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Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

['Adamantios Mamais', 'Cell Biology', 'Gene Expression Section', 'National Institute On Aging', 'National Institutes Of Health', 'Maryland', 'United States Of America', 'Department Of Neurology', 'University Of Florida', 'Gainesville']

Date: 2022-01 

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

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Although these data indicate that Rab8a regulates exocytic and recycling membrane trafficking at the ERC, how phosphorylation of Rab8a affects downstream biology in PD-relevant cell types remains unclear. Here, we identify a novel role of LRRK2 in mediating Rab8a-dependent TfR recycling and show in vivo effects of LRRK2 mutation at the endogenous level on iron homeostasis in microglia. Expression of mutant LRRK2 induces sequestration of Rab8a to lysosomes and dysregulates transferrin recycling in a Rab8a-dependent manner. In induced pluripotent stem cell (iPSC)-derived microglia carrying the G2019S LRRK2 mutation, transferrin is mistrafficked to lysosomes in proinflammatory conditions, and this is accompanied by subtle dysregulation of transferrin clearance compared to wild-type (WT) controls. Finally, we show that G2019S LRRK2 knock-in mice exhibit increased iron accumulation within microglia in the striatum in response to neuroinflammation compared to WT controls. These data support a role of LRRK2 in modulating normal iron uptake and storage in response to proinflammatory stimuli in microglia.

Rab8a is involved in a number of cellular functions including cell morphogenesis, neuronal differentiation, ciliogenesis, and membrane trafficking [ 20 – 22 ]. Rab8a regulates recycling of internalized receptors and membrane trafficking to the endocytic recycling compartment (ERC) [ 21 , 23 ]. Rab8a has been shown to interact directly with the transferrin receptor (TfR) and regulate polarized TfR recycling to cell protrusions [ 24 ] and transfer between cells through tunneling nanotubes [ 25 ]. Furthermore, Rab8a-depleted cells fail to deliver internalized TfR to the ERC [ 24 ]. Rab8a activity is controlled by its association with Rabin8, a guanine exchange factor [ 26 ]. Phosphorylation of Rab8a by LRRK2 inhibits its association with Rabin8 and thereby modulates Rab8a activity [ 8 ]. The G2019S LRRK2 mutation enhances Rab8a phosphorylation, mediating defects on EGFR recycling and endolysosomal transport [ 27 ]. Recently, mutations in LRRK2 were shown to mediate centrosomal deficits through Rab8a signaling [ 28 – 30 ], while ciliogenesis has been placed downstream of LRRK2 activity in different models [ 31 ].

A specific role of LRRK2 in vesicular trafficking has been nominated in recent years, and a number of studies have highlighted how downstream signaling pathways are affected by PD-linked mutations. LRRK2 can phosphorylate a subset of Rab GTPases, including Rab8a, Rab10, and Rab29, at a conserved motif, and this phosphorylation regulates Rab activity and association with effector molecules [ 8 , 9 ]. Rab GTPases control the spatiotemporal regulation of vesicle traffic and are involved in vesicle sorting, motility, and fusion [ 10 ]. Different Rab GTPases exhibit selectivity for different cellular compartments, thus conferring membrane identity that governs secretory and endocytic pathways [ 11 ]. For example, LRRK2 associates with Rab29, a Rab GTPase that is involved in vesicular transport and protein sorting [ 12 , 13 ] and a candidate risk gene for sporadic PD [ 14 ]. Rab29 can mediate recruitment of LRRK2 to the trans-Golgi network, inducing LRRK2 activity and membrane association at least under conditions of overexpression [ 12 , 15 , 16 ]. LRRK2 can phosphorylate Rab8a and Rab10, which may have downstream effects on the endolysosomal system. We, and others, have reported phosphorylation-dependent recruitment of Rab10 onto damaged lysosomes by LRRK2, in a process that orchestrates lysosomal homeostasis [ 17 – 19 ].

Missense mutations on the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson disease (PD), while common genetic variants identified by genome-wide association studies have been linked to idiopathic PD and inflammatory diseases [ 1 – 3 ]. LRRK2 is a multidomain enzyme, and PD-linked mutations are localized to its enzymatic domains, namely the kinase domain and Ras of complex proteins and C-terminal of ROC (ROC-COR) domains. LRRK2 has been linked to a number of cellular pathways including autophagy, lysosomal processing, inflammation, and vesicular trafficking [ 4 ]. LRRK2 is expressed in immune cells, and prior observations suggest a role of LRRK2 in microglial activation and an effect of PD-linked mutations on cytokine release and inflammation [ 5 – 7 ]. The kinase activity of LRRK2 is thought to drive disease pathology and thus is being targeted pharmacologically as a possible PD therapy [ 4 ].

