Fiona Bonello

Sidi-Mohamed Hassoun

François Mouton-Liger

Yea Seul Shin

Adeline Muscat

Christelle Tesson

Suzanne Lesage

Philip M Beart

Alexis Brice

Johannes Krupp

Jean-Christophe Corvol

Olga Corti
email: olga.corti@upmc.fr

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease

Mutations of LRRK2, encoding leucine-rich repeat kinase 2, are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore CCCP. FRET imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a "kinase-dead" LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only.

Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

INTRODUCTION

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the progressive loss of the dopaminergic neurons of the substantia nigra pars compacta. Most cases of PD are sporadic, but about 10% of the cases have a familial origin.

Over the last two decades, disease-causing mutations with autosomal dominant (e.g. PARK1/SNCA, PARK8/LRRK2) or recessive (e.g. PARK2/Parkin, PARK6/PINK1) transmission have been identified in about 20 genes [START_REF] Deng | The genetics of Parkinson disease[END_REF]. Mutations in LRRK2 are the commonest cause of both familial and sporadic PD forms, with reported frequencies of 15-40%, depending on the population studied [START_REF] Nichols | Genetic screening for a single common LRRK2 mutation in familial Parkinson's disease[END_REF][START_REF] Gilks | A common LRRK2 mutation in idiopathic Parkinson's disease[END_REF][START_REF] Lesage | Parkinson's disease-related LRRK2 G2019S mutation results from independent mutational events in humans[END_REF]. Mutations in PARK2 are the commonest cause of early-onset PD, with reported frequencies of 10-20% worldwide [START_REF] Corti | What genetics tells us about the causes and mechanisms of Parkinson's disease[END_REF].

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large multidomain protein of the ROCO family, with both kinase and GTPase activities [START_REF] Ramírez | LRRK2 and Parkinson's Disease: From Lack of Structure to Gain of Function[END_REF]. The most frequent mutation of the gene encoding this protein leads to the G2019S substitution in the kinase domain and has been associated with an increase in kinase activity and with neurotoxicity in cell models [START_REF] West | Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity[END_REF][START_REF] Smith | Kinase activity of mutant LRRK2 mediates neuronal toxicity[END_REF][START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF][START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF]. The precise function of LRRK2 remains unknown, but various roles in vesicle synthesis and trafficking [START_REF] Shin | LRRK2 regulates synaptic vesicle endocytosis[END_REF], microtubule dynamics [START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF][START_REF] Gandhi | The Roc domain of leucine-rich repeat kinase 2 is sufficient for interaction with microtubules[END_REF][START_REF] Gillardon | Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability--a point of convergence in parkinsonian neurodegeneration?[END_REF], inflammation [START_REF] Hakimi | Parkinson's disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures[END_REF][START_REF] Thévenet | Regulation of LRRK2 expression points to a functional role in human monocyte maturation[END_REF][START_REF] Gillardon | Parkinson's disease-linked leucine-rich repeat kinase 2(R1441G) mutation increases proinflammatory cytokine release from activated primary microglial cells and resultant neurotoxicity[END_REF] and autophagy [START_REF] Plowey | Role of autophagy in G2019S-LRRK2associated neurite shortening in differentiated SH-SY5Y cells[END_REF][START_REF] Tong | Loss of leucine-rich repeat kinase 2 causes age-dependent bi-phasic alterations of the autophagy pathway[END_REF][START_REF] Bravo-San Pedro | The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway[END_REF][START_REF] Manzoni | Inhibition of LRRK2 kinase activity stimulates macroautophagy[END_REF] have been described. LRRK2 has also been linked to the regulation of mitochondrial homeostasis.

Several studies have shown that the G2019S substitution disturbs mitochondrial function, decreasing mitochondrial membrane potential, ATP levels and production, and inducing ROS imbalance, and changes in fusion/fission dynamics [START_REF] Mortiboys | Mitochondrial impairment in patients with Parkinson disease with the G2019S mutation in LRRK2[END_REF][START_REF] Papkovskaia | G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization[END_REF][START_REF] Su | Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation[END_REF][START_REF] Cherra | Mutant LRRK2 Elicits Calcium Imbalance and Depletion of Dendritic Mitochondria in Neurons[END_REF]. In particular, several groups have reported a functional interaction with the mitochondrial fission-promoting dynamin-related protein 1 (Drp1) [START_REF] Su | Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation[END_REF][START_REF] Wang | LRRK2 regulates mitochondrial dynamics and function through direct interaction with DLP1[END_REF][START_REF] Niu | Leucine-rich repeat kinase 2 disturbs mitochondrial dynamics via Dynamin-like protein[END_REF][START_REF] Stafa | Functional interaction of Parkinson's diseaseassociated LRRK2 with members of the dynamin GTPase superfamily[END_REF][START_REF] Perez Carrion | The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1[END_REF].

Drp1-dependent mitochondrial fission occurs early in mitophagy and promotes the isolation of the damaged organelle from the healthy network [START_REF] Twig | Fission and selective fusion govern mitochondrial segregation and elimination by autophagy[END_REF]. Two proteins encoded by genes implicated in autosomal recessive PD forms -the E3 ubiquitin protein ligase Parkin and the mitochondrial serine/threonine PINK1 -jointly regulate a mitophagy program triggered by mitochondrial depolarization [START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF][START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF][START_REF] Matsuda | PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy[END_REF][START_REF] Vives-Bauza | PINK1/Parkin direct mitochondria to autophagy[END_REF]. This process involves a sequence of events initiated by the accumulation of PINK1 close to the translocase of outer mitochondrial membrane (TOM complex), followed by the mitochondrial translocation and activation of Parkin, the ubiquitylation of a series of mitochondrial proteins and the recruitment of ubiquitin-binding autophagy adaptors and the autophagy machinery [START_REF] Pickrell | The Roles of PINK1, Parkin, and Mitochondrial Fidelity in Parkinson's Disease[END_REF][START_REF] Mcwilliams | PINK1 and Parkin: emerging themes in mitochondrial homeostasis[END_REF]. We have shown that PINK1 and Parkin cooperate in the recruitment of Drp1 to mitochondria during mitophagy [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF].

We investigated the mechanisms by which LRRK2 affects mitochondrial function, by exploring its potential impact on PINK1/Parkin-dependent mitophagy and the effects of the G2019S substitution. Using cell lines and primary fibroblasts from PD patients, we showed that LRRK2 attenuated mitophagy, and that this effect was exacerbated by the G2019S substitution. We also provide evidence that LRRK2 interferes with protein-protein interactions involving Parkin and Drp1 on the outer mitochondrial membrane (OMM), and that these interactions, together with the defect in PINK1/Parkin-dependent mitophagy, can be rescued by the inhibition of LRRK2 kinase activity.

RESULTS

LRRK2 attenuates PINK1/Parkin-dependent mitophagy in a kinase-dependent manner

We investigated the possible role of LRRK2 in PINK1/Parkin-dependent mitophagy in a model previously validated for the investigation of this process [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF][START_REF] Bertolin | Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10[END_REF]. In this model, Parkin is transiently overexpressed in COS7 cells. The cells are then treated with CCCP (carbonylcyanide m-chlorophenylhydrazone) and mitophagy is followed over time by quantifying two relevant parameters: (1) perinuclear mitochondrial aggregation, an early Parkin-dependent phenomenon preceding mitochondrial degradation [START_REF] Vives-Bauza | PINK1-dependent recruitment of Parkin to mitochondria in mitophagy[END_REF]; and (2) the clearance of mitochondrial markers. We investigated the impact of LRRK2 on mitophagy, by expressing Parkin alone or with full-length LRRK2. Under cotransfection conditions, exogenous Parkin was expressed in the vast majority of the LRRK2-immunopositive cells (95%, data not shown). Moreover, western blots revealed that exogenous LRRK2 expression had no major impact on Parkin levels and that LRRK2 was undetectable in native cells (Supplementary Material, Fig. S1). We then analyzed the mitochondrial network after staining for a marker of the mitochondrial matrix (the β subunit of the mitochondrial processing peptidase, PMPCB) or the outer mitochondrial membrane (the TOMM20 subunit of the translocase of outer mitochondrial membrane, data not shown) in cells immunopositive for Parkin (Parkin-only conditions) or for LRRK2 (cotransfection conditions). After 48 h of CCCP treatment, about 35% of the cells expressing Parkin alone had aggregated mitochondrial networks and 20% had lost all mitochondrial staining. In cells coexpressing LRRK2, the percentage of cells without mitochondrial staining was significantly smaller, but levels of mitochondrial aggregation were similar (Fig. 1A andB). As expected, no mitochondrial aggregation or clearance was observed in the absence of Parkin (Supplementary Material, Fig. S2 and data not shown), regardless of the presence or absence of LRRK2 (Fig. 1A andB). We then investigated the effect of LRRK2 kinase activity on the observed decrease in Parkin-dependent mitophagy, by expressing two LRRK2 variants with altered kinase activity: LRRK2-G2019S, reported to have higher kinase activity [START_REF] West | Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity[END_REF][START_REF] Smith | Kinase activity of mutant LRRK2 mediates neuronal toxicity[END_REF][START_REF] Greggio | Kinase activity is required for the toxic effects of mutant LRRK2/dardarin[END_REF][START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF], and the kinase-dead LRRK2-D1994A variant (40-42) (Fig. 1C, Supplementary Material, Fig. S3).

