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Background Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. Methods We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. Results Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. Conclusion In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia–immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

The inner blood-retina barrier (BRB) resembles the blood-brain barrier (BBB) and is comprised of a single layer endothelium sealed with tight junctions whose abluminal surface is ensheathed by pericytes. The inner BRB is essential for providing a constant and controlled flow of nutrients and molecules to meet metabolic needs. Accordingly, the inner BRB harbors numerous carriers providing the exchange of molecules between blood and retina. One of these carriers is Reduced Folate Carrier 1 (RFC1), which functions as a folate transporter, and has been one of the most consistently detected genes in cerebral pericytes in a recent meta-analysis. The presence of RFC1 protein and its functions in retinal pericytes and microvessels have been overlooked so far despite the similarities between retinal and cerebral microcirculation. In this report, our purposes were to assess the presence of RFC1 protein in retinal microvessels and surrounding pericytes, and modify to increase or decrease RFC1 levels to explore its roles in healthy and ischemic retinal microvessels. Our analyses on whole mount retinas, and trypsin digest preparations from adult Swiss albino mice with concomitant pericyte and endothelial cell markers have shown the presence of RFC1 protein in the inner BRB and colocalization with pericytes. In vivo modifications of RFC1 expression levels were accomplished by intravitreal delivery of genetic and pharmacological agents. Knocking down RFC1 expression via custom-designed short interfering RNA (siRNA) resulted in microvascular dysfunction which was presented by the disintegration of tight junction proteins and the main basal membrane protein collagen-4. This was accompanied by extensive endogenous IgG extravasation suggesting impairment in the barrier functions. On the other hand, delivering RFC1 expressing lentiviral vector to overexpress RFC1 in the retina induced immunosignal increase in tight junction proteins and collagen-4 confirming the role of RFC1 in the inner BRB functions. Administration of methotrexate (MTX), the pharmacological competitive inhibitor of folate, resulted in increased levels of RFC1. In retinal ischemia models, one-hour retinal ischemia increased RFC1 levels and decreased collagen-4 and occludin levels. These effects could be partially reversed with pre-treatment by RFC1 expressing lentiviral vector. All these indicate the importance of RFC1 levels in the pathophysiology of retinal ischemia, a prevalent cause of blindness worldwide. In conclusion, our study clarifies the presence of RFC1 in inner BRB and offers a novel perspective on RFC1, other than being a folate carrier, as a regulator of inner BRB in healthy and ischemic retinas.

Aim Muscle weakness, fatigue and speech problems can occur in neurofibromatosis type 1 (NF1). The pathogenesis of these symptoms is unclear, likely multifactorial. We examined motor function in limb and speech muscles in NF1 patients. Methods We evaluated NF1 and control groups aged 4–18 years for muscle strength, tone and mobility using standard manual testing, joint motion and Beighton score measurements. Speech and language functions were assessed by speech articulation and resonance. As a marker of muscle tissue turnover, we determined collagen degradation products in urine before and after submaximal exercise. Results NF1 patients had reduced strength in proximal limb muscles compared to control subjects. Speech articulation problems and hypernasality were more common in NF1 (47% and 38%, respectively). Collagen products excreted in urine correlated with gluteal and biceps muscle strength. Conclusion Muscle dysfunction can be detected in some children with NF1 and may explain certain clinical features including fatigue, speech and articulation problems. If confirmed by further research, these findings may be relevant to the management of this condition.

Purpose: Muscle weakness and delays in motor development are more common problems in children with neurofibromatosis type 1. Gross Motor Function Measurement-88 is widely used tool to evaluate motor functions in children with developmental disabilities. We aimed to investigate validity of the Gross Motor Function Measurement-88 in a sample of Turkish children with neurofibromatosis type 1. Methods: Aged between 5 to 17 years 40 (20 male/20 female) children with neurofibromatosis type 1. participated in this study. To asses validity of Gross Motor Function Measurement-88, Manual Muscle Test was done seven muscle groups in upper and lower limbs bilaterally by a physical therapist. Results: The mean age was 9.7 ± 3.81 years. A positive moderate to strong correlation was found between Manual Muscle Test and Gross Motor Function Measurement-88 subdomains (r = 0.317-0.668; p <0.05). Conclusion: Gross Motor Function Measurement-88 is a valid measurement for evaluating gross motor functions in children with neurofibromatosis type 1. Identifying motor developmental delays in children with neurofibromatosis type 1 will be a guide for establishing early intervention programs and determining symptom-specific rehabilitation goals. We recommend the use of Gross Motor Function Measurement-88 in children with neurofibromatosis type 1 for evaluating gross motor function.