Results

LRRK2-mediated T72 Rab8a phosphorylation blocks interaction with Rabin8 but not MICAL-L1 The above data suggest that Rab8a can move from its normal location at the ERC to the lysosome after expression of LRRK2 mutations. Given that all LRRK2 mutations are proposed to increase Rab phosphorylation, at T72 for Rab8a [8], we reasoned that the mechanism of relocalization might be related to this phosphorylation event. The pT72 Rab8a antibody that was available during this study was not specific to Rab8a and cross-reacted with Rab3A, Rab10, Rab35, and Rab43, which have a high degree of sequence conservation (for datasheet, refer to ab230260; Abcam). Therefore, using a tagged version of Rab8a, we found that exogenous expression of R1441C, Y1699C, G2019S, or I2020T LRRK2 induced a significant increase in Rab8a phosphorylation at T72 compared to WT or the kinase-dead variant K1906M, and this was blocked by MLi-2 treatment (Fig 3A and 3B). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Rab8a phosphorylation retains interaction with MICAL-L1 that is corecruited in mutant LRRK2 expressing cells. (A) HEK293T cells expressing control (GUS), FLAG WT, or mutant LRRK2 variants were treated with MLi-2 prior to lysis and analysis by western blotting for pS1292 and total LRRK2, as well as pT72 and total Rab8a. (B) Quantification of pT72 Rab8a levels normalized to total Rab8a (B, two-way ANOVA; N = 3 independent experiments; treatment: P < 0.0001, F (1, 28) = 1473, genotype: P < 0.0001, F (6, 28) = 157.1). (C) Structural modeling of T72 phosphorylation on Rab8a in association with Rabin8 or MICAL-L1. T72 is between 2.8–9.9 Å from the closest glutamate on Rabin8 and 6.7–8.4 Å from the closest phenylalanine on MICAL-L1. (D) HEK293T cells expressing FLAG WT, I2020T, or K1906M LRRK2 along with GFP Rab8a, GFP control, or FLAG-GUS control were lysed and proteins immunoprecipitated using GFP beads. Co-immunoprecipitated proteins were analyzed by western blotting probed for MICAL-L1 as well as pT72 and total Rab8a. (E) Cells expressing FLAG WT or I2020T LRRK2 were stained for endogenous Rab8a and MICAL-L1 and analyzed by confocal microscopy. The underlying data can be found in S1 Data. LRRK2, leucine-rich repeat kinase 2; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001480.g003 Having established that Rab8a phosphorylation at T72 is increased by all pathogenic LRRK2 mutations, we next investigated how this affects interactions with known Rab8a partners. We focused on the known activating GEF, Rabin8, and MICAL-L1, which is an established Rab8a-interacting partner [23] that localizes in tubular endosomes and is essential for efficient endocytic trafficking from the ERC to the plasma membrane [21]. Modeling the interaction between Rab8a and Rabin8, we predicted that the Threonine at position 72 on Rab8a resides 2.8 to 3.9 Å away from the neighboring Glutamate (E192) on Rabin8 (Fig 3C). Addition of a phosphate group on T72 will likely hinder the interaction with the negatively charged Glutamate, therefore destabilizing Rab8a–Rabin8 interaction. This prediction is consistent with work by Steger and colleagues who demonstrated biochemically that phosphorylation of Rab8a by LRRK2 can limit its activation by Rabin8 [8]. Threonine 72 of Rab8a is closest to a Phenylalanine (F647) on MICAL-L1 at a 6.7 to 8.4 Å distance, suggesting that phosphorylation at this site will not affect this interaction. To validate this prediction biochemically, we performed co-immunoprecipitation experiments by coexpressing Rab8a with WT, kinase-hyperactive I2020T, and kinase-dead K1906M LRRK2 in cells. In these conditions Rab8a was T72 hyperphosphorylated by I2020T LRRK2, and this modification did not hinder association with endogenous MICAL-L1 (Fig 3D). To investigate the retention of this association in the context of Rab8a recruitment to lysosomes, we stained for Rab8a and MICAL-L1 in cells expressing LRRK2. Cells expressing WT LRRK2 showed colocalization of Rab8a with MICAL-L1 in tubular membranes, while I2020T LRRK2 expression resulted in the recruitment of both proteins to LRRK2-positive structures consistent with the lysosomal phenotype described above (Fig 3E). This was also recapitulated by R1441C and G2019S LRRK2 that recruited endogenous MICAL-L1 and induced association of this trafficking protein with lysosomes (S4 Fig). Our data suggest that mutant LRRK2 can phosphorylate Rab8a inducing recruitment of both Rab8a and MICAL-L1 away from the ERC.

