A STING-CASM-GABARAP Pathway Activates LRRK2 at Lysosomes

Mutations that increase LRRK2 kinase activity have been linked to Parkinson’s disease and Crohn’s disease. LRRK2 is also activated by lysosome damage evoked by chemical and pathogenic stimuli. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through Stimulator of Interferon Genes (STING) as an upstream activator of LRRK2. This LRRK2 activation occurs via the Conjugation of ATG8 to Single Membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM mediates the lipidation of multiple ATG8 protein family members, LRRK2 lysosome recruitment and kinase activation is highly dependent on an interaction with the GABARAP member of this family. Collectively these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation this pathway may be of relevance in both Parkinson’s and Crohn’s diseases.

LRRK2 mutations cause dominantly inherited familial Parkinson's disease and common variants contribute to sporadic Parkinson's disease risk (Taymans et al., 2023).It is also well established that multiple familial Parkinson's disease LRRK2 missense mutations result in an increase in LRRK2 kinase activity (Kalogeropulou et al., 2022).Conversely, LRRK2 variants associated with decreased risk for Parkinson's and Crohn's disease exhibit reduced kinase activity (Wang et al., 2021).Although mutations that increase LRRK2 activity are only found in a subset of Parkinson's disease patients, this strong link between LRRK activity and Parkinson's disease suggests that LRRK2 functions in a disease relevant pathway and that other factors that modulate LRRK2 kinase activity could have disease relevant consequences.This has stimulated great interest in mechanisms that regulate LRRK2 activity.
Recent studies have converged on endosomes and lysosomes as key intracellular sites of LRRK2 activation.LRRK2 is recruited to lysosomes in response to chemical treatments that impair lysosome function and/or that cause lysosome membrane damage, and this is accompanied by an increase in the kinase activity of LRRK2 (Bonet-Ponce et al., 2020;Eguchi et al., 2018;Herbst et al., 2020;Kalogeropulou et al., 2020;Kluss et al., 2022;Radulovic and Stenmark, 2020).LRRK2 is also activated when pathogens such as Mycobacterium tuberculosis, Listeria monocytogenes or Candida albicans rupture lysosomes and LRRK2 also participates in a host defense mechanism that is targeted to Salmonella containing vacuoles (Herbst et al., 2020;Lian et al., 2023).The dynamic recruitment of LRRK2 to damaged membranes requires mechanisms whereby endolysosome status is sensed and communicated to LRRK2.Understanding the basis for this regulation has implications for understanding both the fundamental cellular functions of LRRK2 and potentially for how LRRK2 is aberrantly activated in disease contexts.
Severely damaged lysosomes are cleared by an autophagic process known as lysophagy wherein they are engulfed and delivered to healthy lysosomes for disposal (Maejima et al., 2013).However, cells can also detect and repair lysosome damage before it reaches the point where lysophagy is required (Bohannon and Hanson, 2020).LRRK2 activation at damaged lysosomes has been proposed to promote the restoration of lysosome integrity by phosphorylating specific Rab GTPases that in turn recruit effectors involved in membrane repair (Bonet-Ponce et al., 2020;Herbst et al., 2020;Steger et al., 2016).
Rab GTPases also act upstream of LRRK2 to promote its membrane recruitment and activation (Dhekne et al., 2023;Eguchi et al., 2018;Gomez et al., 2019;Lian et al., 2023;Pfeffer, 2022;Purlyte et al., 2018;Unapanta et al., 2023;Vides et al., 2022;Wang et al., 2023a).However, although Rab GTPases promote LRRK2 kinase activity in specific contexts, recent knockout mouse studies revealed the persistence of partial or even full LRRK2 kinase activity in multiple tissues even after depletion of key Rab proteins implicated in LRRK2 activation (Dhekne et al., 2023;Kalogeropulou et al., 2020).These results suggest the existence of additional factors that contribute to LRRK2 activation.Several Parkinson's disease risk genes, including PINK1, Parkin and VPS13C have been linked to the Stimulator of Interferon Genes (STING) signaling pathway that functions as part of an innate immune response downstream of the sensing of cytoplasmic DNA by cyclic GMP-AMP synthase (cGAS) (Diner et al., 2013;Hancock-Cerutti et al., 2022;Moehlman et al., 2023;Sliter et al., 2018;Wu et al., 2013).Motivated by this convergence of Parkinson's disease genes and the fact that activated STING traffics through cellular compartments (Golgi, Endosomes and Lysosomes) where LRRK2 has been proposed to function, we investigated the relationship between LRRK2 and STING signaling (Erb and Moore, 2020).This led to the discovery of a new pathway wherein STING signals through a process known as Conjugation of ATG8 to Single Membranes (CASM) to activate LRRK2 at lysosomes (Durgan and Florey, 2022).Although CASM results in the lipidation of multiple ATG8 family members, we found that LRRK2 activation is dependent on the GABARAP member of the ATG8 family.This CASM-GABARAP dependent process of LRRK2 activation at lysosomes is required for LRRK2 activation in response to diverse stimuli that perturb the lysosome membrane.

