Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson’s disease-linked LRRK2 kinase
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Dhekne et al report a novel pathway for activation of the multi-domain LRRK2 protein kinase by Rab12 GTPase. LRRK2, which is mutated in Parkinson's Disease phosphorylates a subset of Rab proteins involved in intracellular trafficking, and Parkinson's disease-linked mutations increase this phosphorylation. This work adds an important new layer of understanding of this highly complex pathway by revealing that LRRK2's binding to Rab12 enhances its ability to phosphorylate Rab10. This conclusion is supported by compelling evidence from a wide array of rigorous approaches.
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Abstract
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.
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eLife assessment
Dhekne et al report a novel pathway for activation of the multi-domain LRRK2 protein kinase by Rab12 GTPase. LRRK2, which is mutated in Parkinson's Disease phosphorylates a subset of Rab proteins involved in intracellular trafficking, and Parkinson's disease-linked mutations increase this phosphorylation. This work adds an important new layer of understanding of this highly complex pathway by revealing that LRRK2's binding to Rab12 enhances its ability to phosphorylate Rab10. This conclusion is supported by compelling evidence from a wide array of rigorous approaches.
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Reviewer #1 (Public Review):
In this manuscript, Dhekne et al. sought to identify modulators of LRRK2 activity. Thereto, the authors performed a FACS-based genome-wide pooled CRISPR/Cas9 screen in NIH-3T3 cells monitoring phosphorylation of the Rab GTPase Rab10 which is a well-characterized target of LRRK2. Besides Rab10 and LRRK2, this unbiased screen surprisingly uncovered another Rab GTPase, Rab12, as one of the most significant hits. Validation experiments with Rab12 knockout (KO) NIH-3T3 cells, the LRRK2 inhibitors MLi-2, and Rab12 KO mice unanimously confirmed the dependency of Rab10 phosphorylation on Rab12. Conversely, the authors used A549 LRKK2 and PPM1H KO cells to show that overexpression of Rab12 but not Rab29 which is another LRRK2 modulator leads to elevated phospho-Rab10 levels in a manner dependent on LRRK2 but …
Reviewer #1 (Public Review):
In this manuscript, Dhekne et al. sought to identify modulators of LRRK2 activity. Thereto, the authors performed a FACS-based genome-wide pooled CRISPR/Cas9 screen in NIH-3T3 cells monitoring phosphorylation of the Rab GTPase Rab10 which is a well-characterized target of LRRK2. Besides Rab10 and LRRK2, this unbiased screen surprisingly uncovered another Rab GTPase, Rab12, as one of the most significant hits. Validation experiments with Rab12 knockout (KO) NIH-3T3 cells, the LRRK2 inhibitors MLi-2, and Rab12 KO mice unanimously confirmed the dependency of Rab10 phosphorylation on Rab12. Conversely, the authors used A549 LRKK2 and PPM1H KO cells to show that overexpression of Rab12 but not Rab29 which is another LRRK2 modulator leads to elevated phospho-Rab10 levels in a manner dependent on LRRK2 but insensitive to pathogenic mutations thereof. Moreover, the authors mapped E240 and S244 of LRRK2 as specific binding sites for Rab12 and showed that these residues are important to sustain full LRKK2 activity towards Rab10. Lastly, the authors showed that RAB12-dependent LRRK2 activation occurs under (patho)physiological stress conditions, namely endolysosomal membrane damage. Together, this comprehensive and elegant study of Dhekne and colleagues reveals exciting new mechanistic insights into the regulation of LRRK2 which have therapeutic potential for Parkinson's disease.
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Reviewer #2 (Public Review):
Dhekne and colleagues present an unbiased genome-wide screen by systematic CRISPR-Cas9 gene knock-out in mouse NIH-3T3 fibroblasts to identify regulators of the LRRK2 pathway which is relevant for Parkinson's disease. The screen identified Rab12 as the most potent regulator of the LRRK2 activity. Phosphorylation of the well-established LRRK2 substrate Rab10 has been used as a read-out. To allow a large-scale screen, the authors established a flow cytometry-based assay using phospho-Rab10-specific antibodies. Subsequently, Rab12 has been confirmed as an upstream effector of LRRK2 acting in a similar way as Rab29. Using computational modelling by Alphafold in conjunction with Colabfold the authors could model the Rab12:LRRK2 complex and identify a third Rab binding site within the N-terminal Armadillo repeats …
Reviewer #2 (Public Review):
Dhekne and colleagues present an unbiased genome-wide screen by systematic CRISPR-Cas9 gene knock-out in mouse NIH-3T3 fibroblasts to identify regulators of the LRRK2 pathway which is relevant for Parkinson's disease. The screen identified Rab12 as the most potent regulator of the LRRK2 activity. Phosphorylation of the well-established LRRK2 substrate Rab10 has been used as a read-out. To allow a large-scale screen, the authors established a flow cytometry-based assay using phospho-Rab10-specific antibodies. Subsequently, Rab12 has been confirmed as an upstream effector of LRRK2 acting in a similar way as Rab29. Using computational modelling by Alphafold in conjunction with Colabfold the authors could model the Rab12:LRRK2 complex and identify a third Rab binding site within the N-terminal Armadillo repeats which is distinct from the two sites, previously identified for Rab8a/Rab10 and Rab29. The predicted interaction epitope could be experimentally confirmed by systematic mutational analysis.