Western blot analysis revealed that the abundance of these variants was significantly lower than that of the normal LRRK2 protein in cells (Supplementary Material, Fig. S1). This was not as much due to differences in protein abundance in individual cells as it reflected lower proportions of cells expressing the mutant proteins, possibly linked to toxicity. For the analysis of mitophagy, we took into account only cells with similar levels of LRRK2 immunoreactivity. The proportion of cells with aggregated mitochondria was significantly higher following the expression of LRRK2-G2019S than with LRRK2, at 60% rather than 40%. Moreover, the expression of LRRK2-G2019S further reduced mitochondrial clearance, by approximately 50%. By contrast, LRRK2-D1994A had no effect on mitochondrial aggregation and tended to weaken the impact of LRRK2 on mitochondrial clearance (Fig. 1C, Supplementary Material, Fig. S3; p-value < 0.05 versus Parkin-only condition compared to pvalue < 0.001 versus Parkin-only condition for LRRK2). These results suggest that LRRK2 attenuates Parkin-dependent mitophagy in a kinase-dependent manner, through effects on an early step in this process associated with the formation of mitochondrial aggregates. We explored this relationship further, by performing a kinetic analysis of mitochondrial aggregation in cells expressing Parkin alone (Parkin-only conditions) or together with LRRK2, LRRK2-G2019S or LRRK2-D1994A (Fig. 1D). As expected from our previous experience [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF][START_REF] Bertolin | Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10[END_REF], the proportion of cells with mitochondrial aggregates progressively increased in the presence of overexpressed Parkin, reaching a plateau after 8 h of CCCP treatment. A similar progression was observed in cells coexpressing the kinase-dead LRRK2-D1994A variant. By contrast, the proportion of cells with aggregated mitochondria at each of the time points analyzed was significantly higher in cells coexpressing LRRK2, and higher still in cells coexpressing LRRK2-G2019S. Cell treatment with the well-known LRRK2 kinase inhibitor, LRRK2-IN-1 (43) (5 µM), normalized the aggregation profile in cells coexpressing LRRK2 and significantly decreased the proportion of cells with aggregated mitochondria in cells coexpressing LRRK2-G2019S (Fig. 1E). By contrast, LRRK2-IN-1 had no impact on the aggregation profile in cells expressing Parkin alone or together with LRRK2-D1994A (Fig. 1F). These observations highlight the role of LRRK2 kinase activity in mitochondrial aggregation, an early phenomenon in mitophagy occurring before mitochondrial clearance.

LRRK2 kinase activity interferes with protein-protein interactions involving Parkin and

Drp1 on mitochondria

In previous studies, we used Förster resonance energy transfer (FRET) microscopy with the acceptor photobleaching paradigm to monitor protein-protein interactions on the OMM [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF]. These studies showed that Parkin is recruited to the mitochondria, in close proximity to PINK1 and the TOM complex, early in mitophagy, and that it cooperates with PINK1 in the recruitment to mitochondria of the dynamin-related GTPase Drp1, which is essential for efficient mitophagy [START_REF] Twig | Fission and selective fusion govern mitochondrial segregation and elimination by autophagy[END_REF][START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Tanaka | Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin[END_REF]. All these interactions, detected upon mitochondrial depolarization, were strictly dependent on the presence of PINK1, and were affected by Parkinson's disease-linked PARK2 mutations [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF]. We therefore used this method to explore the impact of LRRK2 on the molecular proximity of Parkin to TOM complex subunits, and of Parkin to Drp1, following CCCP treatment. As previously reported, we detected FRET between the TOMM40 and TOMM70A subunits of the TOM complex and Parkin (Fig. 2A), and between Drp1 and Parkin (Fig. 2D andE) [START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF]. By contrast, no FRET was detected between TOMM20, TOMM22, VDAC1 and Parkin (Fig. 2A). The overexpression of LRRK2 and LRRK2-G2019S abolished FRET between the abovementioned donor/acceptor pairs, whereas FRET was preserved with the kinase-dead LRRK2-D1994A variant (Fig. 2A). The treatment of cells expressing LRRK2 or LRRK2-G2019S with LRRK2-IN-1 restored FRET efficiencies between the investigated protein pairs (Fig. 2B-E, Supplementary Material, Fig. S4). We explored the impact of LRRK2 on the mitochondrial recruitment of Drp1 directly, by investigating the interaction between Drp1 and its mitochondrial receptor, MiD51, which we have previously shown to be required for Parkin-dependent mitophagy [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF]. FRET was detected between Drp1 and MiD51 in cells treated with CCCP, but was abolished in cells overexpressing LRRK2 or LRRK2-G2019S.

Again, the Drp1-MiD51 interaction was preserved in the presence of LRRK2-D1994A and restored by LRRK2-IN-1 treatment in cells expressing active LRRK2 variants (Fig. 2F andG).

Overall, these results suggest that the kinase activity of LRRK2 disrupts protein-protein interactions on the outer mitochondrial membrane occurring early in PINK1/Parkin-dependent mitophagy, including the recruitment of Drp1 to its receptor, MiD51.

A new model for studying Parkin-dependent mitophagy in human fibroblasts

We evaluated the relevance of our findings, by comparing the efficiency of mitophagy in primary fibroblasts from healthy controls and patients carrying the activating G2019S LRRK2 mutation or PARK2 mutations. Our analysis of the mitophagy triggered by CCCP in human fibroblasts with endogenous levels of Parkin led to inconsistent results. Specifically, the severe fragmentation of the mitochondrial network made it impossible to obtain reliable estimates of the modest decreases in the abundance of specific mitochondrial markers.

Previous studies have shown that autophagy can be stimulated by a heat shock [START_REF] Nivon | Autophagy activation by NFkappaB is essential for cell survival after heat shock[END_REF][START_REF] Zhao | Induction of macroautophagy by heat[END_REF][START_REF] Matheoud | Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation[END_REF]. We therefore developed a new mitophagy paradigm based on induction by heat stress, to avoid the massive mitochondrial depolarization triggered by CCCP. We also used a mitophagy reporter consisting of a pH-stable DsRed.T4 fused to a green fluorescent protein sensitive to acidic conditions (pHluorin) and targeted to mitochondria by a classical N-terminal mitochondrial targeting signal (mtRosella) for the quantitative assessment of mitophagy in living cells.

Rosella constructs have been successfully used in the monitoring of autophagy-dependent processes, including mitophagy, in yeast and Caenorhabditis elegans, based on estimation of the ratio of the red-only signal, reflecting the presence of the reporter in the acidic lysosomal compartment, to green/red double fluorescence [START_REF] Rosado | Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast[END_REF][START_REF] Charmpilas | Monitoring Mitophagy During Aging in Caenorhabditis elegans[END_REF]. We here report the first application of this system to mammalian cells. Fibroblasts from control individuals overexpressing mtRosella were shifted to either 42°C or 24°C for 45 minutes and then returned to 37°C for 45 minutes (Fig. 3A andB). The shift to 42°C resulted in an overall compaction of the mitochondrial network relative to basal conditions (cells maintained at 37°C), with no change in the red-only signal. By contrast, the shift to 24°C led to an increase in the abundance of mitochondrial vesicles with red-only fluorescence, resulting in a doubling of mean red-only fluorescence relative to basal conditions. This increase was abolished following treatment with the autophagy inhibitor 3-methyladenine (3-MA) (Fig. 3C), and in cells silenced for ATG5 (Fig. 3D), Parkin (Fig. 4A andB) or PINK1 (Fig. 4C andD), indicating that it was related to PINK1/Parkin-dependent mitophagy. Moreover, the red-only fluorescent mitochondrial vesicles corresponded to mitochondria engulfed by lysosomes, as shown by confocal microscope analyses of co-localization with the lysosomal LysoTracker dye (Supplementary Material, Fig. S5). More than 90% of the red-only signal colocalized with Lysotracker fluorescence, whereas no more than 15% of the green fluorescent mitochondrial staining colocalized with lysosomes. Thus, shifting cells to 24°C triggers Parkin-dependent mitophagy in human fibroblasts.

