Novel dot-blot assay for detection of vascular Notch3 aggregates in patients with CADASIL

Yihong Ma; Mitsuharu Ueda; Akihiko Ueda; Satoru Shinriki; Akihito Nagatoshi; Aito Isoguchi; Masamitsu Okada; Masayoshi Tasaki; Toshiya Nomura; Yasuteru Inoue; Teruaki Masuda; Yohei Misumi; Taro Yamashita; Hirotaka Matsui; Yukio Ando

doi:10.1016/j.jns.2020.116931

Novel dot-blot assay for detection of vascular Notch3 aggregates in patients with CADASIL

J Neurol Sci. 2020 Aug 15:415:116931. doi: 10.1016/j.jns.2020.116931. Epub 2020 May 21.

Authors

Affiliations

¹ Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan.
² Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan. Electronic address: [email protected].
³ Department of Molecular Laboratory Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan.
⁴ Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan; Department of Neurology, Kumamoto General Hospital, Yatsushiro 866-8660, Japan.
⁵ Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan; Department of Morphological and Physiological Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto 862-0976, Japan.
⁶ Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan; Department of Amyloidosis Research, Nagasaki International University, Sasebo 859-3298, Japan.

PMID: 32470649
DOI: 10.1016/j.jns.2020.116931

Abstract

To detect vascular Notch3 extracellular domain aggregates in CADASIL, we developed a novel dot-blot assay with both autopsy and biopsy skin samples. We obtained samples from 11 patients with CADASIL and 12 control patients, and we performed dot-blot analyses by using sequential biochemical tissue extractions with three different antibodies against specific regions of the Notch3 extracellular domain. We also analyzed clinical features and vascular accumulations of Notch3 by immunohistochemistry. Via the dot-blot assay with the antibody against the C-terminal region of the Notch3 extracellular domain, we successfully detected Notch3 extracellular domain aggregates in skin tissue homogenates obtained from patients with CADASIL. Our novel method may therefore aid the diagnosis of CADASIL.

Keywords: Biopsy; CADASIL; Diagnosis; Dot-blot assay; Notch3.

MeSH terms

CADASIL* / diagnosis
Humans
Immunoblotting
Immunohistochemistry
Mutation
Receptor, Notch3 / genetics
Receptors, Notch / genetics
Skin

Substances

NOTCH3 protein, human
Receptor, Notch3
Receptors, Notch