Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

Fernández, Belén; Chittoor-Vinod, Vinita G.; Kluss, Jillian H.; Kelly, Kaela; Bryant, Nicole; Nguyen, An Phu Tran; Bukhari, Syed; Smith, Nathan J.; Ordóñez, Antonio Jesús Lara; Fdez, Elena; Chartier-Harlin, Marie-Christine; Montine, Thomas J.; Wilson, Mark A.; Moore, Darren J.; West, Andrew B.; Cookson, Mark; Nichols, R. Jeremy; Hilfiker, Sabine

doi:10.3233/jpd-213128

Cited by 11 publications

(6 citation statements)

References 60 publications

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“…We previously found with non-selective LRRK2 kinase inhibitors that ∼ 25% inhibition of LRRK2 pSer935 was sufficient to reverse mtDNA damage levels back to healthy control levels [48]. However, we were not able to reliably measure pSer1292 LRRK2 under the same conditions, a barrier in most endogenous contexts [31]. An advantage to the current study is that we utilized stably overexpressing LRRK2 cell lines to compare how pSer1292 and pSer935 LRRK2 dephosphorylation changes with non-selective and selective LRRK2 kinase inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…Measuring endogenous LRRK2 kinase activity by monitoring LRRK2 autophosphorylation at serine 1292 (pSer1292) has been particularly challenging, but can be robustly detected in overexpressed cellular models or with a fractionation-based enrichment technique in G2019S LRRK2 but not wild-type tissue [11, 31, 32]. The phosphorylation of downstream substrates, including Rab GTPase substrates, are difficult to measure reliably due to low stoichiometry [4, 5, 33, 34].…”

Section: Introductionmentioning

confidence: 99%

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G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pena

Gonzalez‐Hunt

Rui

et al. 2022

Preprint

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Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a nonselective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, a novel system that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. Precision medicine is a common approach in modern day cancer research that is not yet routinely applied to neurodegenerative diseases. Abrogation of mtDNA damage with mutant selective tool inhibitor EB-42168 demonstrates the promise of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pena

Gonzalez‐Hunt

Rui

et al. 2022

Preprint

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“…Only the G2019S-LRRK2 mutation increases the LRRK2 kinase activity in vitro [ 18–20 ]. As assessed in cells and tissues, the G2019S-LRRK2 mutation displays increased autophosphorylation at Ser1292 but causes only a modest increase in Rab10 phosphorylation [ 12 , 21 , 22 ]. In contrast, pathogenic mutations in either the ROC or COR domain display modest effects on Ser1292 autophosphorylation but markedly increase Rab10 phosphorylation [ 21 ].…”

Section: Lrrk2 Domain Structure and Kinase Substratesmentioning

confidence: 99%

Insights into the cellular consequences of LRRK2-mediated Rab protein phosphorylation

Fasiczka

Naaldijk

Brahmia

et al. 2023

Biochemical Society Transactions

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Point mutations in leucine-rich repeat kinase 2 (LRRK2) which cause Parkinson's disease increase its kinase activity, and a subset of Rab GTPases have been identified as endogenous LRRK2 kinase substrates. Their phosphorylation correlates with a loss-of-function for the membrane trafficking steps they are normally involved in, but it also allows them to bind to a novel set of effector proteins with dominant cellular consequences. In this brief review, we will summarize novel findings related to the LRRK2-mediated phosphorylation of Rab GTPases and its various cellular consequences in vitro and in the intact brain, and we will highlight major outstanding questions in the field.

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“…We intend to further develop and expand our collaboration with the Michael J. Fox Foundation in publishing replication studies to contribute to enhancing rigor in Parkinson's disease research [8,9]. We are excited by the return of the World Parkinson Congress and look forward to partnering with the organizers and participants to showcase the informative and inspirational content from that unique meeting.…”

Section: Collaborate Collaborate Collaboratementioning

confidence: 99%