Parkin-dependent mitophagy is impaired in fibroblasts from PD patients

We then used the shift to 24°C to investigate Parkin-dependent mitophagy in fibroblasts from control subjects and PD patients with the G2019S LRRK2 mutation, or various compound heterozygous combinations of PARK2 mutations (Table 1). We previously showed that Parkin was detectable in human fibroblasts [START_REF] Gautier | The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations[END_REF]. Western blot analysis showed that LRRK2 was present in cells from each category of subjects (Supplementary Material, Fig. S6). The mtRosella reporter was expressed following transient transfection, and mitophagy was investigated in four independent sessions, each including a different fibroblast line from each category of subjects (Supplementary Material, Table S1 and Table 1). As expected, the overall analysis of these experiments revealed that, in control fibroblasts, the shift to 24°C led to the appearance of numerous mitochondrial vesicles with red-only fluorescence. By contrast, red-only fluorescence was much less abundant in fibroblasts from patients with PARK2 mutations and carriers of the G2019S LRRK2 mutation (Fig. 5A andC). The levels of red-only fluorescence in cells from patients were similar to those in control cells maintained at 37°C (Fig. 3B), suggesting that there was no effect on basal mitophagy. An impaired induction of mitophagy was observed for all patient lines relative to any control line, demonstrating the robustness of these results (Supplementary Material, Table S1). We evaluated the dependence of the apparent impairment of mitophagy induction in LRRK2 patients on the kinase activity of the protein, as suggested by our results in COS7 cells, by treating the cells with LRRK2-IN-1 (Fig. 5B andC). LRRK2-IN-1 restored Parkin-dependent mitophagy in fibroblasts with the G2019S LRRK2 mutation, but not in fibroblasts with PARK2 mutations. The inhibition of LRRK2 kinase activity had no effect on mitophagy induction in fibroblasts from control individuals.

Our results for COS7 cells suggested that LRRK2 overactivity disturbs the mitochondrial recruitment of Drp1 preceding mitophagy. We therefore investigated whether rescue of the mitophagy defect by LRRK2-IN-1 in fibroblasts from LRRK2-G2019S patients was dependent on functional Drp1. We transfected fibroblasts from LRRK2-G2019S patients with the mtRosella construct together with an empty vector or vectors encoding the dominant negative Drp1-K38A variant or functional Drp1 (Fig. 6A andB). We evaluated the impact of LRRK2-IN-1 on the restoration of mitophagy following a shift to 24°C in these conditions.

As previously observed, LRRK2-IN-1 restored mitophagy in fibroblasts from LRRK2-G2019S patients transfected with empty or functional Drp1 vectors. By contrast, mitophagy induction was not rescued in cells expressing Drp1K38A (Fig. 6A andB). Overexpression of Drp1 in cells from LRRK2-G2019S patients did not rescue the mitophagy defect per se, stuggesting that this approach does not restore the mitochondrial recruitment of Drp1. For confirmation of the requirement of Drp1 to rescue mitophagy induction in LRRK2-G2019S fibroblasts, we treated the cells with the cell-permeable Drp1 inhibitor Mdivi-1 (51); again, no correction of the mitophagy defect by LRRK2-IN-1 was observed (Fig. 6C andD). These results suggest that the impairment of Parkin-dependent mitophagy in LRRK2-G2019S patients is at least partly mediated by Drp1. To gain insight into possible mechanisms underlying Drp1 dysregulation by LRRK2-G2019S, we examined intracellular levels of Drp1 or Drp1 forms phosphorylated on serine 616 or serine 637, known respectively to promote mitochondrial translocation and fission [START_REF] Chang | Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1[END_REF] or sequestration of the protein in the cytosol [START_REF] Cereghetti | Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria[END_REF], in fibroblasts from LRRK2-G2019S patients and control subjects (Supplementary Material, Fig. S7). As a control, we also analyzed levels of the profusion GTPase Mfn2. The abundance of total Drp1, Drp1 phosphorylated on serine 616 and Mfn2 were similar in cells from patients and controls, both under basal conditions and following a shift to 24°C. Drp1 phosphorylated on serine 637 was undetectable in these experimental conditions. substitution. This defect was similar to that observed in patients with compound heterozygous PARK2 mutations, and was rescued by a LRRK2 kinase inhibitor. Finally, our results suggest that the effect of LRRK2 kinase hyperactivity on PINK1/Parkin-dependent mitophagy is at least in part mediated by Drp1.

DISCUSSION

Previous studies have already highlighted a functional interplay between LRRK2 and Drp1, albeit with inconsistent results (54) [START_REF] Su | Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation[END_REF][START_REF] Wang | LRRK2 regulates mitochondrial dynamics and function through direct interaction with DLP1[END_REF][START_REF] Niu | Leucine-rich repeat kinase 2 disturbs mitochondrial dynamics via Dynamin-like protein[END_REF][START_REF] Stafa | Functional interaction of Parkinson's diseaseassociated LRRK2 with members of the dynamin GTPase superfamily[END_REF][START_REF] Perez Carrion | The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1[END_REF]. Consistent with our findings, the inhibition of LRRK2 kinase activity has been shown to promote the association of Drp1 with mitochondria and mitochondrial fission [START_REF] Saez-Atienzar | The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrialderived ROS signaling[END_REF], indicating that impairment of the mitochondrial translocation of Drp1 may well underlie the mitophagy defect in cells from patients with the G2019S substitution. Consistent with this possibility, other authors have reported changes in mitochondrial morphology compatible with fission arrest in LRRK2-G2019S knock-in mice and fibroblasts from patients carrying the newly discovered LRRK2 substitution E193K [START_REF] Perez Carrion | The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1[END_REF][START_REF] Yue | Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice[END_REF]. In mice, these alterations were accompanied by a decrease in levels of Drp1 phosphorylated on serine 616 [START_REF] Chang | Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1[END_REF]. The specific mechanisms by which LRRK2 regulates Drp1 to inhibit mitochondria fission before mitophagy remain to be determined. It has been suggested that LRRK2 phosphorylates Drp1 directly on the serine 637 residue, thereby preventing translocation of the protein from the cytosol to the OMM [START_REF] Cereghetti | Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria[END_REF]. We did not detect changes to the phosphorylation state of serine 616 or serine 637 under basal conditions or following thermal stress in total lysates from fibroblasts derived from carriers of the G2019S mutation compared to control cells. This does not exclude a possible impact of LRRK2-G2019S on the mitochondrial pool of Drp1. Such an impact may be indirect and possibly involve other posttranslational modifications known to regulate the mitochondrial association of Drp1 [START_REF] Chang | Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1[END_REF].

The role of LRRK2 in macroautophagy has also been intensively investigated in previous studies [START_REF] Singh | LRRK2 and mitochondria: Recent advances and current views[END_REF], several of which concluded that its effect is proautophagic [START_REF] Macleod | The familial Parkinsonism gene LRRK2 regulates neurite process morphology[END_REF][START_REF] Plowey | Role of autophagy in G2019S-LRRK2associated neurite shortening in differentiated SH-SY5Y cells[END_REF][START_REF] Bravo-San Pedro | The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway[END_REF][START_REF] Schapansky | Membrane recruitment of endogenous LRRK2 precedes its potent regulation of autophagy[END_REF][START_REF] Gómez-Suaga | Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP[END_REF][START_REF] Su | Threonine 56 phosphorylation of Bcl-2 is required for LRRK2 G2019S-induced mitochondrial depolarization and autophagy[END_REF], and others reporting an inhibitory effect [START_REF] Manzoni | Inhibition of LRRK2 kinase activity stimulates macroautophagy[END_REF][START_REF] Sánchez-Danés | Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease[END_REF][START_REF] Alegre-Abarrategui | LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model[END_REF] or no effect [START_REF] Orenstein | Interplay of LRRK2 with chaperone-mediated autophagy[END_REF]. Previous attempts to address the impact of LRRK2 on basal mitophagy [START_REF] Chang | Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1[END_REF][START_REF] Gómez-Suaga | Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP[END_REF], which is unaffected by PINK1 and Parkin [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF][START_REF] Mcwilliams | Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand[END_REF] showed opposing effects, whereas a single study assessing toxin-induced mitophagy in fibroblasts from patients with sporadic and LRRK2-linked PD, using colocalization of TOMM20 and the lysosomal marker LAMP1 as a supposed "mitophagy index" was inconclusive [START_REF] Smith | Fibroblast Biomarkers of Sporadic Parkinson's Disease and LRRK2 Kinase Inhibition[END_REF]. Only one previous study investigated, in detail, the direct impact of LRRK2 on PINK1/Parkin-dependent mitophagy, and its findings are consistent with our observations [START_REF] Hsieh | Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease[END_REF]. This study showed that axonal mitophagy was delayed in induced pluripotent stem cell (iPSC)-derived neurons from patients with sporadic PD and patients with the LRRK2-G2019S mutation, through a combination of approaches, including the colocalization of mitochondria with LC3 or optineurin and the use of the ratiometric mitophagy reporter mt-Keima. This defect was linked to a mechanism converging on the outer mitochondrial membrane protein Miro, a GTPase that anchors mitochondria via molecular motors to the microtubule network and is known to be cleared by the joint action of PINK1 and Parkin before mitophagy. The authors showed that LRRK2 facilitated mitochondrial arrest by promoting the removal of Miro from damaged mitochondria, an effect that was disrupted by the G2019S substitution. Mitochondrial fission and detachment from microtubules are early steps in mitophagy that are thought to require coordination for engulfment of the damaged organelle by the autophagosome. An impact of LRRK2 on mitochondrial arrest is, therefore, not incompatible with an effect on mitochondrial fission during mitophagy, and might be particularly relevant to the efficiency of mitophagy, particularly in highly polarized cells such as neurons.

