Publication: Modeling rare genetic diseases in zebrafish : functional characterization and therapeutic approaches for NEXMIF-related X-linked encephalopathy and IFIH1-mediated type I interferonopathies
Authors
Bernal Bermúdez, Beatriz
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Escuela Internacional de Doctorado
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Mulero Méndez, Victoriano Francisco ; Cayuela Fuentes, María Luisa ; García Moreno, Diana
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
Esta tesis doctoral se centra en el desarrollo y aplicación de modelos en pez cebra (Danio rerio) para investigar los mecanismos moleculares y funcionales subyacentes a dos enfermedades raras genéticamente distintas: el trastorno del desarrollo intelectual ligado al cromosoma X tipo 98 (XLID-98) relacionado con NEXMIF, y el síndrome de Aicardi–Goutières (AGS) mediado por IFIH1, una interferonopatía tipo I. Dada la escasez de modelos vertebrados robustos para estas patologías y la necesidad de plataformas escalables para el descubrimiento terapéutico, el objetivo general de este trabajo fue establecer modelos en pez cebra que permitan elucidar mecanismos patogénicos y acelerar el desarrollo de fármacos dirigidos.
OBJETIVOS DE LA TESIS
Los objetivos específicos incluyeron: (1) generar y caracterizar un modelo de pérdida de función para nexmifa/b, (2) analizar su perfil transcriptómico e identificar modificadores genéticos relevantes, (3) realizar un cribado de compuestos naturales para rescatar fenotipos asociados a NEXMIF, (4) desarrollar un modelo de ganancia de función para IFIH1 y acoplarlo a líneas reporteras de inflamación e interferón, (5) evaluar la inhibición genética y farmacológica de la vía del IFN-I en dicho modelo, y (6) realizar un cribado medio-alto rendimiento de compuestos naturales con capacidad inmunomoduladora.
METODOLOGÍA
Para lograr estos objetivos, se utilizaron múltiples líneas de pez cebra, incluidas líneas transgénicas reporteras de NF-κB e IFN-I (nfkb:eGFP, isg15:eGFP), así como una nueva línea Tg(ifih1_mut) generada mediante clonación Gateway y tecnología Tol2. La edición génica por CRISPR/Cas9 permitió generar mutantes nexmifa/b-/- y silenciar genes clave como entpd3, ddx43, kbtbd7, mavs e ikbke. Se aplicaron técnicas de análisis transcriptómico (RNA-seq), RT-q-PCR convencional y con sondas TaqMan®, análisis de comportamiento (DanioVision), cribados farmacológicos, micro-CT y microscopía de fluorescencia.
RESULTADOS O CONCLUSIONES
Los resultados se presentan en dos bloques principales. En primer lugar, se identificó una nueva variante patogénica en NEXMIF (p.D1161fs*11) en una paciente con retraso psicomotor y epilepsia. El modelo nexmifa/b-/- mostró hiperlocomoción inducida por estímulo, revertida con valproato de sodio y topiramato, validando su utilidad farmacológica. El análisis transcriptómico reveló más de 400 genes diferencialmente expresados, destacando un eje protector entpd3–ddx43 que reduce la excitabilidad neuronal, y la reducción de kbtbd7, que agrava el fenotipo epiléptico. Un cribado de 87 compuestos naturales identificó a kahweol acetato, veratridina y rauwolscina como inductores de entpd3, aunque sin rescatar el fenotipo conductual, lo que sugiere la necesidad de una coactivación multigénica para lograr eficacia terapéutica.
En segundo lugar, se desarrolló un modelo transgénico de interferonopatía mediante la sobreexpresión de ifih1 con la mutación p.R742H (homóloga a la mutación humana p.R779H). Tras la estimulación subóptima con poli I:C, las larvas Tg(ifih1_mut) desarrollaron una firma robusta de IFN-I e inflamación sistémica, visible en tiempo real mediante líneas reporteras. La interrupción genética de mavs o ikbke y el tratamiento con baricitinib inhibieron completamente la respuesta inflamatoria y del interferon. Un cribado de 68 compuestos naturales identificó 26 con capacidad para reducir la expresión de stat1b, siendo swainsonina y aconitina los más eficaces, aunque sin igualar a baricitinib.