The experimental setting and the data presented are overall sound. It should however be considered that the selected cell model is most likely not covering the full set of LRRK2 pathway regulators as these are likely expressed in a tissue and cell-type-specific manner. It could therefore be interesting to also include more disease-relevant models, such as neuronal or immune cells. Nevertheless, Rab12 is an important effector, which is also expressed in cell types relevant to Parkinson's disease.
To validate their computational model of the Rab12 binding epitope within the N-terminal Armadillo domain of LRRK2, the authors determined the binding affinity of Rab12 which is in the lower µM range and similar to the affinities of Rab10 and Rab29 to LRRK2. The authors conducted a mutational screen mutating surface exposed residues within the predicted Rab12 binding epitope in the N-terminus of LRRK2. The study could identify critical residues, which significantly contribute to the affinity of LRRK2 for Rab12. Corresponding alanine mutations could significantly reduce the enhanced LRRK2-mediated Rab10 phosphorylation observed upon Rab12 co-expression. The effect size is similar to the previously identified Rab29 effector. Furthermore, the authors could convincingly demonstrate that Rab12 and Rab29 bind to different LRRK2 epitopes.Noteworthy, besides disrupting mutations targeting the predicted Rab12 binding epitope, the authors also found one mutation enhancing the cellular effect of Rab12 overexpression demonstrated by increased phospho-Rab10 levels. For a better evaluation of the presented computational model of the Rab12:LRRK2 complex, it would be interesting, if the authors could study the binding affinity of that mutant (F283A), as well.
Overall, the authors could convincingly demonstrate that Rab12, previously identified as LRRK2 substrate, acts upstream of LRRK2 similar to Rab29 but via a distinct binding site. The site located within the N-terminal Ankyrin domain has been predicted by a computational 3D model of the complex structure and experimentally validated. The interaction epitope might be an interesting target for the future development of allosteric modulators to treat LRRK2-mediated PD.
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Reviewer #3 (Public Review):
Dhekne et al. set out to identify novel activators of the LRRK2 kinase. They developed a flow cytometry assay to separate pools of unmodified and phosphorylated Rab10 (pRab10) from mouse NIH-3T3 cells. They then used this methodology to perform a CRISPR-based genome-wide screen to identify genes responsible for increased pRab10 levels. Candidates were validated with knock-out experiments. As far as we know, LRRK2 is the only kinase that phosphorylates the Switch II motif in Rab10. Therefore, the genes affecting pRab10 levels were classified into positive and negative LRRK2 regulators. Knocking out a positive LRRK2 regulator led to a decrease in pRab10 while knocking out a negative regulator led to an increase in pRab10. The authors found several interesting, previously unknown modulators of LRRK2 activity, …
Reviewer #3 (Public Review):
Dhekne et al. set out to identify novel activators of the LRRK2 kinase. They developed a flow cytometry assay to separate pools of unmodified and phosphorylated Rab10 (pRab10) from mouse NIH-3T3 cells. They then used this methodology to perform a CRISPR-based genome-wide screen to identify genes responsible for increased pRab10 levels. Candidates were validated with knock-out experiments. As far as we know, LRRK2 is the only kinase that phosphorylates the Switch II motif in Rab10. Therefore, the genes affecting pRab10 levels were classified into positive and negative LRRK2 regulators. Knocking out a positive LRRK2 regulator led to a decrease in pRab10 while knocking out a negative regulator led to an increase in pRab10. The authors found several interesting, previously unknown modulators of LRRK2 activity, including SPTLC2 and CERT1, which are involved in ceramide synthesis.
The major finding of this work is the unexpected effect of Rab12 on pRab10 levels in cells. Knocking out Rab12 resulted in a five-fold decrease in pRab10 levels. This observation was validated in an animal model. Conversely, overexpression of Rab12 led to a ten-fold increase in pRab10 levels. To exclude the possibility that other kinases were responsible for modifying Rab10, the authors overexpressed Rab12 in A549 cells lacking LRRK2; no increase in pRab10 was observed in these cells.
Dhenke et al. then used AlphaFold to model possible interaction between LRRK2 and Rab12 and identified a putative binding site for Rab12 in its Armadillo domain. This is the third Rab binding site in the domain of LRRK2. To validate this interaction, they mutated E240 and S244, both of which are involved in the interface; they observed no changes in pRab10 levels in cells expressing LRRK2 carrying the E240R and S244R mutations. The previously reported site #1 and site #2, both of which also bind Rabs and are involved in feed-forward LRRK2 activation, seem to be unrelated to the binding of Rab12 to site #3. The authors propose that site #3 might open the kinase of LRRK2 to increase its activity.
Finally, the authors point out the important role of Rab12 in lysosomal damage by showing that LLOME- or Nigericin-induced cellular stress increases LRRK2 activity in a Rab12-dependent manner.
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