scholarly journals Pathogenic mutations in LRRK2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

2020 ◽  
Author(s):  
Adamantios Mamais ◽  
Natalie Landeck ◽  
Rebekah G. Langston ◽  
Luis Bonet-Ponce ◽  
Nathan Smith ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Kinase Activity ◽  
Small Gtpase ◽  
Kinase Substrate ◽  
Lrrk2 Kinase ◽  
Effect Of Pd ◽  
Lrrk2 Kinase Activity

AbstractMutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s 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 are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem 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 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.

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

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001480
Author(s):  
Adamantios Mamais ◽  
Jillian H. Kluss ◽  
Luis Bonet-Ponce ◽  
Natalie Landeck ◽  
Rebekah G. Langston ◽  
...  
Keyword(s):  
Parkinson Disease ◽  
Iron Uptake ◽  
Kinase Activity ◽  
Small Gtpase ◽  
Kinase Substrate ◽  
Lrrk2 Kinase ◽  
Effect Of Pd ◽  
Lrrk2 Kinase Activity

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.

Role of LRRK2 kinase activity in the pathogenesis of Parkinson's disease

2012 ◽  
Vol 40 (5) ◽  
pp. 1058-1062 ◽  
Author(s):  
Elisa Greggio
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Complex Molecule ◽  
Disease Onset ◽  
Missense Mutations ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Interest in studying the biology of LRRK2 (leucine-rich repeat kinase 2) started in 2004 when missense mutations in the LRRK2 gene were linked to an inherited form of Parkinson's disease with clinical and pathological presentation resembling the sporadic syndrome. LRRK2 is a complex molecule containing domains implicated in protein interactions, as well as kinase and GTPase activities. The observation that the common G2019S mutation increases kinase activity in vitro suggests that altered phosphorylation of LRRK2 targets may have pathological outcomes. Given that protein kinases are ideal targets for drug therapies, much effort has been directed at understanding the role of LRRK2 kinase activity on disease onset. However, no clear physiological substrates have been identified to date, indicating that much research is still needed to fully understand the signalling pathways orchestrated by LRRK2 and deregulated under pathological conditions.

scholarly journals LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

2007 ◽  
Vol 405 (2) ◽  
pp. 307-317 ◽  
Author(s):  
Mahaboobi Jaleel ◽  
R. Jeremy Nichols ◽  
Maria Deak ◽  
David G. Campbell ◽  
Frank Gillardon ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Late Onset ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Kinase Substrate ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity

Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.

scholarly journals Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

2000 ◽  
Vol 20 (16) ◽  
pp. 5858-5864 ◽  
Author(s):  
Gregory J. Reynard ◽  
William Reynolds ◽  
Rati Verma ◽  
Raymond J. Deshaies
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Multiple Substrates ◽  
Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo

2000 ◽  
Vol 113 (1) ◽  
pp. 135-144 ◽  
Author(s):  
R. Pepperkok ◽  
J.A. Whitney ◽  
M. Gomez ◽  
T.E. Kreis
Keyword(s):  
Protein Transport ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Coated Vesicles ◽  
Gtp Hydrolysis ◽  
Rapid Accumulation ◽  
Gamma S

Microinjection of the slowly hydrolyzable GTP analogue GTP(gamma)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (beta)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(gamma)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(gamma)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.

scholarly journals RIT2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

2020 ◽  
Author(s):  
Julia Obergasteiger ◽  
Anne-Marie Castonguay ◽  
Giulia Frapporti ◽  
Evy Lobbestael ◽  
Veerle Baekelandt ◽  
...  
Keyword(s):  
Risk Factor ◽  
Substantia Nigra ◽  
Dopaminergic Neurons ◽  
Kinase Activity ◽  
Alpha Synuclein ◽  
The Novel ◽  
Lrrk2 Kinase ◽  
Novel Strategy ◽  
Lrrk2 Kinase Activity

AbstractIn Parkinson’s disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear but hypothesized to involve the autophagy lysosome pathways (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, hyperactivate kinase activity and its pharmacological inhibition reduces pS129-aSyn inclusions. We observed selective downregulation of the novel PD risk factor RIT2 in G2019S-LRRK2 expressing cells. Here we studied whether RIT2 could modulate LRRK2 kinase activity. RIT2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of RIT2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, RIT2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. Our data indicate that RIT2 inhibits overactive LRRK2 to ameliorate ALP impairment and counteract aSyn aggregation and related deficits. Targeting RIT2 could represent a novel strategy to combat neuropathology in familial and idiopathic PD.

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

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.

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

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.

The Role of LRRK2 Kinase Activity in Cellular PD Models

2008 ◽  
pp. 423-431 ◽  
Author(s):  
Mark R. Cookson ◽  
Elisa Greggio ◽  
Patrick Lewis
Keyword(s):  
Kinase Activity ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity
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