The Investigation the Role of Reduced Folate Carrier 1 in the Models of Ischemic Stroke and Retinal Ischemia

Abstract

Gurler, G., The Investigation the Role of Reduced Folate Carrier 1 in the Models of Ischemic Stroke and Retinal Ischemia, Hacettepe University, Graduate School of Health Sciences, Neuroscience Program, Doctor of Philosophy Thesis, Ankara, 2023. Reduced Folate Carrier 1 (RFC1, SLC19A1) is the folate transporter in the brain located at the blood-brain barrier (BBB). Polymorphisms of RFC1 in humans are linked to an increased risk of ischemic stroke. Transcriptomic studies have shown an abundant presence of RFC1 mRNA in cerebral microvascular mural cells; however, RFC1 protein had not been demonstrated yet. Our previous studies revealed the importance of microcirculation in stroke pathophysiology. The presence of RFC1 protein and its probable role in ischemic stroke, and retinal ischemia from the perspective of microvessels, remain enigmatic. In this thesis, RFC1 protein in the brain and retinal pericytes as well as in the inner blood-retina barrier (BRB) was characterized in adult mice. RFC1 protein was evaluated by the immunohistochemistry of the whole mount retinas, cerebral sections, isolated microvessels, Western blotting, and cerebral 7 Tesla MRI in separate experiments that include knock down by short interfering RNA (siRNA), and overexpression by Lentiviral Vector (LV), and pharmacological modification by methotrexate. Knocking down retinal RFC1 led to structural abnormalities in microvessels, and decreased in the expression of barrier proteins occludin, claudin-5, ZO-1, and collagen-4, resulting in the functional failure of inner BRB, as seen by an extensive IgG leakage. This failure in the brain was also detected by contrast agent leakage in MRI. Additionally, the detrimental effects of ischemia on the barriers were exacerbated. Pre-treatment with RFC1-LV partially restored collagen-4 and occludin levels in retinal ischemia. In essence, this thesis clarifies the presence of RFC1 protein in the cerebral pericytes and the inner BRB, offering a novel perspective on RFC1 as a dynamic regulator of the BBB and inner BRB under physiologic states, and emphasizes its critical role during ischemia. Key Words: blood-brain barrier, blood-retina barrier, neurovascular unit, cerebral ischemia, retinal ischemia, RFC1, SLC19A1 This study is supported by The Scientific and Technological Research Institution of Turkey (TÜBİTAK; Grant No: 120N690) and Hacettepe University Scientific Research Coordination Unit (Project No: TDK-2020-18590)