Interestingly, the molecular events that initiate mitophagy are integrated by a physical platform corresponding to the interface between the endoplasmic reticulum (ER) and mitochondria that has been reported to be regulated by PINK1 and Parkin [START_REF] Gautier | The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations[END_REF][START_REF] Gelmetti | PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation[END_REF][START_REF] Celardo | Mitofusin-mediated ER stress triggers neurodegeneration in pink1/parkin models of Parkinson's disease[END_REF][START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy[END_REF]. ER tubules mark sites of Drp1 recruitment and mitochondrial fission [START_REF] Friedman | ER tubules mark sites of mitochondrial division[END_REF], and mitochondria are progressively degraded at the ER-mitochondrion interface [START_REF] Yang | Bit-by-bit autophagic removal of parkin-labelled mitochondria[END_REF]. PINK1 and Parkin promote the degradation of the profusion GTPase Mfn2, which tethers the mitochondria to the ER in mammalian cells [START_REF] De Brito | Mitofusin 2 tethers endoplasmic reticulum to mitochondria[END_REF][START_REF] Naon | Reply to Filadi et al.: Does Mitofusin 2 tether or separate endoplasmic reticulum and mitochondria?[END_REF]. Subsequent dissociation of the mitochondria from the ER is essential for efficient mitophagy [START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy[END_REF]. The Miro GTPase Gem1 is an integral component of the ER-mitochondrion interface in yeast, and this role may have been conserved throughout evolution [START_REF] Kornmann | The conserved GTPase Gem1 regulates endoplasmic reticulum-mitochondria connections[END_REF], in which case, PINK1/Parkin-dependent Miro degradation or LRRK2mediated removal from mitochondria during mitophagy may also contribute to organelle separation. Remarkably, mitochondria-lysosome contacts have also been involved in the regulation of Drp1-dependent mitochondrial fission events at sites marked by ER tubules, through GTP hydrolysis by the small Rab GTPase, Rab7, driven by the Rab7 GTPaseactivating protein, TBC1D15 [START_REF] Wong | Mitochondria-lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis[END_REF]. Rab7 and TBC1D15 also regulate autophagosme biogenesis during PINK1/Parkin-dependent mitophagy [START_REF] Yamano | Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy[END_REF], and LRRK2 and its substrate Rab GTPases are recruited to overloaded lysosome to maintain their homeostasis [START_REF] Eguchi | LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis[END_REF].

These observations suggest that, like PINK1 and Parkin, LRRK2 may modulate mitophagy by acting on molecular actors involved in various steps of mitochondrial degradation in a dynamically controlled manner. Further studies are required to explore this hypothesis in greater depth, and to determine the extent to which the regulation of these molecular events corresponds to a physiological role of LRRK2, rather than a gain of function linked to PDcausing mutations. Notably, evidence for LRRK2 hyperactivation was recently provided in brain tissue from patients with idiopathic PD and in two distinct rat models of PD [START_REF] Di Maio | LRRK2 activation in idiopathic Parkinson's disease[END_REF]. This defect was linked to a pathological cascade initiated by oxidative stress, and involving autophosphorylation of LRRK2 and secondary accumulation of signs of mitochondrial and lysosomal dysfunction. Therefore, LRRK2 kinase inhibition may be considered as a potential therapeutic approach for correcting mitochondrial and autophagy defects, possibily not only in patients with LRRK2 mutations but also, more generally, in idiopathic PD.

MATERIALS AND METHODS

Human fibroblasts

Human fibroblasts were obtained from skin biopsy specimens obtained from patients with the G2019S LRRK2 substitution or with PARK2 mutations and healthy individuals, at the Clinical Investigation Center for Neurosciences at Pitié-Salpêtrière hospital. All patients signed an informed consent before the procedure, and the study was approved by a local ethics committee and the regulatory authorities. Patients were screened for PARK2 mutations by exon dosage and bidirectional Sanger sequencing of the entire coding sequence, as previously described [START_REF] Periquet | Parkin mutations are frequent in patients with isolated early-onset parkinsonism[END_REF]. Patients were screened for the LRRK2-G2019S mutation in Taqman assays, with confirmation by bidirectional Sanger sequencing for those testing positive [START_REF] Kay | Validity and utility of a LRRK2 G2019S mutation test for the diagnosis of Parkinson's disease[END_REF].

Plasmids, pharmacological agents, treatments and antibodies

The following plasmids were used for transfection: pcDNA3-HA, pcDNA-HA-Parkin, pDEST53-GFP-LRRK2/LRRK2-G2019S (full-length proteins) (9) and pDEST53-GFP-LRRK2-D1994A (obtained from pDEST53-LRRK2-GFP by site-directed mutagenesis), pCB6-Myc-Drp1 and pCB6-Myc-Drp1-K38A (provided by M. Rojo). The pCINeo-mtRosella vector, encoding a pH-stable DsRed.T4 fused to a green fluorescent protein (pHluorin) linked to the COX VIII N-terminal mitochondrial targeting signal, was designed by Carlos Rosado (Mark Prescott's laboratory, Monash University, Australia). RNA interference approaches involved: ATG5 siRNA (Qiagen, SI02655310), Parkin siRNA (Invitrogen, HSS107593), PINK1 siRNA (Thermo Fisher, HSS127945) and all-star siRNA (control; Qiagen, 1027281), all used at a concentration of 100 nM.

The pharmacological agents used were: 10 µM CCCP (Sigma, C2759) for 48 h (mitophagy analysis) or for 6 h (FRET experiments), 10 mM 3-methyladenine (3-MA, Sigma, M9281) for 3 h (Rosella experiments), 5 µM LRRK2-IN-1 (Millipore, 438193) for 0 to 12 h (mitochondrial aggregation kinetics), 2 h (Rosella experiments) or 6 h (FRET experiments) and 50 µM Mdivi-1 (Sigma, MO199) for 90 min (Rosella experiments). Primary fibroblasts were exposed to heat shock by shifting them from 37°C to 24°C or 42°C for 45 min. They were then returned to 37°C for 45 min before analysis.

The primary antibodies used for western blotting or for immunocytochemistry and FRET experiments are listed in Tables 2 and3, respectively.

Cell culture and transfection

COS7 cells were maintained in DMEM GlutaMAX™ (Dulbecco's modified Eagle's medium; Gibco, 10566016) supplemented with 10% (v/v) fetal bovine serum (Gibco, 10270106) and 1% (v/v) antibiotics (penicillin and streptomycin, Invitrogen, 25030024) at 37°C, under a humidified atmosphere containing 5% CO2. Cells were plated at a density of 30,000 (FRET experiment) or 40,000 cells per well (mitophagy analysis) in 24-well culture plates (Corning, 3527). They were added to the wells on 14-mm glass coverslips precoated with poly-D-lysine (PDL, Sigma, P7886; 250 µg/ml). After incubation at 37°C for 24 hours, the cells were transfected with 1 µg of plasmid per well in the presence of DMRIE-C agent (Invitrogen, 10459-014) in Opti-MEM™ medium (Thermo Fisher Scientific, 31985070) for 5 hours, according to the manufacturer's instructions. In cotransfections, pcDNA3-HA-Parkin and pDEST53-GFP-LRRK2 vectors were used at a ratio of 1:9 by weight. At the end of the fivehour transfection period, the transfection medium was removed and the cells were incubated overnight with complete DMEM GlutaMAX™ at 37°C, under an atmosphere containing 5% CO2. The cells were exposed to pharmacological agents (see above) 24 hours later. They were then fixed by incubation in 4% paraformaldehyde (PFA, Sigma, 252549) for 20 minutes, washed three times with phosphate-buffered saline (PBS) and stored at 4°C. Human fibroblasts were maintained at 80% confluence in complete DMEM GlutaMAX™ medium. Fibroblasts were transfected with the following plasmids: 1 µg of mtRosella plasmid alone, with other plasmids (1 µg) or with siRNAs (100 nM), per 20,000 cells. Cells were transfected by electroporation (2 pulses, 20 ms and 1200 V) with the Neon® Transfection System (Thermo Fisher Scientific, MPK10096) and plated in Ibidi glass-bottomed eight-well chambers (Biovalley, 80827; 20,000 cells per well) or in six-well plates (TPP, 92006; 400,000 cells per well), in phenol red-free medium supplemented with 10% (v/v) FBS and 1% (v/v) glutamine. The medium was replaced after 24 h, and analyses were performed 72 h after transfection.

Immunocytochemistry

Cells were processed with standard immunocytochemical techniques. In brief, cells were permeabilized by incubation in 0.2% Triton X-100 in PBS for 10 minutes and nonspecific binding sites were blocked by incubation for 1 hour at room temperature with 10% normal goat serum (NGS, Thermo Fisher Scientific, 1610064). Cells were incubated with primary antibodies diluted in PBS supplemented with 2% NGS overnight at 4°C, rinsed and incubated with secondary antibodies in PBS supplemented with 2% NGS at room temperature for 1 h.