STING is an endogenous activator of LRRK2
Given previously reported links between cGAS-STING dysregulation and Parkinson's disease-associated genes, we sought to test whether STING controls LRRK2 activity.
We also measured levels of Rab10 phosphorylation as a readout for changes in LRRK2 kinase activity (Kalogeropulou et al., 2022;Lis et al., 2018;Steger et al., 2016).Activation of STING by either agonist led to an increase in phosphorylation of Rab10 and the timing of DMXAA-dependent LRRK2 activation also paralleled STING activation (Fig. 1B-E; Fig. S1A).We also assessed Rab12 phosphorylation and found that cGAMP and DMXAA treatments both increased the phosphorylation of this additional LRRK2 substrate (Fig. S1B and C).The absence of Rab10 phosphorylation following STING activation in LRRK2 KO cells demonstrated the essential role for LRRK2 in Rab phosphorylation downstream of STING activation (Fig. 1D).To further test whether these effects were directly due to LRRK2 kinase activity, we acutely treated cells with the LRRK2 inhibitor MLi-2 and found that it also abolished the STING-mediated increase in Rab10 phosphorylation (Fig. 1F).
We also observed a lack of STING-mediated Rab phosphorylation in cells with a knockin of the inactive LRRK2 T1348N mutant (Ito et al., 2007)(Fig.S1E).Finally, LRRK2 mediated Rab10 phosphorylation following DMXAA treatment occurred with a concentration dependence that paralleled STING activation (Fig. S2A).Altogether, these data demonstrate that STING signaling initiates a response that results in activation of LRRK2 kinase activity.

STING promotes LRRK2 recruitment to lysosomes
Previous studies have shown that upon ligand binding, STING traffics from the endoplasmic reticulum to the Golgi and then to lysosomes where it is internalized and degraded (Balka et al., 2023;Chen et al., 2016;Gonugunta et al., 2017).Consistent with the known requirement for ER to Golgi trafficking as a first step in STING activation, we observed that disruption of such trafficking by treating cells with Brefeldin A prevented STING signaling, as was previously reported (Ishikawa et al., 2009) (Fig. S2B).We also found that Brefeldin A blocked LRRK2 activation downstream of STING (Fig. S2B).
Meanwhile, LRRK2 can be activated at lysosomes in response to various lysosome damaging stimuli (Bonet-Ponce et al., 2020;Eguchi et al., 2018;Herbst et al., 2020;Kalogeropulou et al., 2020).To test the role of lysosomes as sites of STING-dependent LRRK2 activation, we used a previously established method for magnetic isolation of lysosomes from control versus DMXAA stimulated cells that had been pre-loaded with superparamagnetic iron oxide nanoparticles (SPIONs) and found that LRRK2 robustly accumulates on lysosomes in response to STING activation (Amick et al., 2020;Hancock-Cerutti et al., 2022)(Fig.1G-I).

