Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain St
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ORIGINAL PAPER
Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2‑FLAG in Brain Structures Emiliano Tesoro‑Cruz1 · Norma Oviedo1 · Leticia Manuel‑Apolinar2 · Sandra Orozco‑Suárez3 · Miguel Pérez de la Mora4 · Gloria Martínez‑Pérez1 · Francisco Xavier Guerra‑Castillo1 · Charmina Aguirre‑Alvarado1 · Vilma Carolina Bekker‑Méndez1 Published online: 6 February 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIREShrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 μg of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood–Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis. Keywords Ophthalmic administration · Tph2 gene · Serotonin · Brainstem · Depression
Introduction Emiliano Tesoro-Cruz and Norma Oviedo have contributed equally to this work. * Emiliano Tesoro‑Cruz [email protected] * Norma Oviedo [email protected] 1
Unidad de Investigación Biomédica en Infectología e Inmunología, Hospital de Infectología, Centro Médico Nacional “La Raza”, IMSS, Paseo de las Jacarandas s/n esquina Calzada Vallejo, Col. La Raza, C.P. 02990 Mexico City, Mexico
2
Unidad de Investigación Médica en Enfermedades Endocrinas, UMAE, Hospital de Especialidades, Centro Médico Nacional “Siglo XXI”, IMSS, Mexico City, Mexico
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Unidad de Investigación Médica en Enfermedades Neurológicas, UMAE, Hospital de Especialidades, Centro Médico Nacional “Siglo XXI”, IMSS, Mexico City, Mexico
4
Division of Neuroscience, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, UNAM, Mexico City, Mexico
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Serotonin (5-HT) functions include the regulation of mood, appetite, and sleep. It also has some cognitive functions, such as memory and learning. Serotonin deficiency plays a significant role in the pathogenesis of depression. In
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