The coverslips holding the cells were then mounted in ProLong® Gold Antifade Reagent (Thermo Fisher Scientific, P36930) on glass microscope slides. For the analysis of mitophagy in COS7 cells, we performed dual labeling for Parkin (Parkin-only conditions) or LRRK2 (Parkin + LRRK2 conditions) and either TOMM20 or PMPCB. For FRET experiments, the impact of LRRK2 variants on specific protein-protein interactions was assessed by triple labeling for LRRK2 and the specific donor/acceptor pairs.

Epifluorescence and confocal microscopy

We analyzed the effects of LRRK2 on Parkin-dependent mitophagy, by imaging COS7 cells with a Nikon Eclipse Ti-U M7 epifluorescence microscope equipped with HCImage software (20 x objective). We analyzed at least [START_REF] Sánchez-Danés | Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease[END_REF] 3) were acquired from a region of interest (ROI) before and after photobleaching, with an Olympus FluoView FV-100 microscope driven by Olympus FV-1000 3.1 software (63 x oil immersion objective, N. A. 1.35). Care was taken to investigate the effects of different LRRK2 variants in cells with similar immunofluorescence intensities for these proteins.

Before analyzing the effects of LRRK2 on specific protein-protein interactions, we checked that the Alexa Fluor® 405 blue fluorescence did not itself interfere with FRET efficiencies, by comparing specific donor/acceptor pairs in cells co-immunolabeled for GAPDH or tubulin, using Alexa Fluor® 405-coupled secondary antibodies (data not shown). We analyzed seven to 12 cells per condition, in each of at least three independent experiments. We used ImageJ software after photobleaching to correct images for the decrease in fluorescence intensity due to the acquisition process, and quantification was performed as previously described (37): % FRET = [ID postbleach -ID prebleach) / ID postbleach]ROI. For the analysis of mitophagy based on the mtRosella construct in human fibroblasts, cells were captured at 37°C with a Spinning disk CSU-X1 confocal microscope (Leica) equipped with a chamber with controlled temperature and CO2 concentration, for live cell imaging driven by MetaMorph software ( 63x immersion oil objective, N.A. 1.4). We analyzed 12-20 cells per condition in each of at least three independent experiments. For comparative analyses of mitophagy in fibroblasts from control subjects and PD patients, several independent experiments were run, each including cells from a control, and at least one category of patients (Supplementary Material, Table S1).

Images were deconvoluted with Huygens Professional software and analyzed with Fiji Software to quantify the ratio of the mean area of red-only fluorescence to total red fluorescence per cell (%).

Immunoblotting

After 24 h, cells were washed, collected and lysed directly in 1 x sample buffer with DTT (dithiothreitol), protease inhibitors (Thermo Fisher Scientific, 1821281) and phosphatase inhibitors (Thermo Fisher Scientific, 1821279). Proteins were quantified with the Pierce TM BCA protein Assay (Thermo Fisher Scientific, 23225), with absorbance readings at 562 nm.

Whole-cell lysates were heated at 95°C for 5 min, separated by electrophoresis in a 4-20% agarose Tris/glycine gel (Mini-PROTEAN ® TGX™ Precast Gels, BioRad, 4561094), and the proteins were then transferred onto nitrocellulose membrane by wet electroblotting (Amersham Protran, 0.45 µm pores; GE Healthcare, 10600002). The membrane was incubated in 5% non-fat dry milk powder in PBS to block nonspecific binding sites, then overnight at 4°C with primary antibody in 5% non-fat dry milk powder in PBS, and for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (1:50000) in 1% non-fat dry milk powder in PBS. Proteins were visualized by enhanced chemiluminescence (ECL SuperSignal™ West Dura (Thermo Fisher Scientific, 34075) on film (ECL, Amersham Hyperfilm, 28906837). Signals were quantified with MultiGauge software.

Statistical analyses

Statistical analysis was performed with Prism 7 (Graph Pad software). The results are expressed as means ± SD, with the exception of the kinetics analyses based on the evaluation of an interaction factor (time and different conditions). One-or two-way ANOVA followed by Tukey's multiple comparisons post-hoc test was used to evaluate the differences between groups, except for the analysis of the effects of 3-MA treatment, ATG5 siRNA, Parkin siRNA and PINK1 siRNA on mtRosella responses in fibroblasts, for which paired Student's t-tests were used. Values of p < 0.05 were considered significant.

LEGENDS TO FIGURES

  We explored the involvement of LRRK2 in PINK1/Parkin-dependent mitophagy in two complementary model systems: a classical model based on the overexpression of Parkin and the coexpression of LRRK2 variants in a human cell line, and induction of mitochondrial dysfunction using the mitochondrial uncoupler CCCP; and an original model with physiological expression of Parkin and LRRK2 variants, based on the use of primary human fibroblasts from control individuals or PD patients, and thermal stress to trigger mitophagy. A combination of approaches were used to explore mitophagy in these models, including analysis of mitochondrial network aggregation and clearance, investigation of key proteinprotein interactions established during the early phases of the process, and the use of the novel sensitive dual-fluorescence mitophagy reporter, mtRosella. We provide several lines of evidence consistent with a role for LRRK2 kinase hyperactivity in downregulating PINK1/Parkin-dependent mitophagy. First, the overexpression of kinase-active LRRK2 accentuated the perinuclear accumulation of damaged mitochondria and attenuated their Parkin-dependent clearance triggered by CCCP. These effects were exacerbated by the expression of the kinase hyperactive LRRK2-G2019S variant, and mitigated or abolished by expression of the kinase inactive LRRK2-D1994A variant, or treatement of the cells with a pharmacological LRRK2 kinase inhibitor. Second, LRRK2 interfered with the recruitment of Parkin and the fission-promoting GTPase Drp1 to their respective interactors on the OMM, the TOM complex and the receptor MiD51. Again, this effect was abolished by genetic and pharmacological approaches aimed at inhibiting LRRK2 kinase activity. Third, the use of mtRosella, revealed that LRRK2 overactivation compromised the induction of PINK1/Parkindependent mitophagy in fibroblasts from PD patients carrying the LRRK2-G2019S

  cells immunopositive for Parkin (Parkin-only condition) or for LRRK2 or LRRK2 variants (Parkin + LRRK2 conditions) per condition, in each of at least three independent experiments, checking for the presence of aggregated mitochondria or the absence of mitochondrial staining. The effects of different LRRK2 variants were investigated in cells with similar fluorescence immunostaining intensities for the N-terminal GFP moiety encoded by the GFP-LRRK2 constructs. For FRET experiments, we selected cells immunopositive for LRRK2 (Alexa Fluor® 405 blue fluorescence) and containing mitochondrial aggregates reflecting the presence of Parkin. Images of donor (Alexa Fluor® 488 green) and acceptor (Alexa Fluor® 568 red) fluorescence (see Table

Figure 1 :

 1 Figure 1: LRRK2 attenuates Parkin-dependent mitophagy

Figure 2 :

 2 Figure 2: LRRK2 kinase activity disturbs the protein-protein interactions induced by

Figure 3 :

 3 Figure 3: Thermal stress triggers mitophagy in human fibroblasts

Figure 4 :

 4 Figure 4: The mitophagy triggered by thermal stress depends on PINK1 and Parkin

ACKNOWLEDGEMENTS

We would like to thank S. Forlani (DNA and Cell Bank, Hôpital de la Pitié-Salpêtrière) for human cell collection and banking. We also thank S. Bardien for providing LRRK2/LRRK2-G2019S vectors (Stellenbosch University, South Africa). We are grateful to S. Fouquet for providing us with access to the imaging facilities at Institut de la Vision.

FUNDING

This work was supported by funding from INSERM, Ipsen Laboratories, Association France Parkinson, the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115568, including financial contributions from the European Union Seventh Framework Programme (FP7/2007-2013), EFPIA and "Investissements d'Avenir" ANR-10-IAIHU-06. F.B. received financial support from Association Nationale Recherche Technologie and Association France Parkinson. Y.S.S. received support from the International Union of Biochemistry and Molecular Biology (IUBMB, Wood-Whelan Research Fellowhsip).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online

TABLES Table 1. Age, sex and genotype of the donors of the skin fibroblasts used in this study

Red-only fluorescence to total red fluorescence ratio (%) expressed as a "Mitophagy index"

for each subject (Table 1) included in the experiments presented in Fig. 4A (mean of the 4 independent experiments).


Anna Kotova
email: akotova@irap.omp.eu

E Roussos

P Kollmann

N Krupp

I Dandouras

Galactic Cosmic Rays access to the magnetosphere of Saturn

The MeV proton radiation belts of Saturn are isolated from the middle and outer magnetosphere and the source of these high energy protons is thought to be linked to the access of Galactic Cosmic Rays (GCRs) in the system.

