Cellular Protein Trafficking of Notch3 in Vascular Dementia CADASIL

Abstract

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy) is a late onset, dominantly inherited genetic disease associated with vascular smooth muscle cell (VSMC) degeneration, recurrent stroke, and vascular dementia. CADASIL mutants map to the NOTCH3 gene, a signalling receptor that regulates the differentiation and maturation of VSMCs. Canonical Notch signal activation follows ligand binding, which initiates proteolytic cleavages that remove the extracellular domain (ECD) by an ADAM10-dependent mechanism, followed by γ-secretase release of the intracellular domain (ICD). CADASIL mutants result in an odd number of Cysteines in Epidermal Growth Factor (EGF) repeats that comprise the Notch3 ECD. These mutations are associated with an accumulation of detached Notch3 ECD by an unknown mechanism. There is no consensus on how CADASIL arises, and loss of signal, gain of signal, and a toxicity arising from ECD accumulations, have all been proposed. Here I investigate a hypothesis that an altered basal, ligand-independent, Notch3 signalling mechanism, involving its endocytic trafficking, is a common underlying consequence of CADASIL mutants. Using an over expression cell model I showed that ECD detachment from ICD is initiated in the early endosome, with ICD subsequently trafficking separately in late endosome and lysosome. I found that CADASIL mutants were associated with a subtle increase in Notch3 accumulation in the cell, and in ECD separation from ICD. Basal Notch3 signalling was affected by endocytic trafficking regulators, Dtx2, WWP1, WWP2 and VPS4 and increased by three different CADASIL mutants. The latter include Notch3C455R, which, in contrast, disrupts ligand-dependent signalling. One mutant, Notch3R90C, shifted the basal activation mechanism from an ADAM10-dependent to an ADAM10-independent form, similar to that of Notch1. Notch3R90C also showed an altered band pattern on western blots, a biochemical change that I suggest is linked to the altered mechanism. I also investigated endogenous Notch3 staining in MCF-7 breast cancer cell line and in VSMCs differentiated from a human Embryonic Stem (hES) cells. The results supported Notch processing in the endocytic pathway and establish hES cells as a model system for future work to investigate the interplay between endocytic trafficking, mutations of Notch, and cell differentiation.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Baron (Supervisor), Keith Brennan (Supervisor) & Tao Wang (Supervisor)