Role of SETD5 in Moyamoya Disease Pathogenesis

摘要

ABSTRACT Moyamoya disease (MMD) occurs when the distal internal carotid arteries are progressively narrowed and eventually occluded, and is a common cause of pediatric stroke. Numerous pathogenic genetic variants have been identified to cause MMD, but a common mechanism of pathogenesis has yet to be defined. Pathology from affected vessels shows the occlusive lesions are comprised of fibroproliferative cells that stain positive for smooth muscle cell (SMC)-specific α-actin (SMA); we therefore propose that SMC migration and proliferation may be drivers of the disease. Multiple genes encoding proteins that participate in chromatin remodeling have been identified to cause MMD, including heterozygous loss of function (LOF) variants in the gene SETD5. SETD5 interacts with the nuclear receptor-corepressor (Ncor) complex to regulate histone acetylation. Our previous work on MMD-causing pathogenic variants in ACTA2 showed that these variants impair SMC differentiation, and the incompletely differentiated cells have increased proliferation and migration and rely on glycolysis for cellular energy production. Importantly, treatments that boost oxidative phosphorylation restored differentiation and reduced migration in mouse SMCs with MMD-causing Acta2 variants, suggesting a potential therapeutic strategy. Based on these results and the list of identified genetic triggers for MMD, we propose a common pathogenic mechanism: aberrant chromatin remodeling during SMC specification leads to cells that proliferate and migrate to occlude the vessels. Here, we will test this hypothesis in cells with LOF variants in SETD5 in two specific aims. 1) We will assess whether LOF variants in SETD5 impact SMC differentiation and phenotype. We will use Crispr/Cas9 gene editing to introduce SETD5 LOF alleles into human induced pluripotent stem cells (iPSCs). We will differentiate these iPSCs alongside isogenic controls into neural crest progenitors and then into SMCs, and will characterize the differentiation, proliferation, migration, and metabolism of the resulting cells. 2) We will assess whether SETD5 impacts chromatin remodeling at loci critical for SMC differentiation. We will introduce a 3xFlag tag at the C-terminus of the SETD5 protein using targeted Crispr/Cas9 gene editing in human iPSCs and will use these cells to identify genomic loci where SETD5 is acting in iPSCs, neural crest progenitors, and SMCs by chromatin immunoprecipitation sequencing. We will assess whether LOF variants in SETD5 affect histone acetylation and gene transcription at the identified loci. Completion of these aims will link SETD5- dependent chromatin remodeling with SMC phenotype and elucidate the molecular mechanisms by which LOF variants in SETD5 cause MMD. The results have the potential to identify therapeutic strategies to treat or prevent MMD in patients with SETD5 LOF variants. Finally, these data will dramatically advance our understanding of a potential common pathway for MMD pathogenesis.