En conjunto, este trabajo establece nuevos modelos vertebrados para el estudio de enfermedades raras, aporta conocimiento sobre sus mecanismos fisiopatológicos y consolida al pez cebra como una plataforma versátil para el cribado genético y farmacológico orientado al fenotipo.
This PhD thesis focuses on the development and application of zebrafish (Danio rerio) models to investigate the molecular and functional mechanisms underlying two genetically distinct rare diseases: NEXMIF-related X-linked intellectual developmental disorder (XLID-98) and IFIH1-driven Aicardi–Goutières syndrome (AGS), a type I interferonopathy. Due to the limited availability of robust vertebrate models for these disorders and the need for scalable platforms for drug discovery, this work aimed to establish zebrafish models to elucidate disease mechanisms and accelerate mechanism-based therapeutic screening. THESIS OBJECTIVES The specific objectives included: (1) generating and characterizing a loss-of-function zebrafish model for nexmifa/b, (2) analyzing its transcriptomic profile and identifying genetic modifiers involved in disease-relevant pathways, (3) performing a medium-throughput compound screen to identify approved drugs that rescue NEXMIF-related phenotypes, (4) developing a gain-of-function model of IFIH1-driven interferonopathy integrated with inflammation and interferon reporter lines, (5) testing genetic and pharmacological inhibition of the IFN-I pathway in this model, and (6) conducting a medium-throughput screen of natural compounds with immunomodulatory potential. MATERIALS AND METHODS Multiple zebrafish lines were used, including transgenic inflammation and IFN-I reporters (nfkb:eGFP, isg15:eGFP) and Tg(ifih1_mut) line, generated using Gateway® cloning and Tol2-mediated transgenesis. CRISPR/Cas9 gene editing was employed to generate nexmifa/b-/- mutants and knock down target genes (entpd3, ddx43, kbtbd7, mavs, ikbke). Techniques included RNA-sequencing, RT-q-PCR (SYBR and TaqMan®), behavioral testing (DanioVision), small molecule screening, micro-CT imaging, and fluorescence microscopy. RESULTS OR CONCLUSIONS Results are presented in two main chapters. First, a novel pathogenic NEXMIF variant (p.D1161fs*11) was identified in a female patient with psychomotor delay and epilepsy. A nexmifa/b-/- double mutant zebrafish line was generated and displayed stimulus-evoked hyperlocomotion and increased body length. Notably, the behavioral phenotype was rescued by sodium valproate and topiramate, supporting the model’s pharmacological relevance. Transcriptomic analysis of 7 dpf larval heads revealed over 400 differentially expressed genes enriched in MAPK signaling, sodium transport, and cell adhesion pathways. Functional analyses revealed a neuroprotective entpd3–ddx43 axis that mitigates excitability, while kbtbd7 downregulation intensified hyperactive behavior. A targeted screen of 87 compounds from the SCREEN-WELL® Natural Product Library identified kahweol acetate, veratridine, and rauwolscine as strong entpd3 inducers. However, their failure to rescue the behavioral phenotype indicated that co-modulation of multiple targets may be necessary for therapeutic efficacy. Second, a gain-of-function transgenic zebrafish line overexpressing ifih1 p.R742H (orthologous to the human p.R779H mutation) was developed. Upon suboptimal poly I:C stimulation, Tg(ifih1_mut) larvae exhibited a strong interferon signature and systemic inflammation, mimicking the clinical features of Aicardi-Goutières síndrome (AGS). Combining this model with fluorescent reporter lines enabled real-time, whole-organism visualization of innate immune activation. CRISPR disruption of mavs and ikbke, as well as treatment with baricitinib (a JAK1/JAK2 inhibitor), effectively suppressed inflammatory and IFN-I responses. A focused screen of 68 natural compounds identified 26 that downregulated stat1b expression, with swainsonine and aconitine showing the strongest effects, although baricitinib remained the most potent. Together, this work establishes novel vertebrate models for two rare diseases, sheds light on their underlying pathophysiological mechanisms and provides a versatile zebrafish-based platform for phenotype-driven genetic and pharmacological screening in the context of neurodevelopmental and autoinflammatory disorders.