TBK1 and IKKe are not required for LRRK2 activation by STING
We next implemented a series of genetic perturbations to understand how STING signaling leads to LRRK2 activation.STING KO cells were defective in STING agonistmediated LRRK2 activation and this was rescued following re-expression of STING (Fig. 2A).Following agonist binding, STING recruits and activates the closely related TBK1 and IKKe kinases (Balka et al., 2020;Chen et al., 2016)(Fig.1A).Interestingly, TBK1 and IKKe were previously shown to phosphorylate LRRK2 which led us to speculate that this could be part of the pathway whereby STING activates LRRK2 (Dzamko et al., 2012).To test the requirement for TBK1 and IKKe in LRRK2 activation, we generated TBK1 KO, IKKe KO, and TBK1 + IKKe double KO RAW 264.7 cells and measured their ability to activate LRRK2 in response to STING agonist treatment.In each of these KO lines, STING still triggered LRRK2-mediated Rab10 phosphorylation to a degree that was statistically indistinguishable from WT cells (Fig. 2B and C).Furthermore, we still observed LRRK2-mediated Rab10 phosphorylation after rescuing the STING KO cells with a truncated form of STING (amino acids 1-339) that cannot bind and activate TBK1/IKKe (Gui et al., 2019)(Fig.2A).These results indicate that STING activates LRRK2 independent of signaling through TBK1 and IKKe kinases.

STING activates LRRK2 via the CASM pathway
In addition to TBK1-IKKe activation, STING also independently activates CASM (also known as V-ATPase-ATG16L1-induced LC3B lipidation or VAIL) (Durgan and Florey, 2022;Fischer et al., 2020;Gui et al., 2019)(Fig.3A).Consistent with this, we observed that LRRK2 activation by STING ligands was also accompanied by LC3B and GABARAP lipidation (Fig. 3B).ATG16L1 is a scaffold protein that is required for this lipidation reaction (Durgan and Florey, 2022).We therefore generated ATG16L1 KO RAW 264.7 cells, tested their response to STING activation, and found that LRRK2-mediated Rab phosphorylation was abolished (Fig. 3C).This observation led us to focus more deeply on a potential requirement for CASM in LRRK2 activation.
The v-ATPase plays a critical role in the CASM pathway and strategies have been defined that target the v-ATPase to activate or inhibit CASM (Hooper et al., 2022)(Fig.3A).Saliphenylhalamide (Salip) activates CASM by inhibiting the v-ATPase and stabilizing assembly of the V1 and V0 v-ATPase subunits (Hooper et al., 2022;Xie et al., 2004).
Consistent with a role for CASM in activating LRRK2, Salip treatment increased LRRK2mediated Rab phosphorylation (Fig. 3D and E).In contrast, treatment of cells with folimycin, a v-ATPase inhibitor that binds to a different site than Salip and inhibits CASM (but not the induction of macroautophagy), blocked the activation of LRRK2 downstream of STING (Fig. 3F).Finally, we generated a cell line that stably expresses the Salmonella effector protein SopF which inhibits CASM by ADP-ribosylating the v-ATPase V0 subunit (Fischer et al., 2020;Hooper et al., 2022;Xu et al., 2019) and found that this abolished STING-dependent LRRK2 activation (Fig. 3G and H).These tests distinguished between ATG8 family lipidation associated with macroautophagy versus CASM and collectively support an essential role for CASM in mediating LRRK2 activation in response to multiple stimuli.

Multiple chemicals activate LRRK2 via CASM
Multiple chemical stimuli that perturb lysosomes have previously been separately shown to activate LRRK2 and to activate CASM.This includes L-leucyl-L-leucine methyl ester (LLOME), a chemical that selectively damages lysosome membranes following its processing by cathepsin C (Bonet-Ponce et al., 2020;Durgan and Florey, 2022;Kalogeropulou et al., 2020;Thiele and Lipsky, 1990).We confirmed that LLOME activates LRRK2 and established that LLOME also strongly triggers endogenous LRRK2 accumulation on lysosomes in RAW 264.7 cells (Fig. 4A-C).ML SA1 is an activator of the TRPML1 lysosomal cation channel and a known activator of CASM but was not previously shown to activate LRRK2 (Durgan and Florey, 2022;Shen et al., 2012).We found that ML SA1 treatment also activated LRRK2 and that this effect required ATG16L1 (Fig. 4D-F).Nigericin, a proton-potassium ionophore that disrupts ion balances across cellular membranes, is also known to activate both CASM and LRRK2 (Herbst et al., 2020;Hooper et al., 2022;Ito et al., 2016;Jacquin et al., 2017;Kalogeropulou et al., 2020).We confirmed this robust relationship between Nigericin and LRRK2 kinase activity (Fig. 4G).
Importantly, the KO of ATG16L1 blocked the ability of Nigericin to activate LRRK2 (Fig. 4H and I).Collectively, these results establish a role for CASM in the activation of LRRK2 by STING as well as by multiple chemical stimuli that perturb lysosomes.

