Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease

Author Summary

The most common hereditary cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor, a cell surface protein implicated in a fundamental cell signaling pathway involved in development. CADASIL pathology is characterized by the degeneration of smooth muscle cells in blood vessels and by the accumulation of electron-dense vascular deposits termed granular osmiophilic material (GOM). The mechanism by which the Notch 3 mutations implicated in CADASIL affect small vessels and lead to the accumulation of GOM remains unclear. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two distinct Notch 3 mutations, C455R and R1031C, respectively associated with early- and late-onset stroke, by using a combination of transgenic mouse models, postmortem human tissue, and cell culture-based assays (Fig. 1). The mutations each lead to single amino acid replacements in the Notch 3 protein. We demonstrate that the two mutations define reduced activity states of Notch 3, a property linked to stroke susceptibility in the mouse models we generated. Importantly, these mice develop granular deposits and other age-related changes that are remarkably similar to those observed in individuals with CADASIL. Proteomic analysis of blood vessels from the brains of people carrying the same CADASIL-related mutations helped identify components of the GOM, including the proteins clusterin and collagen 18 α1/endostatin. Our findings link the loss of Notch signaling with ischemic cerebral small-vessel disease. We find that CADASIL pathophysiology is associated with reduced Notch 3 activity in vascular smooth muscle cells, and implicate the age-dependent accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.

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Fig. 1.

Experimental design and key findings. Transgenic mouse models conditionally expressing two mutant forms of the Notch 3 receptor identified in Colombian families afflicted with CADASIL, each defining a particular phenotype of the disease, were generated. They were used to examine the activity of Notch 3 receptors carrying the C455R or R1031C mutations by using functional assays in vivo, ultrastructural analysis, and a ligand-dependent assay of Notch 3 receptor activity in cell culture. Both mutations are loss-of-function mutations: the early-onset C455R mutation represents a more severe loss of Notch 3 receptor function than the late-onset R1031C mutation, and both result in age-dependent phenotypes in the transgenic animal models, including accumulation of deposits in brain vessels. In parallel, brain blood vessels were collected from postmortem tissue carrying the R1031C mutation and analyzed by using MS. The proteomic analysis identified several proteins enriched in CADASIL vessels, including clusterin and collagen 18 α1/endostatin. Both proteins showed abnormal distribution in brain vessels of patients with several CADASIL mutations and, moreover, their expression was misregulated in the transgenic animal models expressing the mutant Notch 3 receptors. These findings point to remarkable similarity between the human disease and the transgenic animal models characterized in this study.

The Notch cell surface receptors play crucial roles in the development and homeostasis of metazoans. Mammals harbor four Notch receptors, of which Notch 3, the receptor associated with CADASIL, is expressed in smooth muscle cells that form a part of the blood vessel wall. Thus, far, more than 100 Notch 3 mutations underlying CADASIL have been molecularly characterized, and most, if not all, affect single cysteine residues in the extracellular domain of the Notch receptor protein (1–3). However, the functional significance of these mutations remains unknown. For example, we do not know if the CADASIL mutations lead to a loss or gain of function, this knowledge being essential for contemplating therapeutic approaches for the condition. The difficulty in answering such fundamental questions has been partly because of the lack of appropriate CADASIL animal models and sensitive quantitative assays for Notch receptor activity.

Our study was aimed at developing such models, with the goal of probing the functionality of Notch 3 receptors carrying CADASIL mutations and examining the nature of the GOM deposits that characterize CADASIL. To address these issues, we adopted an experimental approach that combines transgenic animal models with cellular assays while taking advantage of our access to postmortem tissue donated by members of two large Colombian families afflicted with CADASIL. The mutations were chosen for our study not only because of our access to this tissue and the associated detailed clinical information about them, but also because each mutation defines a particular phenotype of the disease. Individuals carrying the C455R mutation in the extracellular domain of Notch 3 show an apparently more severe phenotype than individuals carrying the R1031C mutation, such that the median age at onset of stroke in individuals with the C455R mutation precedes by more than two decades that of individuals with the R1031C mutation. The early-onset phenotype is accompanied by extensive white matter abnormalities that can be visualized by MRI.

We generated two transgenic mouse models that carry these two Notch 3 mutations and examined the functionality of the mutant receptors. More generally, we attempted to develop experimental systems that would allow us to better study the cellular and molecular mechanisms underlying this catastrophic disease. We have previously shown that mice in which the Notch 3 gene had been knocked out, leading to a complete loss of Notch 3 activity in vascular smooth muscle cells, are strikingly susceptible to experimentally induced brain injury (4). This phenotype can be reversed by expressing the WT Notch 3 receptor in vascular smooth muscle cells, thus demonstrating that Notch3 is necessary and sufficient for this particular physiological function. When this experimental paradigm is used to examine the activity of Notch 3 receptors carrying the C455R or R1031C mutations, we find that the CADASIL mutations impair Notch signaling in vascular smooth muscle cells. The early-onset C455R mutation represents a more severe loss of Notch 3 receptor function than the late onset R1031C mutation. This genotype–phenotype correlation is also observed in patients. The conclusion that the CADASIL mutations impair Notch signaling was corroborated through cell-based assays developed to address receptor activity. These assays enabled a quantitative comparison of receptor activity and helped us demonstrate that the mutants represent partial loss-of-function states of the receptor. Moreover, further validating the analogy between the transgenic models and the human disease, we find that the susceptibility of the mice carrying CADASIL mutations to induced stroke becomes more severe with age, indicating that the models undergo age-related degeneration reminiscent of that observed in patients with CADASIL.

To further characterize CADASIL pathology and gain insight into the composition of the GOM seen in patients with CADASIL, we used a laser capture microdissection technique to collect brain blood vessels from postmortem tissue carrying the R1031C mutation and subsequently analyzed them by using MS. The proteomic analysis revealed several proteins enriched in CADASIL vessels. Two proteins, namely clusterin and collagen 18 α1/endostatin, stood out. COL18A1 is cleaved following its translation to give rise to endostatin, a C-terminal fragment with strong antiangiogenic properties, whereas clusterin functions as a molecular chaperone, variants of which have been associated with Alzheimer disease and with extracellular protein deposits in several disorders. Further immunocytochemical analysis of postmortem CADASIL brains from patients carrying the R1031C or C455R mutation indicates that both proteins can indeed be detected in the GOM deposits.

Importantly, we find that the age-dependent phenotypes associated with our CADASIL mouse models include the accumulation of deposits in brain vessels. The link, if indeed such a link exists, between Notch activity and the deposits is unclear. We thus took advantage of these transgenic models to examine whether disrupting Notch 3 activity by expressing the R1031C CADASIL receptor in vascular smooth muscle cells affects the expression of the two GOM components identified in our proteomics analysis. We find that the expression of the mutant Notch 3 receptor results in the misregulation of expression of clusterin and COL18A1/endostatin, raising the possibility of a functional link between Notch signaling and the expression of these proteins.

The remarkable similarity between the human disease and the models we developed suggests that these models may help researchers further explore the mechanisms underlying this devastating condition.