Analysis of LRRK2 localization towards understanding the pathogenic mechanisms underlying Parkinson's disease

Résumé

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.