GABARAP is required for CASM-dependent LRRK2 activation
In order define specific cellular machinery that is required for CASM-mediated LRRK2 activation, we performed a targeted siRNA screen focusing on the ATG8-related proteins that are lipidated as part of the CASM pathway.As expected, LRRK2 activity (measured by Rab10 phosphorylation) was reduced by the knockdown of LRRK2 and Rab10 itself as well as by knockdown of Rab12, a recently identified regulator of LRRK2 activity (Dhekne et al., 2023;Wang et al., 2023a)(Fig.5A).Consistent with a requirement for lipidation of Atg8 family members, depletion of ATG3 and ATG16L1 also reduced the phosphorylation of Rab10 by LRRK2 (Fig. 5A).Interestingly, out of the ATG8 family members annotated in the mouse genome (LC3A, LC3B, Gabarap, GabarapL1, and GabarapL2) only GABARAP was identified as critical for CASM-mediated LRRK2 activation (Fig. 5A).This requirement for GABARAP in LRRK2 activation was independently validated through assays in genome edited GABARAP KO cells where neither DMXAA nor ML SA1 nor Nigericin were able to activate LRRK2 (Fig. 5B-E).
Analysis of SPION purified lysosomes furthermore revealed that GABARAP is enriched on lysosomes following STING activation and is required for LRRK2 recruitment to lysosomes (Fig. 5 F and G).Consistent with a direct role for GABARAP in promoting LRRK2 recruitment to lysosomes, HALO-tagged LRRK2 was present in HA-GABARAP immunoprecipitations and this interaction increased in response to STING activation (Fig. 5H and I). Figure S2C shows that the HALO-tagged LRRK2 is functional based on ability to support both basal and DMXAA-stimulated Rab10 phosphorylation when stably expressed in LRRK2 KO cells.Altogether this data defines a major role for GABARAP in CASM-dependent LRRK2 activation at lysosomes.

