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Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/48377

Title: Analysis of LRRK2 localization towards understanding the pathogenic mechanisms underlying Parkinson's Disease
Authors: Blanca Ramírez, Marian
Direction: Navarro Hilfiker, Sabine Nicole
Collaborator: Universidad de Granada. Programa Oficial de Doctorado en Biomedicina
Consejo Superior de Investigaciones Científicas (CSIC). Instituto de Parasitología y Biomedicina López-Neyra
Issue Date: 2017
Submitted Date: 5-Jul-2017
Abstract: En esta tesis, primero demostramos que la mayoría de los mutantes patogénicos, así como su versión farmacológicamente inhibida de la actividad kinasa, intensifican la asociación de LRRK2 con un grupo de MTs estables, mostrando un fenotipo filamentoso. Esto se contrapone con wildtype LRRK2, que muestra una localización predominantemente citosólica. Segundo, encontramos que esta asociación puede ser modulada mediante la alteración de los niveles de tubulina destirosinada, mientras que el estado de acetilación de los MTs no parece jugar un papel de manera directa. Esta asociación puede desembocar en la desestabilización de los MTs. Tercero, dilucidamos que los determinantes moleculares de esta interacción requieren la unión de GTP. Encontramos que dos mutantes sintéticos (R1398L; R1398L/T1343V), así como una variante de riesgo protectora para EP (R1398H) dismininuyen la unión a GTP, lo cual revierte este fenotipo. El tratamiento con dos nuevos inhibidores de GTP también revierte la localización alterada mostrada por LRRK2 patogénico y kinasa inactivo. Finalmente, esta localización alterada es inducida por analógos de GTP, lo cual representa una prueba formal de que la alteración de la unión de GTP a LRRK2 es la causa de esta alteración en su localización subcelular.
Mutations in leucine rich repeat kinase 2 (LRRK2) represent the most common cause of familial Parkinson's Disease (PD), and variants in this gene modify risk for sporadic PD. Thus, the study of LRRK2 is key towards elucidating the mechanism(s) underlying both familial and sporadic disease entities. Towards this goal, previous studies have reported an interaction between LRRK2 and microtubules (MTs). However, the determinants within LRRK2 responsible for such interactions, and the possible downstream alterations in MT-mediated transport events remain unknown. Here, we first we demonstrate that most pathogenic LRRK2 mutants as well as pharmacological LRRK2 kinase inhibition causes an enhanced association of LRRK2 with a subset of stable MTs, displaying a filamentous phenotype. This is in contrast to wildtype LRRK2, which displays a largely cytosolic localization. Second, we find that this association can be modulated upon altering the levels of detyrosinated tubulin, whereas the MT acetylation status does not seem to play a direct role. Such association may cause subsequent MT destabilization. Third, we elucidate the molecular determinants of this interaction to be regulated by LRRK2 GTP binding. We find that two synthetic mutants (R1398L; R1398L/T1343V) as well as a protective risk variant for PD (R1398H) decrease GTP binding which causes a rescue of this phenotype. Treatment with two novel GTP binding inhibitors also reverts such altered localization of pathogenic or kinase-inhibited LRRK2. Finally, such altered subcellular localization is induced by GTP analogs, providing formal proof-of-concept that altered LRRK2 GTP binding causes such altered subcellular localization. Altogether, our findings indicate a preferential association of pathogenic mutant and pharmacologically kinase-inhibited LRRK2 with stable MTs, which may directly or indirectly impact upon various MT-mediated vesicular trafficking events.
Sponsorship: Tesis Univ. Granada. Programa Oficial de Doctorado en Biomedicina
Publisher: Universidad de Granada
Keywords: Parkinson
Microbiología médica
Microtúbulos
Patógenos
Proteínas quinasas
Genes de la GTPasa
Leucine rich repeat kinase 2 (LRRK2)
UDC: 61
2490
URI: http://hdl.handle.net/10481/48377
ISBN: 9788491635567
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Citation: Blanca Ramírez, M. Analysis of LRRK2 localization towards understanding the pathogenic mechanisms underlying Parkinson's Disease. Granada: Universidad de Granada, 2017. []
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