To validate this hypothesis it is first of all necessary to determine the realistic spectrum of GCRs at Saturn. Previously only analytical attempts were performed in order to calculate the GCR spectra. In this letter we provide for the first time the numerical solution for the determination of the GCR access to the upper atmosphere and rings of Saturn. The proposed method is based on the charged particle tracing technique. As a result the GCRs access energies to the atmosphere and to the equatorial plane of the magnetosphere as a function of radial distance to the planet were obtained and the GCRs spectra were reconstructed. Dependencies of the spectral parameters such as the time or the incidence direction, were also obtained offering all necessary information for simulating the interaction of GCRs with the saturnian system during different phases of the Cassini mission.

Keypoints:

• We provide GCR access energies to the Saturnian atmosphere and across the magnetosphere

• GCR spectra and integrated flux to Saturn and to the equatorial plane of the magnetosphere are calculated • Advantages of a particle tracing approach over the analytical solution are demonstrated

Introduction

The presence of stable radiation belts of Saturn between the outer edge of the A ring (2.27 R S , where R S is the radius of Saturn) and Tethys' orbit (4.8 R S ) was confirmed already during the first visits of this planet by Pioneer 11, Voyager 1 and 2 [Fillius et al., 1980;[START_REF] Krimigis | Two-component proton spectra in the inner Saturnian magnetosphere[END_REF][START_REF] Simpson | Saturnian trapped radiation and its absorption by satellites and rings: The first results from Pioneer 11[END_REF][START_REF] Vogt | Energetic charged particles in Saturn's magnetosphere: Voyager 2 results[END_REF]. Rings and numerous moons of Saturn shape the unique configuration of the Saturnian radiation belts. Bouncing between mirror points the energetic charged particles regularly cross the equatorial plane of the magnetosphere. Owing to the nearly symmetric magnetic field and their almost circular and equatorial orbits, the large moons of Saturn effectively absorb the trapped energetic particles that follow the magnetic field lines of those moons' L-shells along their motion around the planet. These sweeping corridors that form behind the moons barely replenish, indicating that radial diffusion cannot supply the radiation belts with a sufficient portion of new energetic particles from the middle magnetosphere.

Cassini observations confirmed, that energetic ions along these L-shells are absent in all magnetospheric local times and latitudes, independently from the location of the moons.

Similarly absorption of the charged particles happens along the L-shells connected to the Main Rings. In the same time, outside the Tethys orbit proton fluxes are changing a lot from one orbit to another, but the particle flux inside the radiation belts remains unchanged [START_REF] Roussos | Discovery of a transient radiation belt at Saturn[END_REF][START_REF] Paranicas | Sources and losses of energetic protons in Saturn's magnetosphere[END_REF]. Independently from the solar cycle phase only a weak modulation on the time scale of years was observed by Cassini MIMI/LEMMS in the > 10 MeV protons flux of the Saturnian radiation belts [Roussos c 2018American Geophysical Union. All Rights Reserved. et al., 2011;[START_REF] Kollmann | The evolution of saturn's radiation belts modulated by changes in radial diffusion[END_REF]. This indicated that the source for these energetic ions should be local, most likely being connected to the interaction of Galactic Cosmic Rays (GCR) with Saturn and its rings through the CRAND (Cosmic Ray Albedo Neutron Decay) process [START_REF] Blake | On the injection of cosmic ray secondaries into the inner saturnian magnetosphere: 1. protons from the crand process[END_REF][START_REF] Cooper | Nuclear cascades in saturn's rings: Cosmic ray albedo neutron decay and origins of trapped protons in the inner magnetosphere[END_REF][START_REF] Cooper | The secondary radiation under saturn's a-b-c rings produced by cosmic ray interactions[END_REF]. Existence of the energetic protons population within Saturn's innermost radiation belt, located in the narrow zone between the Saturn's atmosphere and the Main Rings, was recently confirmed during Cassini Proximal orbits in 2017. This population can originate also only from CRAND [Roussos et al., 2018a;[START_REF] Kollmann | Saturn's innermost radiation belt throughout and inward of the d-ring[END_REF].

At Saturn this process was described by several authors including Fillius et al. [1980]; [START_REF] Cooper | Nuclear cascades in saturn's rings: Cosmic ray albedo neutron decay and origins of trapped protons in the inner magnetosphere[END_REF]; Fillius and McIlwain [1980]; [START_REF] Van Allen | Sources and sinks of energetic electrons and protons in Saturn's magnetosphere[END_REF]; [START_REF] Randall | Energetic electrons in the magnetosphere of Saturn[END_REF]; [START_REF] Kollmann | Processes forming and sustaining Saturn's proton radiation belts[END_REF]. A GCR proton with a sufficient energy enters the magnetosphere, reaches the planet and undergoes collisions with atmospheric particles or ring matter and produces cascades of secondaries, also known as cosmic ray showers. These secondaries include neutrons, protons, electrons, neutrinos, pions, muons, various antiparticles and photons, partly at much lower energies. Charged secondary particles will be trapped by the magnetic field and most probably will be absorbed during their bounce motion in a short time. Neutrons in contrast can freely escape from their production region. A free neutron has a mean lifetime of 885.7 ± 0.8 sec [START_REF] Nakamura | Review of particle physics[END_REF]. Beta decay of a neutron leads to the production of a proton, an electron and an electron antineutrino. c 2018 American Geophysical Union. All Rights Reserved.

The difference in atomic mass of neutron and proton leads to the release of 782 keV of the kinetic energy, which is shared between the electron and the antineutrino, and all the kinetic energy of a parent neutron goes to the daughter proton [START_REF] Blake | On the injection of cosmic ray secondaries into the inner saturnian magnetosphere: 1. protons from the crand process[END_REF]. Energetic (keV-GeV) neutrons decay with low probability inside the magnetosphere across a wide range of L-shells and thus populate the radiation belts with a newborn energetic proton and electron.

In order to estimate the CRAND input on the radiation belts population it is essential first of all to determine the incoming GCR flux onto the Saturnian atmosphere and rings.

Saturn has a strong magnetic field which can deflect high energy GCRs. However, if the GCR has enough energy, it still can get into the atmosphere (or on the planetary rings).

Therefore, for any given location and arrival direction it is necessary to determine an energy, which can overcome the deflection by the planetary magnetic field and reach the atmosphere (or rings). We will name this energy "access energy". There exist analytical and numerical solutions for its determination. However most of them were developed for Earth and do not take into account the specificities of other planets, such as their non-dipolar magnetic field. Particularly for Saturn only analytical calculations were performed so far by the above-mentioned authors, providing uncertain results. Therefore the comprehensive study of the CRAND process at Saturn essentially requires using a numerical method is essential. c 2018 American Geophysical Union. All Rights Reserved.

In this work we use particle tracing to determine the incoming GCR flux at a given location. This method can provide a solution for many different questions in the study of the GCR impact on the magnetosphere of Saturn.

Analytical Solution Versus Tracing Technique

Analytical Solution Based on Störmer Theory

The Störmer theory for the calculation of the particle's energy needed to access the planetary surface (or atmosphere) is based on the concept of magnetic rigidity, which describes the resistance of a charged particle to change its direction of motion under the influence of a magnetic force [START_REF] Kallenrode | Space Physics. An Introduction to Plasmas and Particles in the Heliosphere and Magnetospheres[END_REF]. Similarly the GCR cutoff rigidity is a quantitative measure of planetary magnetic field shielding [START_REF] Smart | A review of geomagnetic cutoff rigidities for earth-orbiting spacecraft[END_REF] and is proportional to the momentum needed for a cosmic ray to reach the planet at a specific location as a function of arrival direction (see the Support Information for formulas).

The analytical approach is a relatively fast method for the derivation of the GCR cutoff energy and can be sufficient as a first approximation for some problems, but cannot sustain an accurate and precise solution. Analytically derived values for GCR access energies do not fit the observed ones at Earth, because the real planetary magnetic field is not a static dipole magnetic field and because the planetary magnetosphere is deformed by a series of processes changing its shape with solar wind conditions. Satellite observations show a marked variation of the detected GCR flux with changes of the magnetic activity within a magnetosphere and suggest that the GCR access energy c 2018 American Geophysical Union. All Rights Reserved.

decreases as the magnetic activity increases [START_REF] Reid | The influence of the geomagnetic tail on lowenergy cosmic-ray cutoffs[END_REF]. The dipole model lacks the higher order moments (quadrupole, octopole, etc.) of the magnetic field, which makes a significant difference for the GCR access energies at lower latitudes and at higher energies [START_REF] Weygand | Cosmic ray cutoff prediction using magnetic field from global magnetosphere MHD simulations[END_REF][START_REF] Beer | Geosphere[END_REF]. Since the contributions of higher order moments of the magnetic field vary as a function of space and time, the GCR cutoff energy varies correspondingly.

Various currents in the magnetosphere, especially the ring current and the cross-tail current, are important contributors to the decrease of the cutoff energies, compared to analytically calculated values [START_REF] Reid | The influence of the geomagnetic tail on lowenergy cosmic-ray cutoffs[END_REF][START_REF] Dorman | Cosmic Rays in Magnetosphere of the Earth and other Planets[END_REF]. The complex shape of the magnetosphere influences the GCR access and in particular, certain particles with lower energy still can access the planet through the tail.