Discussion
Collectively, our data defines a pathway for the activation of LRRK2 at lysosomes wherein STING signaling and stimuli that perturb lysosome integrity converge on CASM-mediated GABARAP lipidation to promote LRRK2 lysosome recruitment and activation of LRRK2 kinase activity.Although there has been great progress in elucidating components of the CASM pathway, the physiological functions downstream of CASM have remained elusive (Durgan and Florey, 2022;Hooper et al., 2022;Jacquin et al., 2017;Ulferts et al., 2021;Xu et al., 2019).Our identification of CASM-mediated, GABARAP-dependent, LRRK2 activation provides an important new insight into a functional output of this pathway.
Given that GABARAP lipidation onto intracellular membranes occurs during both conventional macroautophagy and CASM, but only CASM activates LRRK2, an additional factor (or factors) must contribute to the specificity of LRRK2 activation.Candidate contributors to a coincidence detection mechanism to ensure spatial control of LRRK2 activation at GABARAP-positive lysosomes include Rab GTPase proteins that have been demonstrated to mediate LRRK2 membrane recruitment and kinase activation (Pfeffer, 2022).These include Rab10, Rab12 and Rab29 (Dhekne et al., 2023;Purlyte et al., 2018;Vides et al., 2022;Wang et al., 2023a;Zhu et al., 2023).It remains to be determined whether GABARAP acts in parallel with any of these Rabs to ensure that LRRK2 is recruited to and activated at the correct intracellular membranes.It was also very recently discovered that LRRK2 can assemble into helical polymers on membranes that are enriched in acidic lipid head groups (Wang et al., 2023b).Such LRRK2-membrane interactions could also act together with GABARAP to specify sites of LRRK2 activation.
Although our data is consistent a model (Fig. S3) wherein lipidated GABARAP directly interacts with LRRK2 to help recruit it to lysosomal membranes, our coimmunoprecipitation data does not rule out the possibility that additional proteins are involved in this regulated interaction.Future studies should focus on reconstitution of this interaction with purified proteins and mapping the regions on LRRK2 and GABARAP that mediate their interaction selectively under conditions when GABARAP has been lipidated downstream of CASM.
The importance of LRRK2 kinase activity for Parkinson's disease is well supported by the genetics of both sporadic and familial forms of the disease (Bandres-Ciga et al., 2020;Taylor and Alessi, 2020).The identification of the STING pathway as a robust endogenous activator of LRRK2 suggests that upstream stimuli that activate cGAS-STING may also contribute to Parkinson's disease risk.This includes activation of cGAS in response to release of endogenous DNA from damaged mitochondria and disruptions to nuclear integrity as well as exogenous DNA from pathogen infections (Gulen et al., 2023;Lan et al., 2019;Motwani et al., 2019).Recent findings suggest that in the aging brain such effects are likely to be of particular importance in microglia (Gulen et al., 2023).
However, failure of neuronal lysosomes to efficiently degrade nuclear and mitochondrial DNA can also result in STING activation following DNA leakage into the cytoplasm (Lan et al., 2014;Van Acker et al., 2023).
The role for CASM in LRRK2 activation provides a unifying mechanism that explains how diverse stimuli that perturb lysosome integrity and/or ion balance result in LRRK2 activation.In fact, the recent discovery that STING itself is a proton channel suggests that it may activate CASM by disrupting endo-lysosomal pH (Liu et al., 2023).The identification of CASM as a convergence point for multiple stimuli that activate LRRK2 provides a foundation for further investigation of how lysosome dysfunction contributes to Parkinson's disease.CASM-dependent LRRK2 activation may also be relevant for Crohn's disease where human genetics has established roles for both ATG16L1 and LRRK2 and our data now places these genes into a common cellular pathway (Hampe et al., 2007;Van Limbergen et al., 2009).
In conclusion, we have defined a STING-CASM-GABARAP pathway for LRRK2 activation at lysosomes (Fig. S3).This pathway more broadly helps to explain how multiple additional lysosome perturbations activate LRRK2.By establishing a site and machinery for LRRK2 activation, these results provide a new avenue for investigating the regulation of LRRK2 activity in the context of normal physiology as well as human diseases such as Parkinson's disease, Crohn's disease and microbial pathogenesis.

Stable Cell Line Generation.
A piggybac transposase strategy was used for stable cell line generation.Briefly, 2.5 x 10 5 cells were plated per well in a 6-well dish.The following day, cells were transfected using Lipofectamine 2000 (Invitrogen, 11668019) or FuGene HD (Promega, E2311), as per the manufacturer's protocols, using a 1:2 ratio of Piggybac transposon plasmid:geneof-interest plasmid (total 1 ug DNA).After 48 hours, the media was changed.After 72 hours, puromycin (Gibco A11138-03) or blasticidin (Invivogen ant-bl) were used at concentrations of 3.5 µg/mL and 10 µg/mL, respectively.After 48-72 hours, media containing puromycin and blasticidin was washed out with fresh media.After cells had recovered from selection, single cells were plated into 96-well dishes to obtain clonal cell lines.Cell lines were confirmed via immunoblotting.Plasmid information is summarized in Supplemental Table 3.

Genome edited cell lines.
STING KO, TBK1 KO, IKKe KO, TBK1 KO, IKKee KO, ATG16L1 KO, and GABARAP KO cells were created using the Synthego CRISPR Gene Knockout V2 Mouse Kits for each respective target.Briefly, 2.5 x 10 5 cells were plated into a 6-well dish.The following day, cells were transfected with ribonucleoprotein particles using Lipofectamine CRISPRiMAX (ThermoFisher Scientific, CMAX00003), gene specific sgRNA and recombinant Cas9 (Synthego).After 48 hours, the media was changed.After 72 hours, single cells were plated into 96-well dishes to obtain clonal populations.After expansion of clonal populations, KO clones were identified by immunoblotting.

