β-hydroxylase (AAH) in cortex and white matter from 3 CADASIL and 6 control brains. We assessed CADASIL-associated cell loss by measuring mRNA corresponding to neurons, oligodendroglia, and astrocytes, and indices of vascular degeneration by measuring smooth muscle actin (SMA) and endothelin-1 expression in isolated vessels. Immunohistochemical staining was used to assess SMA degeneration. Significant abnormalities, including reduced cerebral white matter mRNA levels of Notch 1, Notch 3, AAH, SMA, IGF receptors, myelin-associated glycoproteins, and glial fibrillary acidic protein, and reduced vascular expression of SMA, IGF receptors, Notch 1, and Notch 3 were detected in CADASIL-lesioned brains. In addition, we found CADASIL-associated reductions in SMA, and increases in ubiquitin immunoreactivity in the media of white matter and meningeal vessels. No abnormalities in gene expression or immunoreactivity were observed in CADASIL cerebral cortex. In conclusion, molecular abnormalities in CADASIL are largely restricted to white matter and white matter vessels, corresponding to the distribution of neuropathological lesions. These preliminary findings suggest that CADASIL is mediated by both glial and vascular degeneration with reduced expression of IGF receptors and AAH, which regulate Notch expression and function.

Figure 1

Altered SMA immunoreactivity in CADASIL white matter vessels. Histological sections of (A) control and (B–H) CADASIL brains were immunostained with monoclonal antibodies to SMA. Immunoreactivity was detected with biotinylated secondary antibody, followed by horseradish peroxidase conjugated avidin-biotin complexes and diaminobenzidine substrate. Sections were counterstained lightly with hematoxylin. Note uniform dense labeling for SMA in control vessels and variable degrees of fragmented and incomplete labeling of CADASIL vessels. Panel I shows a negative control reaction in which the primary antibody was omitted from the reaction.

Figure 2

Increased ubiquitin immunoreactivity in CADASIL white matter vessels. Paraffin-embedded histological sections of (A–D) CADASIL and (E, F) control frontal lobe white matter were immunostained with monoclonal antibodies to ubiquitin. Immunoreactivity was detected with biotinylated secondary antibody, followed by horseradish peroxidase conjugated avidin-biotin complexes and diaminobenzidine substrate. Sections were counterstained lightly with hematoxylin. Note ubiquitin immunoreactivity in the media of (A) medium-size and (B, C) small CADASIL white matter vessels, as well as in (C, D) glial cells. (E, F) Control brain vessels exhibited focal labeling of endothelial cells, which appeared to be non-specific, whereas ubiquitin immunoreactivity was not detected in the media of control white matter vessels.