Compression of the magnetosphere under the changing solar wind conditions and during extreme coronal mass ejections [START_REF] Adriani | PAMELA's measurements of geomagnetic cutoff variations during the 14 December 2006 storm[END_REF], solar modulation of the GCR flux in the Solar system, related to the solar cycle, seasonal changes in the magnetosphere along the motion of the planet around the Sun and other periodical changes of the conditions in the magnetosphere and in the ambient GCR flux are all reflected in the final dose of GCR reaching the planet.

Moreover, particularly at Saturn, the presence of rings plays an important role: GCRs can pass through Saturn's rings. While the rings are not massive enough to be an obstacle for the entire GCR flux, they contain large enough bodies, which could be hit by the c 2018 American Geophysical Union. All Rights Reserved.

GCRs producing secondaries showers and such a passage would drive the CRAND process. All these factors cannot be taken into account in an analytical formula. In contrast, the trajectory tracing method can overcome the aforementioned restrictions and provide a more accurate and comprehensive base for the CRAND study on Saturn.

GCR Tracing

GCR trajectory tracing is an extensively used method for the determination of the GCR access energies [START_REF] Smart | Geomagnetic cutoffs: A review for space dosimetry applications[END_REF][START_REF] Selesnick | Cosmic ray access to Jupiter's magnetosphere[END_REF][START_REF] Kress | Dynamic modeling of geomagnetic cutoff for the 23-24 November 2001 solar energetic particle event[END_REF][START_REF] Smart | A review of geomagnetic cutoff rigidities for earth-orbiting spacecraft[END_REF][START_REF] Weygand | Cosmic ray cutoff prediction using magnetic field from global magnetosphere MHD simulations[END_REF][START_REF] Chu | The geomagnetic cutoff rigidities at high latitudes for different solar wind and geomagnetic conditions[END_REF][START_REF] Adriani | PAMELA's measurements of geomagnetic cutoff variations during the 14 December 2006 storm[END_REF].

The common approach is to launch particles from a given latitude and longitude sequentially at zenith angles from -90 • to 90 • and at corresponding phase angles in order to cover the whole range of possible arrival directions, to trace these particles backwards in time and determine, if they escape the magnetosphere or not (here we set the borders of the magnetosphere at the radial distance of 25R S ). Reversal in time implies that this particle can also enter the magnetosphere and hit the planet.

Figure 1 shows trajectories of five GCRs launched backwards in time from the top of Saturn's atmosphere. All five particles were launched from the same location and at the same pitch and phase angle, the only difference is their energy: 1 to 81 GeV. On this Figure it is seen, that only the 81-GeV particle can escape the magnetosphere, since its trajectory is "allowed", and the others are "forbidden", meaning the access energy for that given location and arrival direction is between 61 GeV and 81 GeV. c 2018 American Geophysical Union. All Rights Reserved.

The ECPT (Energetic Charged Particle Tracing) code was developed especially for the purpose of this project and was validated using the Cassini MIMI/LEMMS observations during Rhea and Dione flybys [START_REF] Kotova | Modeling of the energetic ion observations in the vicinity of Rhea and Dione[END_REF]. As a magnetospheric model we used here the dipole model with a shift of the magnetic equator towards north by 0.036R S and also the Khurana model, which today is the only global magnetospheric model of Saturn available to us [START_REF] Khurana | A Model of Saturn's Magnetospheric Field Based on Latest Cassini Observations[END_REF]. It includes the internal spherical harmonic field, the ring currents and the magnetotail current system, the field from the radial current system, the shielding fields from the magnetopause and the interconnection magnetic field from the solar wind IMF. On the base of this model we performed simulations for different seasons: Pre-equinox (July 2004), Equinox (August 2009) and Post-Equinox (September 2017). As a numerical method for tracing of GeV particles we used the Vay method [START_REF] Vay | Simulation of beams or plasmas crossing at relativistic velocity[END_REF] -a modification of the Boris method [START_REF] Boris | Relativistic plasma simulation -optimization of a hybrid code[END_REF] for relativistic particles, since it shows the best performance compared to the 4 th -order Runge-Kutta method and to the classical Boris scheme. GeV for some directions. Since significant difference is found on all latitudes, it suggests, that the calculations on the base of realistic magnetospheric models are more preferable.

GCR Access Energies

Access Energy Mapped on Saturn

The values of access energies for various fractions of the arriving directions calculated on the base of the Khurana model demonstrated on Figure 4 can be found in Table S3 in the Supporting Information. Another important aspect in the evaluation of the GCR interaction with Saturn is the rings crossing rate. Every time the GCR particle crosses the rings it might stop or lose some of its energy, changing its trajectory. Moreover, every passage through the rings will produce a cascade of secondaries, triggering the CRAND process.

We calculated the percentage of particles per number of ring crossings averaged across longitudes for different latitudes. It can be noticed, that at latitudes below ∼ 35 • , in both hemispheres, most of the allowed trajectories cross the rings at least 3 times on their way.

And for middle latitudes (∼ 40 • ) for some very few arrival directions particles can cross the rings up to 12 times. For the current study the assumption is, that a GeV-particle does not lose energy when crossing the rings, but in reality particles either stop or lose a few percent of their energy per crossing, depending on the thickness of the rings. The ring crossing rate should be taken into account in the estimation of the GCR flux at lower latitudes of Saturn and for the evaluation of the CRAND production rate.

To illustrate the possible impact of the non-transparent rings we calculated GCR access energies assuming that particle lose 50% of its energy in every passage through the rings (determined at a distance from the centre of the planet 66 900 km -140 180 km). On the upper panel of Figure 5 on top of the already shown in Figure 4 access energies in fractions of the arrival directions we plot contours, which shows access energy sufficient for the same portion of the arrival directions, but obtained with assumption, that rings are not transparent. Dashed green line highlights the access energy for the 50% of the arrival directions and dash-point red line -minimum access energy. On the bottom panel we show c 2018 American Geophysical Union. All Rights Reserved.

for how much percent increases access energy for every fraction. Here we see, that access energy for half of arrival directions increases for 40 -60% at the middle and low latitudes, minimum access energy increases only for few percent and the ultimate access energy, which assure that particles can arrive from every direction, increases up to several times, depending on the latitude. More precise calculation of the rings impact on the GCR access to the Saturnian atmosphere considering different scenarios is left for the future work.

East-West asymmetry

The predicted by [START_REF] Rossi | On the magnetic deflection of cosmic rays[END_REF] so-called "East-West effect" occurs also at Saturn, but in reversed direction compared to the Earth, because of the opposite orientation of the magnetic moment of the planet dipole. The access energy depends on the arrival direction. To reach the point at the top of the atmosphere from the west the GCR should have much more energy, than from the east, and the difference is bigger at lower latitudes. For instance, the 65 GeV GCR managed to arrive to equator from the east, while from the west the allowed trajectories started only from 350 GeV, since the planet shadows the lower energy trajectories. This example is depicted on the upper panel of Figure 6.

The bottom panel of Figure 6 demonstrates the dependence of the access energy on arrival direction for different latitudes. The arrival direction at Saturn's surface in local horizon coordinates can be described by two angles: azimuth (Az, from 0 • 0 to 360 • ) and zenith (Zen, from 0 • to 90 • ). Therefore for the projection to the plane x = cos(Az)sin(Zen) points indicate higher access energy for this particular arrival direction for the current latitude.

Access Energies to the Saturn's Magnetosphere

We used the same approach to evaluate the GCR access to the equatorial plane of the magnetosphere, which can uncover the intensity of the GCR interaction with the Saturnian rings, neutral gas cloud and moons. One may also use these results to understand how easily ions from ICME events may populate the magnetosphere [Roussos et al., 2018b]. The trajectory tracing was performed from points located at distances from 1.05R S (Saturn radii) up to 20R S from Saturn towards the Sun in the equatorial plane. For every point 211 arrival directions were evaluated. Figure 7 S5 in the Supporting Information.

GCR Spectra and Integral Flux

The fractional distribution allows reconstruction of the GCR spectra at Saturn. Due to lack of detailed GCR measurements on Saturn we used, as a parent (before entering into the magnetosphere) GCR spectrum, the estimation of a general GCR flux on that distance from the Sun. For that the CREME (Cosmic Ray Effects on Micro-Electronics code) data for general GCR spectra in the vicinity of the Earth [START_REF] Tylka | CREME96: A Revision of the Cosmic Ray Effects on Micro-Electronics Code[END_REF][START_REF] Weller | Monte carlo simulation of single event effects[END_REF][START_REF] Mendenhall | A probability-conserving cross-section biasing mechanism for variance reduction in monte carlo particle transport calculations[END_REF] were multiplied by a factor of 1.425 in order to take into account the radial gradient of 5% per AU [START_REF] Mcdonald | A comparative study of cosmic ray radial and latitudinal gradients in the inner and outer heliosphere[END_REF]. By applying the GCR fraction distribution, one can model the GCR spectra to the particular location on the planet or in the magnetosphere. Upper left panel of Figure 8 shows the initial (parent) GCR spectrum and the obtained modeled spectra, averaged along every 5 • of latitudes. Upper right panel demonstrates the modeled GCR spectra for radial distances up to 20 R S from the planet. For the evaluation of the CRAND production rate from the Main Rings, the fluxes corresponding to the radial distances of the rings are most important, therefore we marked the spectra at the distances of the D-ring (innermost ring) and the F-ring (the outermost ring). These spectra can be used directly as an input for the evaluation of the CRAND production rate, noting however that they do not include effects from GCR multiple ring crossings. These spectra can be found in the Support Information. c 2018 American Geophysical Union. All Rights Reserved.