Generation and use of Superparamagnetic Iron Oxide Nanoparticles (SPIONs).
SPIONS were generated based on an established protocol (Rodriguez-Paris et al., 1993).
Briefly, 10 mL of 1.2M FeCl2 (Sigma-Aldrich, 220299) and 10 mL of 1.8M FeCl3 (Sigma-Aldrich, 157740) were combined slowly by stirring.Then, 10 mL of 30% NH4OH (Sigma-Aldrich, 320145) was slowly added while stirring for 5 minutes.The resulting particles were then washed with 100 mL of water three times.The particles were resuspended in 80 mL of 0.3M HCl (J.T. Baker, 9535) and stirred for 30 minutes.Then, 4g of dextran (Sigma-Aldrich D1662) was added and stirred for 30 minutes.The particles were transferred into dialysis tubing and dialyzed with ddH2O for at least 2 days with multiple water changes.The particles were centrifuged at 26,900g for 30 minutes to remove large aggregates and stored at 4°C.An established SPION protocol for mouse macrophages was followed for lysosome purification (Rofe and Pryor, 2016) with the exception of using a dounce homogenizer for cell lysis.

Immunoprecipitation.
For cell lysis to generate whole cell lysates, the protocol in immunoblotting was followed with few alterations.One 80% confluent 15-cm plate was used per sample.RAW 264.7 LRRK2 KO cells rescued with stably expressed HALO-human LRRK2 (but no HA-GABARAP) were used as a negative control for non-specific LRRK2 interactions with the anti-HA beads.RAW 264.7 LRRK2 KO cells rescued with HALO-LRRK2 and stably expressing HA-tagged human GABARAP were used to test LRRK2-GABARAP interaction.Cells were washed 2X with PBS, scraped in ice-cold lysis buffer, and centrifuged at 14,000 RPM (4°C) for 8 minutes.Protein concentrations were measured using Coomassie Plus Protein Assay Reagent (ThermoFisher Scientific, 23236) as per manufacturer's protocol.Lysates were then immunoprecipitated using a mix of 15 µl anti-HA beads (Thermo Fisher Scientific, 88837) that were pre-washed 3X with lysis buffer.
The same amount of protein was used for each sample.Where needed, samples were supplemented with lysis buffer to maintain the same protein concentration and volume.
Lysates were incubated with beads rotating end-over-end for 1 hour at 4°C.Beads were washed 3X with 0.1% TBST and 1X with mqH20, as per the manufacturer's protocol.
Proteins were eluted by incubating beads with Laemmli Buffer and boiling at 95°C for 3 minutes.The sample was then transferred to another microcentrifuge tube and supplemented with 6.187% fresh B-mercaptoethanol (Sigma-Aldrich, M3148).Samples were then subjected to electrophoresis, immunoblotting, and chemiluminescence as previously described above.
siRNA knockdowns were accomplished using Horizon Biosciences siGENOME pooled siRNAs.Briefly, 2.5 x 10 5 RAW 264.7 cells were plated per well in a 6-well dish.The following day, cells were transfected using Lipofectamine RNAiMAX (Invitrogen, 2448190) as per the manufacturer's protocols using 100 nM siRNA pool.After 48 hours, cells were treated with drugs or lysed and subjected to immunoblotting.siRNAs used in this study are summarized in Supplemental Table 6.

Statistical analysis.
Statistical analysis was performed with Prism 10 software, with specific details about the statistical tests conducted, the number of independent experiments, and P values provided in the corresponding figure legends.

Figure 2 :
Figure 2: TBK1 and IKKe are not required for LRRK2 activation by STING.(A)

Figure 3 :
Figure 3: CASM is required for LRRK2 activation by STING.(A) Schematic diagram

Figure
Figure S1 provides additional evidence in support of STING-dependent activation of

Figure
Figure S2 demonstrates concentration dependence of LRRK2 activation, Brefeldin A

Figure
FigureS3contains a schematic diagram that summarizes a STING-CASM- Figure 1 A B