LRRK2 Kinase Activity Does Not Alter Cell-Autonomous Tau Pathology Development in Primary Neurons

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD) and are also associated with genetic risk in idiopathic PD. Mutations in LRRK2, including the most common p.G2019S lead to elevated kinase activity, making LRRK2 kinase inhibitors prime targets for therapeutic development. However, the role of LRRK2 kinase activity in PD pathogenesis has remained unclear. While essentially all LRRK2-PD patients exhibit dopaminergic neuron loss, many of these patients to not have α-synuclein Lewy bodies in their brains. So, what is the neuropathological substrate of LRRK2-PD? Tau has emerged as a possible candidate due to the presence of tau pathology in the majority of LRRK2 mutation carriers and reports of hyperphosphorylated tau in LRRK2 animal models. Objective: In the current study, we aim to address whether a mutation in LRRK2 changes the cell-autonomous seeding of tau pathology in primary neurons. We also aim to assess whether LRRK2 kinase inhibitors are able to modulate tau pathology. Methods/Results: Treatment of primary neurons with LRRK2 kinase inhibitors leads to prolonged kinase inhibition but does not alter tau pathology induction. The lack of an effect of LRRK2 kinase activity was further confirmed in primary neurons expressing LRRK2G2019S and with two different forms of pathogenic tau. In no case was there more than a minor change in tau pathology induction. Conclusion: Together, our results indicate that LRRK2 kinase activity is not playing a major role in the induction of tau pathology in individual neurons. Understanding the impact of LRRK2 kinase inhibitors on pathology generation is important as kinase inhibitors move forward in clinical trials.

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The activities of LRRK2 and GCase are positively correlated in clinical biospecimens and experimental models of Parkinson’s disease

10.1101/2021.09.27.461935 ◽

2021 ◽

Author(s):

Maria Kedariti ◽

Emanuele Frattini ◽

Pascale Baden ◽

Susanna Cogo ◽

Laura Civiero ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Activity ◽

Experimental Models ◽

Cellular Functions ◽

Gene Encoding ◽

Cellular Models ◽

Lrrk2 Kinase ◽

The Impact ◽

Lrrk2 Kinase Activity

AbstractLRRK2 is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways and vesicle trafficking. Mutations in LRRK2 cause autosomal dominant forms of Parkinson’s disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we investigated GCase levels and activity in LRRK2 G2019S knockin mice, in clinical biospecimens from PD patients carrying this mutation and in patient-derived cellular models. In these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase.

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A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115606707 ◽

2015 ◽

Vol 21 (2) ◽

pp. 145-155 ◽

Cited By ~ 21

Author(s):

Melanie Leveridge ◽

Lee Collier ◽

Colin Edge ◽

Phil Hardwicke ◽

Bill Leavens ◽

...

Keyword(s):

Mass Spectrometry ◽

Parkinson’S Disease ◽

Parkinson's Disease ◽

High Throughput ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Label Free ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson’s disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.

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Inhibition of LRRK2 kinase activity promotes anterograde axonal transport and presynaptic targeting of α-synuclein

10.1101/2021.10.04.463043 ◽

2021 ◽

Author(s):

Charlotte F Brzozowski ◽

Baraa A Hijaz ◽

Vijay Singh ◽

Nolwazi Z Gcwensa ◽

Kaela Kelly ◽

...

Keyword(s):

Axonal Transport ◽

Kinase Activity ◽

Alpha Synuclein ◽

Presynaptic Terminal ◽

Lewy Pathology ◽

Anterograde Axonal Transport ◽

Lrrk2 Kinase ◽

The Impact ◽

Lrrk2 Kinase Activity

Pathologic inclusions composed of alpha-synuclein called Lewy pathology are hallmarks of Parkinson Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and alpha-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of alpha-synuclein, and enhances formation of pathologic alpha-synuclein, particularly when synaptic activity is increased. alpha-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence alpha-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences alpha-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of alpha-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of alpha-synuclein. Live imaging of axonal transport of alpha-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on alpha-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases alpha-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of alpha-synuclein-GFP. In vivo, LRRK2 inhibition increases alpha-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of alpha-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of alpha-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD.

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Nanobodies as allosteric modulators of Parkinson′s disease-associated LRRK2

10.1101/2021.08.30.458082 ◽

2021 ◽

Author(s):

Ranjan K. Singh ◽

Ahmed Soliman ◽

Giambattista Guaitoli ◽

Eliza Störmer ◽

Felix von Zweydorf ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Common Disease ◽

Type I ◽

Rab Protein ◽

Lrrk2 Kinase ◽

Inhibitory Drug ◽

Lrrk2 Kinase Activity

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson′s disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

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Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson’s disease

Scientific Reports ◽

10.1038/s41598-020-74195-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

C. P. Gonzalez-Hunt ◽

E. A. Thacker ◽

C. M. Toste ◽

S. Boularand ◽

S. Deprets ◽

...

Keyword(s):

Dna Damage ◽

Clinical Trials ◽

Mitochondrial Dna ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Mtdna Damage ◽

Mitochondrial Dna Damage ◽

Lrrk2 G2019s ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Abstract Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson’s disease (PD) and LRRK2 kinase inhibitors are currently being tested in early phase clinical trials. In order to ensure the highest chance of success, a biomarker-guided entry into clinical trials is key. LRRK2 phosphorylation, and phosphorylation of the LRRK2 substrate Rab10, have been proposed as target engagement biomarkers for LRRK2 kinase inhibition. However, a pharmacodynamic biomarker to demonstrate that a biological response has occurred is lacking. We previously discovered that the LRRK2 G2019S mutation causes mitochondrial DNA (mtDNA) damage and is LRRK2 kinase activity-dependent. Here, we have explored the possibility that measurement of mtDNA damage is a “surrogate” for LRRK2 kinase activity and consequently of kinase inhibitor activity. Mitochondrial DNA damage was robustly increased in PD patient-derived immune cells with LRRK2 G2019S mutations as compared with controls. Following treatment with multiple classes of LRRK2 kinase inhibitors, a full reversal of mtDNA damage to healthy control levels was observed and correlated with measures of LRRK2 dephosphorylation. Taken together, assessment of mtDNA damage levels may be a sensitive measure of altered kinase activity and provide an extended profile of LRRK2 kinase modulation in clinical studies.

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Allosteric inhibition of LRRK2, where are we now

Biochemical Society Transactions ◽

10.1042/bst20200424 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2185-2194

Author(s):

Ahmed Soliman ◽

Fatma Nihan Cankara ◽

Arjan Kortholt

Keyword(s):

Recent Progress ◽

Kinase Activity ◽

Kinase Inhibitors ◽

High Selectivity ◽

Gtpase Activity ◽

Leucine Rich Repeat ◽

Allosteric Inhibitors ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Parkinson's disease (PD) is the second most common neurodegenerative disease. In recent years, it has been shown that leucine-rich repeat kinase 2 (LRRK2) has a crucial function in both familial and sporadic forms of PD. LRRK2 pathogenic mutations are thought to result in an increase in LRRK2 kinase activity. Thus, inhibiting LRRK2 kinase activity has become a main therapeutic target. Many compounds capable of inhibiting LRRK2 kinase activity with high selectivity and brain availability have been described. However, the safety of long-term use of these ATP-competitive LRRK2 kinase inhibitors has been challenged by several studies. Therefore, alternative ways of targeting LRRK2 activity will have a great benefit. In this review, we discuss the recent progress in the development of allosteric inhibitors of LRRK2, mainly via interfering with GTPase activity, and propose potential new intra and interprotein interactions targets that can lead to open doors toward new therapeutics.

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