Using the modeled GCR spectra it might be interesting to calculate the GCR integrated flux, which is shown on the lower left panel of Figure 8 for the atmosphere of Saturn and on the lower right panel for the equatorial plane of the magnetosphere. These results based on simulations using the Khurana model for Preequinox and Equinox. The decrease in the integrated flux at the atmosphere towards the equatorial zone is a direct consequence of an increase in the access energy at the corresponding latitudes, demonstrated in Figure 2. Here the differences in the two flux levels for different seasons arise from differences in initial GCR fluxes for these particular solar cycle phases.

On Figure 8 it is seen that the integral GCR flux to the rings and atmosphere below ∼ 70 • is independent of solar cycle phases, which supports the [START_REF] Kollmann | The evolution of saturn's radiation belts modulated by changes in radial diffusion[END_REF] results saying that variations in radiation belts have to come from other processes, e.g.

diffusion.

As it was reported by Roussos et al. [2018b] the background GCR noise in the various channels of the Cassini MIMI/LEMMS instrument decreased notably during the close approach to the planet. On panel D) of Figure 8 we compared the LEMMS E7 count rates from Saturn Orbit Insertion (SOI) normalized to integral flux at 12R S , with the integrated above 300 MeV modeled flux profile for Preequinox. The E7 data are shown with red diamonds. The point at L = 1 (orange diamond) is the "1 count upper limit" inside L = 1.03, assuming that 1 count would have been counted in the total 600 sec that Cassini spent inward of that region. In reality, during the Proximal orbits channel E7 measured 0 counts, consistent with our prediction of even lower GCR fluxes than the 1 c 2018 American Geophysical Union. All Rights Reserved. count upper limit. The drop in the GCR noise in LEMMS measurements starts developing at about 6 Rs, as predicted in the simulations, and is proportional to our modeled and integrated GCR flux dropout in the close vicinity of Saturn, which supports the idea of GCR origin of this background signal.

Summary

The MeV proton radiation belts of Saturn are proved to be isolated from the outer magnetosphere, and the source of these high energy protons should be related to the GCRs. Therefore a realistic flux of GCRs in the Saturnian system is required and, in this letter, we propose a numerical solution for the calculation of the GCR access energies to Saturnian magnetosphere and demonstrate its advantage over the analytical approach.

While to a first order, the Störmer theory predicts well the access of GCRs at the rings and the low latitude atmosphere, depending on the accuracy of the problem studied, this approximation may be less or more appropriate. Since, for instance, LEMMS measurements indicate that CRAND is a very well defined and quasi-stable process at Saturn, the results from particle tracing will be important for reducing the small but non-negligible errors introduced by the analytical theory, if we want to reconstruct the CRAND spectra with good precision.

As a result we obtained the GCR access energies to the atmosphere of the planet and its rings, using two different magnetospheric models: a Dipole model and the Khurana model. We demonstrate that numerical solution provides more detailed information about c 2018 American Geophysical Union. All Rights Reserved.

the GCR access energies, and is more preferable for future usage than the Störmer vertical cutoff as a mean value.

We analysed the variations of the access energy in many different aspects and calculated the GCR spectra and GCR integrated flux to the atmosphere of Saturn as a function of latitude, and to the equatorial distance of the Saturnian magnetosphere as a function of distance from the planet.

Having determined the incoming GCR flux, it is possible now to perform a simulation of the cascades of particles resulting from GCR penetration through the rings and GCR impact on the Saturn's atmosphere, and to calculate the CRAND input to the ion populations of the radiation belts.

Figure 2

 2 Figure 2 reports the GCR access energy mapped onto Saturn. Here the first row shows

Figure 4

 4 Figure 4 shows the comparison of access energies obtained by the analytical Störmer

  3.1.2. Impact of the Main Ringsc 2018 American Geophysical Union. All Rights Reserved.

  and y = sin(Az)sin(Zen). For instance, for North those angles would be Zen = 90 • and Az = 0 • . Here each point corresponds to one of 221 arrival directions. More reddish c 2018 American Geophysical Union. All Rights Reserved.

  reports the modeling results on the basis of the Dipole model (upper panel) and the Khurana model for Preequinox. The different contours represent the fractions of allowed arrival directions as a function of energy and distance from the planet. The grey zone indicates the location of the Main Rings and is zoomed in separately on Figure S2 in Support Information. Also these values are available in the Table S4 in the Supporting Information. It should be noted, that some of the arrival directions produce extremely high access energy values in the close vicinity to the planet, since we evaluated them isotropically distributed in a whole volume of incidence angles and about the half of arrival directions very close to the planet are simply forbidden because of the presence of the planet. The biggest difference in the two plots at Figure 7 can be observed at distances outside 7 R S , since the dipole model can well describe the equatorial magnetic field only close to Saturn. Here values for GCR access energy on the base of the Khurana model are about c 2018 American Geophysical Union. All Rights Reserved. twice lower for the same arrival directions. The values of access energies for various fractions of the arriving directions calculated on the base of the Khurana model demonstrated on bottom panel of Figure 7 can be found in Table

Figure 1 .

 1 Figure 1. Simulated backwards in time trajectories of five protons of different energies. Only

Figure 2 .

 2 Figure 2. GCR access energy mapped to Saturn using the Dipole model (left column) and

Figure 3 .

 3 Figure 3. GCR access energy to Saturn: deviation of minimum access energy along latitudes,

Figure 4 .

 4 Figure 4. GCR access energy in fractions of arrival directions as a function of energy and latitude, in comparison with Störmer cutoff energies: Westward (point-dash indigo line), vertical (dashed navy line) and Eastward (solid blue line). Contours represent the different fractions of allowed arrival directions, obtained by particle tracing, the solid red line highlights the absolute minimum access energy, the solid green line -energy needed for 50% of arrival directions.

Figure 5 .

 5 Figure 5. Changes in the GCR access energy if rings are not transparent for particles. Upper panel:GCR access energy in fractions of arrival directions as a function of energy and latitude calculated assuming rings are transparent for GCRs (solid coloured zone) and contours of the same colour indicate, how the access energy for corresponding fraction of arrival directions will increase, if we assume GCR lose 50% of its energy in every passage through the rings. Bottom panel: percentage of the GCR access energy increase for corresponding fractions of arrival directions if the rings assumed not transparent as a function of latitude.

Figure 6 .

 6 Figure 6. Minimum access energy per arrival direction for different latitudes. X-axis is the cosine and y-axis is the sine of the arrival direction. West is on the left and East on the right.

Figure 7 .

 7 Figure 7. GCR proton access energy in fractions of arrival directions as a function of energy and distance from the planet (Rs -is the Saturn radii) in the equatorial plane: comparison between the Dipole model (upper panel) and Khurana model for Preequinox (lower panel). The location of the Main Rings is indicated by the grey zone. Different contours represent fractions of arrival directions as a function of access energy, a pink zone indicates no access of particles with such energy to the corresponding point and a white zone indicates the full access to the point from all arrival directions.

Figure 8 .

 8 Figure 8. Panel (A): The estimated GCR spectra in the vicinity of Saturn and modeled spectra

Acknowledgments. The authors like to thank K. Khurana for the magnetospheric model code and L. Regoli for the help with a particle tracing code. Funding was provided by CNES (AK, ID), German BMWi through the German Space Agency DLR under contracts 50OH0103, 50OH0801, 50OH0802, 50OH1101, 50OH1502 and by the Max Planck Society (AK, IR, NK). PK was partially supported by the NASA Office of Space Science under task order 003 of contract NAS5-97271 between NASA/GSFC and JHU. The Cassini/MIMI data are available through NASA's Planetary Data System at https://pdsppi.igpp.ucla.edu/mission/Cassini-Huygens/CO/MIMI. c 2018 American Geophysical Union. All Rights Reserved.

In the Supporting Information we provide the look-up tables with GCR access energies to the planet and to the equatorial plane of the magnetosphere, calculated on the base of the Khurana model, the modeled GCR spectra as a function of latitude and as a function of distance from Saturn. Also we provide some details about the Störmer cutoff rigidities calculation and additional figures demonstrating changes in the minimum access energy along latitudes and showing GCR access energies to Saturnian rings.