This PhD thesis focuses on the development and application of zebrafish (Danio rerio) models to investigate the molecular and functional mechanisms underlying two genetically distinct rare diseases: NEXMIF-related X-linked intellectual developmental disorder (XLID-98) and IFIH1-driven Aicardi–Goutières syndrome (AGS), a type I interferonopathy. Due to the limited availability of robust vertebrate models for these disorders and the need for scalable platforms for drug discovery, this work aimed to establish zebrafish models to elucidate disease mechanisms and accelerate mechanism-based therapeutic screening. THESIS OBJECTIVES The specific objectives included: (1) generating and characterizing a loss-of-function zebrafish model for nexmifa/b, (2) analyzing its transcriptomic profile and identifying genetic modifiers involved in disease-relevant pathways, (3) performing a medium-throughput compound screen to identify approved drugs that rescue NEXMIF-related phenotypes, (4) developing a gain-of-function model of IFIH1-driven interferonopathy integrated with inflammation and interferon reporter lines, (5) testing genetic and pharmacological inhibition of the IFN-I pathway in this model, and (6) conducting a medium-throughput screen of natural compounds with immunomodulatory potential. MATERIALS AND METHODS Multiple zebrafish lines were used, including transgenic inflammation and IFN-I reporters (nfkb:eGFP, isg15:eGFP) and Tg(ifih1_mut) line, generated using Gateway® cloning and Tol2-mediated transgenesis. CRISPR/Cas9 gene editing was employed to generate nexmifa/b-/- mutants and knock down target genes (entpd3, ddx43, kbtbd7, mavs, ikbke). Techniques included RNA-sequencing, RT-q-PCR (SYBR and TaqMan®), behavioral testing (DanioVision), small molecule screening, micro-CT imaging, and fluorescence microscopy. RESULTS OR CONCLUSIONS Results are presented in two main chapters. First, a novel pathogenic NEXMIF variant (p.D1161fs*11) was identified in a female patient with psychomotor delay and epilepsy. A nexmifa/b-/- double mutant zebrafish line was generated and displayed stimulus-evoked hyperlocomotion and increased body length. Notably, the behavioral phenotype was rescued by sodium valproate and topiramate, supporting the model’s pharmacological relevance. Transcriptomic analysis of 7 dpf larval heads revealed over 400 differentially expressed genes enriched in MAPK signaling, sodium transport, and cell adhesion pathways. Functional analyses revealed a neuroprotective entpd3–ddx43 axis that mitigates excitability, while kbtbd7 downregulation intensified hyperactive behavior. A targeted screen of 87 compounds from the SCREEN-WELL® Natural Product Library identified kahweol acetate, veratridine, and rauwolscine as strong entpd3 inducers. However, their failure to rescue the behavioral phenotype indicated that co-modulation of multiple targets may be necessary for therapeutic efficacy. Second, a gain-of-function transgenic zebrafish line overexpressing ifih1 p.R742H (orthologous to the human p.R779H mutation) was developed. Upon suboptimal poly I:C stimulation, Tg(ifih1_mut) larvae exhibited a strong interferon signature and systemic inflammation, mimicking the clinical features of Aicardi-Goutières síndrome (AGS). Combining this model with fluorescent reporter lines enabled real-time, whole-organism visualization of innate immune activation. CRISPR disruption of mavs and ikbke, as well as treatment with baricitinib (a JAK1/JAK2 inhibitor), effectively suppressed inflammatory and IFN-I responses. A focused screen of 68 natural compounds identified 26 that downregulated stat1b expression, with swainsonine and aconitine showing the strongest effects, although baricitinib remained the most potent. Together, this work establishes novel vertebrate models for two rare diseases, sheds light on their underlying pathophysiological mechanisms and provides a versatile zebrafish-based platform for phenotype-driven genetic and pharmacological screening in the context of neurodevelopmental and autoinflammatory disorders.
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22-nov-2026
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