Evaluación del potencial del hámster GASH/Sal como modelo traslacional de amiloidosis por transtiretina mediante análisis in silico e histológico

Abstract

[ES]Introducción: La amiloidosis por transtiretina (ATTR) es una enfermedad sistémica progresiva causada por el depósito extracelular de fibrillas derivadas de transtiretina (TTR) mal plegada. La desestabilización del tetrámero de TTR (por mutaciones autosómicas dominantes o por envejecimiento sin mutación identificada) constituye el paso limitante de la amiloidogénesis. El hámster GASH/Sal (Genetic Audiogenic Seizure Hamster/Salamanca), modelo validado de epilepsia audiogénica genética, presenta dos mutaciones puntuales en el gen Ttr (sustitución prolinaàleucina en posición 63 (P63L) y valinaàisoleucina en posición 85 (V85I) de la secuencia precursora) y sobreexpresión de TTR en el colículo inferior, lo que plantea su potencial como modelo traslacional de ATTR. Objetivos: Evaluar si el GASH/Sal constituye un modelo traslacional de amiloidosis por mutaciones en Ttr, mediante: (1) caracterización in silico del impacto estructural y termodinámico de las mutaciones P63L y V85I; y (2) análisis histológico de tejido cardíaco y renal en busca de depósitos amiloides. Material y métodos: El análisis bioinformático, utilizando diversos programas, incluyó el modelado estructural de la TTR wild-type (WT) y de la variante mutada GASH/Sal mediante AlphaFold3, la caracterización de interacciones interatómicas con Arpeggio y la estimación del cambio en energía libre de Gibbs (ΔΔG) mediante DynaMut2. Para el estudio histológico, se procesaron muestras cardiacas y renales de tres hámsteres machos (GASH/Sal de 5 y 12 meses y control de 5 meses), obteniéndose secciones de 6 µm teñidas con Rojo Congo, y evaluadas mediante microscopía de campo claro y luz polarizada cruzada. Resultados: El análisis comparativo de interacciones no covalentes reveló que la variante GASH/Sal presenta una remodelación significativa del perfil estructural respecto a la TTR WT, con la pérdida neta de un puente de hidrógeno fuerte, y un empeoramiento de la calidad estructural, volviéndose la proteína inestable, adoptando una conformación físicamente desfavorable, compatible con desestabilización termodinámica. La mutación V85I debilita la red de puentes de hidrógeno en el bucle proteico, comprometiendo la estabilidad cuaternaria del tetrámero; mientras que P63L altera la geometría de la hebra C al eliminar la restricción conformacional de la prolina, generando nuevos contactos hidrofóbicos locales. Ambas variantes se localizan en regiones estructuralmente análogas a áreas de alta densidad mutacional descritas en la ATTR humana. En el análisis histológico, no se identificaron depósitos con birrefringencia verde-manzana en tejido cardíaco ni renal de ninguno de los grupos estudiados. Conclusiones: Las mutaciones P63L y V85I confieren propiedades amiloidogénicas a la TTR del GASH/Sal mediante mecanismos estructurales análogos a los descritos en variantes patogénicas de TTR humana. La ausencia de depósitos congófilos no excluye patología amiloide, dado que los modelos murinos de ATTR presentan predominantemente depósitos no congófilos a edades equivalentes y no se aplicaron técnicas de mayor sensibilidad diagnóstica. Los resultados obtenidos sugieren el potencial traslacional del hámster GASH/Sal como modelo de ATTR, si bien su validación definitiva requiere estudios con mayor sensibilidad metodológica, cohortes más amplias y seguimiento longitudinal a edades más avanzadas.

