NOTCH3 CADASIL Variant Receptor Aggregation Requires NOTCH3 Wild-Type Receptors: Identification of Highly Selective Inhibitors That Block the Process.

Cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) is a monogenic autosomal dominant small vessel disease clinically characterized by a broad spectrum of symptoms including recurrent ischemic strokes, cognitive dysfunction, and premature dementia. It is caused (predominantly) by cysteine-altering mutations in exons encoding the epidermal growth factor-like repeats (EGFrs) of the extracellular domain of the NOTCH3 receptor, which are associated with pathological impairment of the cerebral vasculature. Whilst the pathophysiological features of the condition are well established, the precise molecular mechanisms by which NOTCH3 mutations trigger the disease have remained unclear. In this study, we have biochemically interrogated the impact of different pathogenic NOTCH3 CADASIL variants on NOTCH3 receptor aggregation (and signaling) using novel in vitro analyses and cell-based assays. By these means, we found that CADASIL mutant receptors alone could not self-associate into high molecular mass assemblies, but did promote NOTCH receptor aggregation only in combination with wild-type receptors. Moreover, pathogenic CADASIL variants localized to the EGFr 4 of NOTCH3 specifically and significantly enhanced JAGGED1 (JAG1)-dependent NOTCH3 transactivation. Importantly, we show that unique, paralogue-specific NOTCH3 inhibitors can block both receptor aggregation and CADASIL mutant gain-of-function NOTCH3 signaling, which could represent a new therapeutic approach to treating the disease.