A novel nuclear role for smooth muscle alpha-actin

ABSTRACT Pathogenic variants in ACTA2, which encodes smooth muscle α-actin (SMA), are a major cause of heritable thoracic aortic disease, and missense variants affecting two specific amino acids of ACTA2 (arginines 179 and 258; R179 and R258) also predispose to moyamoya disease (MMD). MMD is characterized by narrowing and occlusion of the distal internal carotid arteries, and is a common cause of pediatric-onset strokes. Multiple genetic factors predisposing to MMD have been found, but there is no identified common mechanism of pathogenesis. However, pathology from affected vessels suggests that smooth muscle cell (SMC) proliferation and migration may drive the disease. We found a common molecular defect between R179 and R258 ACTA2 variants not shared by other ACTA2 variants: a loss of nuclear SMA function. Based on our preliminary data, we hypothesize that nuclear SMA is critical for differentiation of cerebrovascular SMCs from neuro-ectodermal progenitor cells, and that incomplete differentiation of SMCs primes the cells for the excessive proliferation and migration that causes MMD. We are focusing on ACTA2 p.R179 variants because these patients have the most severe and earliest-onset disease. We will test our hypothesis with the following specific aims: 1) Using our conditional smooth muscle-specific knock-in Acta2SMC-R179C/+ mouse, we will assess cell fate trajectories of wild-type and heterozygous Acta2 p.R179C mutant SMCs over developmental time using single cell RNA- sequencing. 2) We will use Crispr/Cas9 to correct the pathogenic variant in induced pluripotent stem cells (iPSCs) from ACTA2 p.R179 patients (n=3) and assess whether gene editing restores the SMC differentiation potential of these cells; we will also generate sequencing and integrated bioinformatics analyses to interrogate chromatin remodeling changes during differentiation that are reliant on nuclear SMA. 3) We will investigate the role of chromatin remodeling protein EZH2 in the incomplete differentiation phenotype of Acta2 p.R179 cells, including testing an FDA- approved inhibitor of EZH2 as a potential therapeutic in our mouse model. Successful completion of these aims will provide clear data on epigenetic remodeling required for SMC development and how that development is perturbed by ACTA2 p.R179 variants. These data will also establish the potential efficacy of EZH2 inhibitors as a treatment for patients with ACTA2 p.R179 variants, and future studies will explore applicability of this treatment for patients with other pediatric MMD- associated pathogenic variants. Finally, these data should identify molecular regulators of SMC cell fate specification, which could lead to promising avenues for future research on the maintenance of SMC identity across multiple vascular diseases.