Cellular effects mediated by pathogenic LRRK2: homing in on Rab-mediated processes

Leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease. Mutations in LRRK2 are associated with increased kinase activity that correlates with cytotoxicity, indicating that kinase inhibitors may comprise promising diseasemodifying compounds. However, before embarking on such strategies, detailed knowledge of the cellular deficits mediated by pathogenic LRRK2 in the context of defined and pathologically relevant kinase substrates is essential. LRRK2 has been consistently shown to impair various intracellular vesicular trafficking events, and recent studies have shown that LRRK2 can phosphorylate a subset of proteins that are intricately implicated in those processes. In light of these findings, we here review the link between cellular deficits in intracellular trafficking pathways and the LRRK2-mediated phosphorylation of those newly identified substrates

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

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

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.Tesis Univ. Granada. Programa Oficial de Doctorado en Biomedicin

A Link between Autophagy and the Pathophysiology of LRRK2 in Parkinson’s Disease

Parkinson's disease is a debilitating neurodegenerative disorder, and its molecular etiopathogenesis remains poorly understood. The discovery of monogenic forms has significantly advanced our understanding of the molecular mechanisms underlying PD, as it allows generation of cellular and animal models carrying the mutant gene to define pathological pathways. Mutations in leucine-rich repeat kinase 2 (LRRK2) cause dominantly inherited PD, and variations increase risk, indicating that LRRK2 is an important player in both genetic and sporadic forms of the disease. G2019S, the most prominent pathogenic mutation, maps to the kinase domain and enhances enzymatic activity of LRRK2, which in turn seems to correlate with cytotoxicity. Since kinases are druggable targets, this has raised great hopes that disease-modifying therapies may be developed around modifying LRRK2 enzymatic activity. Apart from cytotoxicity, changes in autophagy have been consistently reported in the context of G2019S mutant LRRK2. Here, we will discuss current knowledge about mechanism(s) by which mutant LRRK2 may regulate autophagy, which highlights additional putative therapeutic targets.This work is supported by Grants from the Spanish Ministry of Economy and Competitiveness (BFU2011-29899), the Junta de Andaluc´ıa (CTS 6816), and the Michael J. Fox Foundation.Peer reviewe

LRRK2: from kinase to GTPase to microtubules and back

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are intimately linked to both familial and sporadic Parkinson's disease. LRRK2 is a large protein kinase able to bind and hydrolyse GTP. A wealth of in vitro studies have established that the distinct pathogenic LRRK2 mutants differentially affect those enzymatic activities, either causing an increase in kinase activity without altering GTP binding/GTP hydrolysis, or displaying no change in kinase activity but increased GTP binding/decreased GTP hydrolysis. Importantly, recent studies have shown that all pathogenic LRRK2 mutants display increased kinase activity towards select kinase substrates when analysed in intact cells. To understand those apparently discrepant results, better insight into the cellular role(s) of normal and pathogenic LRRK2 is crucial. Various studies indicate that LRRK2 regulates numerous intracellular vesicular trafficking pathways, but the mechanism(s) by which the distinct pathogenic mutants may equally interfere with such pathways has largely remained elusive. Here, we summarize the known alterations in the catalytic activities of the distinct pathogenic LRRK2 mutants and propose a testable working hypothesis by which the various mutants may affect membrane trafficking events in identical ways by culminating in increased phosphorylation of select substrate proteins known to be crucial for membrane trafficking between specific cellular compartments.Work in the laboratory is supported by FEDER, grants from the Spanish Ministry of Economy and Competitiveness [grant number SAF2014-58653-R], the BBVA Foundation and the Michael J. Fox Foundation (MJFF).Peer reviewe