[ENIntroduction: Transthyretin amyloidosis (ATTR) is a progressive systemic disease caused by the extracellular deposition of fibrils derived from misfolded transthyretin (TTR). The destabilization of the TTR tetramer (due to autosomal dominant mutations or aging without an identified mutation) constitutes the rate-limiting step in amyloidogenesis. The GASH/Sal (Genetic Audiogenic Seizure Hamster/Salamanca) hamster, a validated model of genetic audiogenic epilepsy, carries two point mutations in the Ttr gene (proline à leucine substitution at position 63 (P63L) and valine à isoleucine substitution at position 85 (V85I) in the precursor sequence) and TTR overexpression in the inferior colliculus, suggesting its potential as a translational model of ATTR. Objectives: To evaluate whether GASH/Sal constitutes a translational model of amyloidosis caused by Ttr mutations, through: (1) in silico characterization of the structural and thermodynamic impact of the P63L and V85I mutations; and (2) histological analysis of cardiac and renal tissue for amyloid deposits. Materials and Methods: The bioinformatic analysis, which utilized various software programs, included structural modeling of wild-type (WT) TTR and the GASH/Sal mutant variant using AlphaFold3, characterization of interatomic interactions using Arpeggio, and estimation of the change in Gibbs free energy (ΔΔG) using DynaMut2. For the histological study, cardiac and renal samples from three male hamsters (5- and 12-month-old GASH/Sal and a 5-month-old control) were processed, yielding 6-µm sections stained with Congo Red and evaluated using brightfield and crossed-polarized light microscopy. Results: Comparative analysis of noncovalent interactions revealed that the GASH/Sal variant exhibits a significant restructuring of its structural profile compared to WT TTR, with the net loss of a strong hydrogen bond and a deterioration in structural quality, causing the protein to become unstable and adopt an unfavorable conformation., consistent with thermodynamic destabilization. The V85I mutation weakens the hydrogen bond network in the protein loop, compromising the quaternary stability of the tetramer; whereas P63L alters the geometry of strand C by removing the conformational constraint of proline, generating new local hydrophobic contacts, including a de novo carbon–π bond. Both variants are located in regions structurally analogous to areas of high mutational density described in human ATTR. In the histological analysis, no applegreen birefringent deposits were identified in cardiac or renal tissue from any of the study groups. Conclusions: The P63L and V85I mutations confer amyloidogenic properties on GASH/Sal TTR through structural mechanisms analogous to those described in pathogenic variants of human TTR. The absence of congophilic deposits does not rule out amyloid pathology, given that murine models of ATTR predominantly exhibit noncongophilic deposits at equivalent ages and techniques with higher diagnostic sensitivity were not applied. The results obtained suggest the translational potential of the GASH/Sal hamster as a model for ATTR, although its definitive validation requires studies with greater methodological sensitivity, larger cohorts, and longitudinal follow-up at older ages.

[ENIntroduction: Transthyretin amyloidosis (ATTR) is a progressive systemic disease caused by the extracellular deposition of fibrils derived from misfolded transthyretin (TTR). The destabilization of the TTR tetramer (due to autosomal dominant mutations or aging without an identified mutation) constitutes the rate-limiting step in amyloidogenesis. The GASH/Sal (Genetic Audiogenic Seizure Hamster/Salamanca) hamster, a validated model of genetic audiogenic epilepsy, carries two point mutations in the Ttr gene (proline à leucine substitution at position 63 (P63L) and valine à isoleucine substitution at position 85 (V85I) in the precursor sequence) and TTR overexpression in the inferior colliculus, suggesting its potential as a translational model of ATTR. Objectives: To evaluate whether GASH/Sal constitutes a translational model of amyloidosis caused by Ttr mutations, through: (1) in silico characterization of the structural and thermodynamic impact of the P63L and V85I mutations; and (2) histological analysis of cardiac and renal tissue for amyloid deposits. Materials and Methods: The bioinformatic analysis, which utilized various software programs, included structural modeling of wild-type (WT) TTR and the GASH/Sal mutant variant using AlphaFold3, characterization of interatomic interactions using Arpeggio, and estimation of the change in Gibbs free energy (ΔΔG) using DynaMut2. For the histological study, cardiac and renal samples from three male hamsters (5- and 12-month-old GASH/Sal and a 5-month-old control) were processed, yielding 6-µm sections stained with Congo Red and evaluated using brightfield and crossed-polarized light microscopy. Results: Comparative analysis of noncovalent interactions revealed that the GASH/Sal variant exhibits a significant restructuring of its structural profile compared to WT TTR, with the net loss of a strong hydrogen bond and a deterioration in structural quality, causing the protein to become unstable and adopt an unfavorable conformation., consistent with thermodynamic destabilization. The V85I mutation weakens the hydrogen bond network in the protein loop, compromising the quaternary stability of the tetramer; whereas P63L alters the geometry of strand C by removing the conformational constraint of proline, generating new local hydrophobic contacts, including a de novo carbon–π bond. Both variants are located in regions structurally analogous to areas of high mutational density described in human ATTR. In the histological analysis, no applegreen birefringent deposits were identified in cardiac or renal tissue from any of the study groups. Conclusions: The P63L and V85I mutations confer amyloidogenic properties on GASH/Sal TTR through structural mechanisms analogous to those described in pathogenic variants of human TTR. The absence of congophilic deposits does not rule out amyloid pathology, given that murine models of ATTR predominantly exhibit noncongophilic deposits at equivalent ages and techniques with higher diagnostic sensitivity were not applied. The results obtained suggest the translational potential of the GASH/Sal hamster as a model for ATTR, although its definitive validation requires studies with greater methodological sensitivity, larger cohorts, and longitudinal follow-up at older ages.