RNF213 variant contributes to dysfunction of endothelial cells through impaired autophagy in Moyamoya disease Moyamoya disease (MMD) is a unique cerebrovascular disorder marked by the progressive constriction or blockage of the distal internal carotid arteries. This abnormal constriction of the major arteries that supply blood to the brain triggers ischemic reactions, resulting in a decrease in cerebral blood flow and subsequent damage to the endothelial cells and neurons. Although the majority of MMD cases are predominantly sporadic in nature, there is a subset of approximately 15% of MMD patients in East Asia region who have a familial history, prompting researchers to explore potential genetic influences that may play a role in development of MMD. The identification of Ring finger protein 213 (RNF213) as a gene that can potentially elevate a vulnerability to MMD has been established. This gene is predominantly recognized for its role in the involvement in blood vessel development and the formation of blood vessels. The RNF213 p.R4810K variant, also known as rs112735431, c.14429 G>A, is the most frequently observed genetic variant among patients who have been diagnosed with MMD in the East Asia region. This variation in a single nucleotide can be found within the E3 ligase domain of the RNF213 protein, and it is recognized for its ability to reduce the enzymatic activity of the E3 ligase, resulting in a notable impairment in the ubiquitylation process. However, the specific role and mechanism of the RNF213 contributes to the development and advancement of MMD are not fully understood. Autophagy is a significant cellular process that is essential for the maintenance of normal physiological cellular states. It exhibits improved activity levels, notably in circumstances of stress like deprivation of nutrients or oxygen. Autophagy helps to break down and eliminate abnormal proteins and damaged organelles, thereby ensuring cellular health and function under cellular stress conditions. It was hypothesized that the presence of the RNF213 p.R4810K variant, which has been found to have diminished E3 ligase activity, could potentially have a negative impact on the process of protein degradation within cells, leading to a disruption in cellular clearance mechanisms. This study aimed to explore the relationship between the RNF213 p.R4810K variant and autophagy, a cellular process activated in reaction to stressful conditions, in the context of MMD pathology. The motivation for conducting this study stemmed from a particular case involving a patient diagnosed with MMD who possessed the RNF213 p.R4810K genetic variant. The patient displayed the shrinkage of cerebral vessels, decreased cerebral blood flow (CBF), and cognitive function as result of malnutrition. As a result of this observation, it was concluded that individuals diagnosed with MMD who possess genetic variants that render them more susceptible may potentially undergo a deterioration in their cerebrovascular health in response to alterations in their environment, such as deficiencies in nutrients. The main objective of the study was to investigate the processes triggered by the RNF213 p.R4810K variation, as well as to clarify the role and processes of the RNF213 p.R4810K variant in endothelial cells during ischemic situations that imitate the vascular condition of progressive MMD. Patients diagnosed with MMD were selected for participation in the study based on the genotyping related to the RNF213 p.R4810K variant. In vascular disorders, endothelial cells are typically the first to be affected, leading to the utilization of flow- mediated dilation (FMD) was measured as an indicator endothelial cell function. A significant decrease in FMD levels was observed in the RNF213 p.R4810K variant group of MMD patients compared to non-MMD patients, indicating impaired endothelial function. After conducting a thorough, analysis of perfusion CT scans from 45 patients revealed a significant reduction in CBF. Analysis of protein expression in peripheral blood mononuclear cells (PBMCs) isolated from MMD patients demonstrated increased RNF213 expression and elevated levels of autophagy markers (SQSTM1/p62 and LC3- II) following exposure to oxygen-glucose deprivation (OGD), suggesting impaired autophagy in the individuals carrying the RNF213 p.R4810K variant carriers. Additionally, following exposure to OGD, there were significant impairments in marker proteins related to autophagy (SQSTM1/p62 and LC3-II). Investigations in human umbilical vein endothelial cells (HUVECs) overexpressing the RNF213 p.R4810K variant and exposure to OGD. The assay showed an increase in levels of the RNF213 protein, as well as levels of autophagy marker proteins. An increase in the number of autophagic vesicles was also observed using transmission electron microscopy (TEM) analysis. Tube formation assays revealed impaired endothelial function in HUVECs transfected with the RNF213 p.R4810K variant under OGD exposure conditions. The administration of rapamycin (RA), a commonly used substance known to stimulate autophagy, successfully restored the autophagy process in HUVECs that possess the RNF213 p.R4810K genetic variant when exposed to OGD. RA treatment also improved endothelial function, as assessed by the tube formation assay in HUVECs that were transfected with the RNF213 p.R4810K variant under OGD exposure. Similarly, the administration of cilostazol (CSZ), a phosphodiesterase III inhibitor known for its vasodilatory properties, was observed to induce autophagy in a manner comparable to RA treatment. Additionally, CSZ effectively improved the endothelial function in HUVECs that were transfected with the RNF213 p.R4810K variant under OGD exposure. To summarize, the findings presented in this thesis suggest that autophagy plays an important role in the progression of endothelial dysfunction in MMD patients carrying the RNF213 p.R4810K variant. Consequently, therapeutic strategies aimed at modulating autophagy may offer significant advantages by addressing the interplay between genetic factors and environmental factors that contribute the progression of Keywords: Moyamoya disease, Ring finger protein 213, Autophagy, Cilostazol, Endothelial dysfunction
