In vivo susceptibility to energy failure parkinsonism and LRRK2 kinase activity

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

10.1101/2020.07.27.219501 ◽

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.

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

10.1101/2020.10.21.348144 ◽

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.

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

PLoS Biology ◽

10.1371/journal.pbio.3001480 ◽

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.

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LRRK2 and the Endolysosomal System in Parkinson’s Disease

Journal of Parkinson s Disease ◽

10.3233/jpd-202138 ◽

2020 ◽

Vol 10 (4) ◽

pp. 1271-1291

Author(s):

Madalynn L. Erb ◽

Darren J. Moore

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Activity ◽

Neuronal Damage ◽

Distinct Cell Type ◽

In Vivo Models ