Modelling the functional genomics of Parkinson’s in Caenorhabditis elegans: LRRK2 and beyond

For decades, Parkinson’s disease (PD) cases have been genetically categorised into familial, when caused by mutations in single genes with a clear inheritance pattern in affected families, or idiopathic, in the absence of an evident monogenic determinant. Recently, genome-wide association studies (GWAS) have revealed how common genetic variability can explain up to 36% of PD heritability and that PD manifestation is often determined by multiple variants at different genetic loci. Thus, one of the current challenges in PD research stands in modelling the complex genetic architecture of this condition and translating this into functional studies. Caenorhabditis elegans provide a profound advantage as a reductionist, economical model for PD research, with a short lifecycle, straightforward genome engineering and high conservation of PD relevant neural, cellular and molecular pathways. Functional models of PD genes utilising C. elegans, show many phenotypes recapitulating pathologies observed in PD. When contrasted with mammalian in vivo and in vitro models, these are frequently validated, suggesting relevance of C. elegans in the development of novel PD functional models. This review will discuss how the nematode C. elegans PD models have contributed to the uncovering of molecular and cellular mechanisms of disease, with a focus on the genes most commonly found as causative in familial PD and risk factors in idiopathic PD. Specifically, we will examine the current knowledge on a central player in both familial and idiopathic PD, Leucine-rich repeat kinase 2 (LRRK2) and how it connects to multiple PD associated GWAS candidates and Mendelian disease-causing genes

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate

Background Lrrk2, a gene linked to Parkinson\u2019s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Results Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Conclusions Given that the most common Parkinson\u2019s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF

The Roc domain of LRRK2 as a hub for protein-protein interactions:a focus on PAK6 and its impact on RAB phosphorylation

Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson’s disease

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson’s disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration

Cytosolic sequestration of spatacsin by Protein Kinase A and 14-3-3 proteins

Mutations in SPG11, encoding spatacsin, constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal and axonal transport impairments. However, the upstream mechanisms that regulate spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of spatacsin. The interaction is modulated by Protein Kinase A (PKA)-dependent phosphorylation of spatacsin at Ser1955, which initiates spatacsin trafficking from the plasma membrane to the intracellular space. Our study provides novel insight in understanding spatacsin physio- pathological roles with mechanistic dissection of its associated pathways

PAK6 phosphorylates 14-3-3γ to regulate steady state phosphorylation of LRRK2

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain

Investigating the role of the Roc/GTPase domain of the Parkinson's disease kinase LRRK2 in regulating protein function and activity

Parkinson’s disease (PD) is the second most common neurodegenerative disease of the modern era. Although PD aetiology is still uncertain, approximately 10% of patients suffer from a monogenic form of PD. Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal dominant, late onset familial PD and increase PD risk. LRRK2 possesses dual Roc/GTPase and kinase domains, bridged by a COR scaffold. Given the robust association with PD and the presence of a “druggable” kinase activity, substantial efforts have been made to explore LRRK2 functions in health and disease. The current understanding of LRRK2 biology comes from the characterisation of knockout models or the manipulation of its kinase activity, especially since the most common pathological mutation is the G2019S in the kinase domain and kinase activity is associated with increased cellular toxicity. Conversely, Roc has received less attention, probably because of the challenges related to the measurement of in vitro GTPase activity. Nevertheless, the cross-talk between the two enzymatic modules and the signalling properties of Roc make the GTPase domain a key element in determining the biochemical and cellular properties of LRRK2. Therefore, a thorough characterisation of the intramolecular mechanisms of LRRK2 regulation as well as the signalling cascades orchestrated by the kinase is of high priority to provide alternative therapeutic targets in those cases where kinase inhibition proves badly-tolerated or ineffective. In this scenario, this project has focused on a comprehensive characterisation of the role of LRRK2-Roc in regulating protein biochemistry and the binding with the previously identified interactor p21-activated kinase 6 (PAK6). As a functional readout, two pathways convincingly linked to LRRK2, i.e. autophagy and neurite remodelling, have been investigated. Specifically, we characterised a murine cellular model where endogenous Lrrk2 has been genetically engineered to disrupt guanine nucleotides binding in terms of Lrrk2 expression levels, basal autophagy and the ability to respond to specific autophagic stimuli. We observed that lack of nucleotide binding in Roc affects protein steady-state levels and possibly the response to autophagy-inducing treatments. The second part of the study was dedicated to the characterisation of the interaction between LRRK2 and PAK6. PAK6 regulates actin-cytoskeletal dynamics and it was demonstrated by our group to interact with Roc and to promote neurite outgrowth in vivo through its kinase activity in a LRRK2- and GTP-dependent manner. More recently, we demonstrated that the two proteins bidirectionally modulate each other and overexpression of PAK6 is able to rescue the defects in neurite outgrowth associated with the G2019S pathological mutation. We then went a step further and, in collaboration with Dr. Patrick Lewis at University of Reading, we evaluated the impact of PAK6 pharmacological inhibition on autophagy, given the established role of LRRK2 and the importance of actin cytoskeleton in assisting the process, as well as the involvement of PAK6 homolog PAK1 in promoting autophagy through the AKT/mTOR/ULK1 axis. Our results show clear alterations in the autophagic markers analysed after treatment with an inhibitor of PAK6 kinase activity. Second, we characterised the effects of a de novo substitution in PAK6 in terms of kinase activity, localisation and neurite development as well as interaction with LRRK2, and the consequences of a PD mutation in Roc in terms of binding with PAK6. While the PAK6 variant does not affect the properties of the protein, the presence of a mutation in LRRK2-Roc impairs the interaction with PAK6, with possible consequences on downstream pathways. Overall, our data suggest that any alterations in Roc have severe implications for the steady-state levels of the protein, its activities and binding with partners, with a predicted impact on its subcellular localisation and downstream signalling.La malattia di Parkinson (MP) è la seconda malattia neurodegenerativa più comune dell’era moderna. Nonostante un’eziologia incerta, il 10% dei pazienti soffre di una forma monogenica di MP. Mutazioni nel gene Leucine-rich repeat kinase 2 (LRRK2) sono la causa più comune di MP autosomica dominante ad insorgenza tardiva e aumentano il rischio di sviluppare la MP. LRRK2 possiede una duplice attività enzimatica: GTPasica nel dominio Roc e chinasica, collegate da un dominio COR. Date la robusta associazione con la MP e la presenza di un’attività chinasica modulabile dal punto di vista farmacologico, sono stati compiuti notevoli sforzi per esplorare il ruolo fisiopatologico di LRRK2. L’attuale comprensione della biologia di LRRK2 proviene dallo studio di modelli knockout o dalla manipolazione dell’attività chinasica, dato che la mutazione patologica più comune è la G2019S nel dominio chinasico e l’attività chinasica è associata ad aumentata tossicità cellulare. Al contrario, Roc ha ricevuto minore attenzione, probabilmente a seguito delle difficoltà nel misurare l’attività GTPasica in vitro. Tuttavia, l’interazione tra i due moduli enzimatici e le proprietà di signalling di Roc rendono il dominio GTPasico un elemento chiave nel determinare le proprietà biochimiche e cellulari di LRRK2. Una caratterizzazione completa dei meccanismi intramolecolari di regolazione e delle cascate di segnale orchestrate dal dominio chinasico è dunque un requisito essenziale per individuare strategie terapeutiche alternative qualora l’inibizione dell’attività chinasica fosse mal tollerata o inefficace. Questo progetto si è focalizzato su una caratterizzazione globale del ruolo di Roc nel regolare le proprietà biochimiche di LRRK2 ed il legame con un interattore precedentemente identificato, p21-activated kinase 6 (PAK6). A livello funzionale, sono stati investigati due processi associati a LRRK2 in modo convincente: autofagia e rimodellamento dei neuriti. Più precisamente, abbiamo caratterizzato un modello cellulare murino, in cui Lrrk2 endogena è stata ingegnerizzata geneticamente per impedire il legame dei nucleotidi guaninici, in termini di stabilità della proteina, autofagia basale e capacità di rispondere a stimoli autofagici. Abbiamo osservato che l’assenza di legame coi nucleotidi nel Roc influenza i livelli di Lrrk2 e la risposta all’induzione del flusso autofagico. La seconda parte dello studio riguarda la caratterizzazione dell’interazione LRRK2-PAK6. PAK6 regola le dinamiche del citoscheletro di actina. Il nostro gruppo ha dimostrato che PAK6 interagisce con Roc e promuove la crescita dei neuriti in vivo grazie alla sua attività chinasica in dipendenza da LRRK2 e dal GTP. Di recente, abbiamo dimostrato che le due proteine esercitano una vicendevole modulazione e la sovra-espressione di PAK6 può recuperare i difetti nella crescita dei neuriti associati alla mutazione G2019S in LRRK2. Abbiamo quindi fatto un passo ulteriore e, in collaborazione con il Dr. Patrick Lewis all’Università di Reading, abbiamo valutato l’impatto dell’inibizione farmacologica di PAK6 sull’autofagia, visto il ruolo assodato di LRRK2 e l’importanza del citoscheletro di actina nel processo, così come il coinvolgimento dell’omologo di PAK6, PAK1, nel promuovere l’autofagia tramite l’asse AKT/mTOR/ULK1. I nostri risultati mostrano chiare alterazioni nei markers autofagici analizzati a seguito del trattamento con un inibitore dell’attività chinasica di PAK6. Abbiamo poi caratterizzato gli effetti di una sostituzione de novo in PAK6 in termini di attività chinasica, localizzazione, sviluppo dei neuriti ed interazione con LRRK2, e le conseguenze di una mutazione patologica nel Roc in termini di legame con PAK6. Mentre la variante di PAK6 non influenza le proprietà della proteina, la mutazione nel Roc riduce l’interazione con PAK6. Globalmente, i nostri dati suggeriscono che qualunque alterazione di Roc abbia severe conseguenze per i livelli basali di LRRK2, l’attività della proteina ed il legame con gli interattori, con un probabile impatto sulla localizzazione subcellulare e le cascate di segnale a valle

On the corrosion, stress corrosion and cytocompatibility performances of ALD TiO2 and ZrO2 coated magnesium alloys

Magnesium alloys are increasingly studied as materials for temporary implants. However, their high corrosion rate and susceptibility to corrosion-assisted cracking phenomena, such as stress corrosion cracking (SCC), continue to prevent their mainstream use. Recently, coatings have been considered to provide an effective solution to these issues and researchers have focused their attention on Atomic Layer Deposition (ALD). ALD stands out as a coating technology due to the outstanding film conformality and density achievable, and has shown encouraging preliminary results in terms of reduced corrosion rate and reduced SCC susceptibility. Here, we contribute to the ongoing interest in ALD-coated Mg alloys, providing a comprehensive characterisation of the effect of 100 nm thick ALD TiO2 and ZrO2 coatings on the corrosion behaviour and SCC susceptibility of AZ31 alloy. Moreover, we also investigate the effect of these coatings on the induced biological response. Our results suggest that the ALD coatings can improve the corrosion and SCC resistance of the Mg alloy, with the ZrO2 ALD coating showing the best improvements. We suggest that the different corrosion behaviours are the cause of the cytocompatibility results (only the ZrO2 ALD coating was found to meet the demands for cellular applications). Finally, we leverage on considerations about the coatings’ wettability, electrochemical stability and surface integrity to justify the different results

The role of LRRK2 in cytoskeletal dynamics

Leucine-rich repeat kinase 2 (LRRK2), a complex kinase/GTPase mutated in Parkinson's disease, has been shown to physically and functionally interact with cytoskeletal-related components in different brain cells. Neurons greatly rely on a functional cytoskeleton for many homeostatic processes such as local and long-distance vesicle transport, synaptic plasticity, and dendrites/axons growth and remodeling. Here, we will review the available data linking LRRK2 and the cytoskeleton, and discuss how this may be functionally relevant for the well-established roles of LRRK2 in intracellular trafficking pathways and outgrowth of neuronal processes in health and disease conditions