The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization

The Parkinson's-disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that regulators of the LRRK2 signaling pathway including vps35 and PPM1H converge upon causing centrosomal deficits. The cohesion alterations do not require the presence of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43 or the presence of RILPL2. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendage of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab proteins to cause the centrosomal defects. The centrosomal alterations impair cell polarization as monitored by scratch wound assays which is reverted by LRRK2 kinase inhibition. These data reveal a common molecular pathway by which enhanced LRRK2 kinase activity impacts upon centrosome-related events to alter the normal biology of a cell

Cellular effects mediated by pathogenic LRRK2: homing in on Rab-mediated processes

Leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease. Mutations in LRRK2 are associated with increased kinase activity that correlates with cytotoxicity, indicating that kinase inhibitors may comprise promising disease-modifying compounds. However, before embarking on such strategies, detailed knowledge of the cellular deficits mediated by pathogenic LRRK2 in the context of defined and pathologically relevant kinase substrates is essential. LRRK2 has been consistently shown to impair various intracellular vesicular trafficking events, and recent studies have shown that LRRK2 can phosphorylate a subset of proteins that are intricately implicated in those processes. In light of these findings, we here review the link between cellular deficits in intracellular trafficking pathways and the LRRK2-mediated phosphorylation of those newly identified substrates.Work in the laboratory is supported by FEDER, grants from the Spanish Ministry of Economy and Competitiveness [grant no. SAF2014-58653-R], the Banco Bilbao Vizcaya Argentaria (BBVA) Foundation and the Michael J. Fox Foundation (MJFF).Peer reviewe

LRRK2 and Parkinson's Disease: From Lack of Structure to Gain of Function

Mutations in LRRK2 comprise the most common cause for familial Parkinson's disease (PD), and variations increase risk for sporadic disease, implicating LRRK2 in the entire disease spectrum. LRRK2 is a large protein harbouring both GTPase and kinase domains which display measurable catalytic activity. Most pathogenic mutations increase the kinase activity, with increased activity being cytotoxic under certain conditions. These findings have spurred great interest in drug development approaches, and various specific LRRK2 kinase inhibitors have been developed. However, LRRK2 is a largely ubiquitously expressed protein, and inhibiting its function in some non-neuronal tissues has raised safety liability issues for kinase inhibitor approaches. Therefore, understanding the cellular and cell type-specific role(s) of LRRK2 has become of paramount importance. This review will highlight current knowledge on the precise biochemical activities of normal and pathogenic LRRK2, and highlight the most common proposed cellular roles so as to gain a better understanding of the cell type-specific effects of LRRK2 modulators.Work in the laboratory is funded by FEDER, the Spanish Ministry of Economy and Competitiveness (SAF2014-58653-R), the Junta de Andalucia (CTS-6816), the BBVA Foundation and the Michael J. Fox Foundation. B.F. was funded by a Juan de la Cierva Fellowship (MINECO; JCI2010-07703).Peer reviewe

Impaired α-tubulin re-tyrosination leads to synaptic dysfunction and is a feature of Alzheimer’s disease

SUMMARY In neurons, dynamic microtubules play regulatory roles in neurotransmission and synaptic plasticity. While stable microtubules contain detyrosinated tubulin, dynamic microtubules are composed of tyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination (Tyr/deTyr) cycle modulates microtubule dynamics and synaptic function. In the Tyr/deTyr cycle, the C-terminal tyrosine of α-tubulin is re-added by tubulin-tyrosine-ligase (TTL). Here we show that TTL +/− mice exhibit decreased tyrosinated microtubules, synaptic plasticity and memory deficits, and that reduced TTL expression is a feature of sporadic and familial Alzheimer’s disease (AD), with human APPV717I neurons having less dynamic microtubules. We find that spines visited by dynamic microtubules are more resistant to Amyloidβ 1-42 and that TTL, by promoting microtubule entry into spines, prevents Aβ 1-42 -induced spine pruning. Our results demonstrate that the Tyr/deTyr cycle regulates synaptic plasticity, is protective against spine injury, and that tubulin re-tyrosination is lost in AD, providing evidence that a defective Tyr/deTyr cycle may contribute to neurodegeneration