Mutant LRRK2 sequesters Rab8a leading to transferrin mistrafficking and accumulation of intracellular iron Given the above data showing that LRRK2 phosphorylation prevents binding of Rab8a to its effectors and redirects Rab8a and MICAL-L1 away from the ERC, we speculated that these events would then lead to a defect in Rab8a-mediated recycling. To evaluate this hypothesis, we examined the recycling of the transferrin receptor 1 (TfR), which is known to depend on Rab8a function [24]. Cells expressing LRRK2 genetic variants were stained for endogenous TfR and analyzed. TfR localized in distinct cytoplasmic vesicles in cells expressing WT LRRK2, whereas LRRK2 pathogenic mutants showed a clustered TfR localization that associated with LRRK2. The distribution of TfR vesicles in LRRK2-expressing cells was analyzed in Imaris (Bitplane, Zürich, Switzerland) where vesicles were rendered to spots throughout z-stack 3D reconstructed images and distances between them were measured (Fig 4A, right-hand panels). TfR vesicles were significantly more clustered in cells expressing LRRK2 mutants compared to WT LRRK2 expressing cells (Fig 4B and 4C). To test whether the coalescent TfR staining that we observe with mutant LRRK2 associates with lysosomes, we analyzed the fraction of TfR colocalizing with the late endosome/lysosome marker Lamp2 (S5 Fig). We found a significant increase in colocalization between TfR and Lamp2 in cells expressing R1441C (approximately 50% of whole-cell TfR staining) and G2019S LRRK2 (approximately 40%) compared to WT LRRK2 expressing cells (S5A and S3B Fig). Our data are consistent with prior reports of perinuclear localization of TfR in cells expressing kinase hyperactive mutant LRRK2 [31]. Our data suggest that TfR is recruited to damaged lysosomes by mutant LRRK2. To further test this hypothesis, we treated cells with LLOMe and analyzed intracellular localization by Airyscan (S5C Fig). We observe TfR recruitment in LRRK2-positive and Rab8a-positive membranes consistent with damaged lysosomes as previously described [18]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Mutant LRRK2 sequesters TfR and dysregulates transferrin recycling. (A) HEK293T cells exogenously expressing WT and mutant LRRK2 constructs were stained for TfR and visualized by superresolution microscopy. TfR vesicles were analyzed using the Imaris Spot Detection module, and the mean and minimum distances between spots were plotted (B, C). (N > 2,000 vesicles were counted in at least 20 cells per construct from 2 independent experiments, *P < 0.05, ****P < 0.0001, one-way ANOVA with Tukey post hoc, B: F(4, 13334) = 46.14, C: F (4, 13336) = 194.1). (D) HEK293FT cells expressing LRRK2 genetic variants were incubated with Alexa Fluor 568–conjugated transferrin, fixed at 20 minutes of incubation and stained for endogenous Rab8a and FLAG LRRK2 (D). (E) Uptake of Alexa Fluor 568–conjugated transferrin was monitored by high-content imaging, and transferrin levels per cell were plotted at different time points (E: T = 20 minutes, N = 3 technical replicates per construct (>800 cells/well) one-way ANOVA, Tukey post hoc, *P < 0.05, ***P = 0.0005, ****P = < 0.0001). (F) HEK293T cells exogenously expressing FLAG WT or G2019S LRRK2 constructs were incubated in DMEM supplemented with 1 μM MLi-2 or DMSO for 45 minutes prior to addition of Alexa Fluor 568–conjugated transferrin in the same media, and high-content imaging was used to monitor Tf uptake at different time points (F: N = 3 technical replicates per construct (>800 cells/well), two-way ANOVA, genotype: P < 0.05, F (2,30) = 3.575; Time: P < 0.0001, F (4, 30) = 44.96). (G) Cells were treated with MLi-2 as in (F), and incubated with Alexa Fluor–conjugated transferrin for 30 minutes, prior to changing to fresh media containing MLi-2 and monitoring transferrin release by high-content imaging (G; at T10: N = 3 technical replicates per construct (>800 cells/well), one-way ANOVA, Tukey post hoc, P < 0.05, F (2, 30) = 3.575). (H) HEK293T cells transiently expressing WT or I2020T LRRK2 were stained for Tf, Lamp2, and LRRK2 (FLAG) and analyzed by confocal microscopy. (I, J) Cells were transfected with Rab8a siRNA or scrambled sequence constructs (control) and 24 hours later were transfected with I2020T LRRK2 that was expressed overnight, before fixation and staining for Tf, Rab8a, and LRRK2 (FLAG). (N = 27 cells for NTC siRNA, N = 41 cells for Rab8a siRNA imaged across 2 independent experiments, two-tailed Mann–Whitney U test, ****P < 0.0001). Intracellular iron levels were analyzed by ICP-MS in cells stably expressing GFP WT or mutant LRRK2 constructs (K) (N = 4 confluent plates of cells per construct, two-way ANOVA with Tukey post hoc, *P = 0.018, F (3, 11) = 6.225). [SD bars are shown]. The underlying data can be found in S1 Data. ICP-MS, inductively coupled plasma mass spectrometry; LRRK2, leucine-rich repeat kinase 2; siRNA, small interfering RNA; TfR, transferrin receptor; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001480.g004 The observed altered TfR intracellular distribution in cells transiently expressing mutant LRRK2 prompted us to test whether LRRK2 functionally affected Rab8a-dependent Tf recycling. Cells expressing WT or mutant LRRK2 were preloaded with Alexa Fluor 568-Tf and visualized at T10 minutes of recycling in fresh media. Mutant LRRK2 expressing cells exhibited association of Tf with enlarged vesicles that were labeled with LRRK2 and Rab8a (Fig 4D). Tf vesicles were clustered in G2019S LRRK2 expressing cells compared to WT LRRK2, while this was rescued by treatment with MLi-2 prior to Tf loading (S5D Fig). In Tf uptake time course experiments, LRRK2 mutant expressing cells plateaued at higher Tf levels compared to WT at T15 to T20 minutes as monitored by high-throughput imaging, pointing to Tf accumulation driven by dysregulated recycling (Fig 4E). The modest increase in net transferrin levels detected in G2019S LRRK2 expressing cells was partly reversed in cells treated with MLi-2 (Fig 4F). We further evaluated a possible impairment of Tf recycling employing pulse-chase experiments to monitor Tf clearance. Tf recycling assays revealed higher initial transferrin levels at T0 following 45 minutes of loading in G2019S LRRK2 expressing cells compared to WT, while this difference was not evident at later time points (Fig 4G). In parallel experiments, we detected no significant difference in the total levels or surface-bound levels of TfR in cells expressing G2019S LRRK2 and WT LRRK2 (S5E Fig). These data cumulatively suggest a subtle dysregulation of transferrin clearance driven by transient expression of mutant LRRK2. The intracellular localization of internalized Tf in the context of LRRK2 mutations was explored further. In cells overexpressing I2020T LRRK2, we observed partial colocalization of internalized Tf with Lamp2 (Fig 4H), suggesting that excess Tf may associate with lysosomes. To test whether this phenotype was Rab8a dependent, the localization of internalized Tf in I2020T LRRK2 expressing cells was investigated following Rab8a siRNA knock-down. Knock-down of Rab8a decreased the proportion of Tf that colocalized with mutant LRRK2 relative to total internalized Tf (Fig 4I and 4J). Lastly, inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed elevated iron levels in cells stably expressing G2019S LRRK2 compared to WT LRRK2, while the pathogenic R1441C and Y1699C mutants do not show detectable alterations in iron levels, at least as measured by ICP-MS (Fig 4K). Collectively, these data suggest subtle dysregulation of Tf-mediated iron uptake and iron homeostasis by LRRK2 mutations as a consequence of altered Rab8a localization away from the ERC and toward lysosomes.

Endolysosomal and iron-binding gene expression in microglia is modulated by inflammation, in vitro and in vivo LRRK2 is expressed in microglia and has been linked to inflammation, cytokine release, and phagocytosis. Our data presented here show that LRRK2 associates with lysosomes and that LRRK2 mutations dysregulate Tf-dependent iron uptake mechanisms. Glial cells are known to be iron rich, and microglia activation state is integrally linked to brain iron content [33]. Proinflammatory stimuli are known to induce uptake of extracellular iron by microglia, and, conversely, the iron status of their environment can modulate their activation. Given the role of LRRK2 in inflammation, we asked whether endolysosomal processes and iron homeostasis might converge in proinflammatory conditions. We addressed this hypothesis using 2 genome-scale approaches. First, we mined our previously described RNA-Seq dataset [34] of primary microglia treated with lipopolysaccharide (LPS) or preformed α-synuclein fibrils in culture (Fig 5A). Analysis of the shared hits between the 2 treatments using gene ontology revealed enrichment for endosomal and lysosomal pathways (Fig 5B and 5C). Unsupervised hierarchical clustering of differential gene expression separated the controls from the 2 treatment groups (Fig 5D). These data demonstrate that genes involved in the endolysosomal system are consistently regulated by inflammatory stimuli. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Neuroinflammation remodels endolysosomal gene expression in microglia, in vitro and in vivo. (A) Outline of RNA-Seq experiment: Primary mouse microglia cultures were incubated with LPS or α-synuclein fibrils, and transcriptomic profiles were analyzed by RNA-Seq. (B) Common and distinct hits were detected between the LPS and PFF-treated groups. (C) Bubble plot showing GO:CC term enrichment in the shared hits from LPS and PFF-treated primary microglia highlights enrichment for endolysosomal processes. (D) Unsupervised hierarchical clustering shows that the treated groups cluster together suggesting common transcriptomic profiles. (E) Schematic of the in vivo experiment where LPS striatal injections were administered to WT and LRRK2 KO mice, followed by microglia isolation and single-cell RNA-Seq. (F) UMAP plot showing separation of the retrieved microglia in distinct groups of activation states. (G) Microglia from LPS-injected animals spanned the activation states, while PBS-injected animals gave predominantly resting microglia. (H) Lysosomal and endocytic mechanisms as well as cytoskeletal pathways are enriched in the cumulative data. (I) Heatmap showing clustering of microglia in distinct activation states highlighting increase in lysosomal and iron-related gene expression by inflammation. The underlying data have been deposited in NCBI’s GEO [75] and are accessible through GEO accession numbers GSE186483 and GSE186559. GEO, Gene Expression Omnibus; KO, knockout; LRRK2, leucine-rich repeat kinase 2; LPS, lipopolysaccharide; PFF, preformed fibril; scRNA-Seq, single-cell RNA-Seq; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001480.g005 We next asked whether the same regulation could be confirmed in vivo. Either WT or Lrrk2 knockout (KO) mice were given a single intrastriatal injection of LPS or vehicle control and 3 days later adult microglia were isolated using CD11b microbeads (Fig 5E). Between 3,000 and 5,000 cells were recovered from each of the 4 groups and analyzed using single-cell RNA-Seq (scRNA-Seq). We identified 5 distinct clusters of cells based on transcriptome similarity that represent resting microglia and 4 activation states (Fig 5F), with PBS-injected animals exhibiting predominantly resting microglia whereas the LPS-injected animals spanned the range of activation states (Fig 5G). Notably, we did not see strong differences between genotypes other than the KO animals tended to have fewer activated microglia, suggesting that Lrrk2 influences how microglia respond to proinflammatory stimuli [35]. Gene ontology showed enrichment for lysosomal and endocytic membrane processes (Fig 5H). These analyses identified genes involved in iron storage and uptake (FTH1; ferritin heavy chain), vesicular transport (SNAP23), acidification of vesicles (ATP6V1G1), and the late endosomal pathway (Rab7) (Fig 5I). These data demonstrate that processes of vesicular trafficking and iron homeostasis are modulated in a synchronized manner by inflammation. Given the importance of iron uptake in microglia activation, we hypothesized that LRRK2 may play a role in trafficking of TfR and that PD-linked mutations may affect iron uptake and accumulation in inflammatory conditions. Thus, we next sought to examine the effect of LRRK2 mutations on Tf recycling and iron uptake in proinflammatory conditions.

[END]

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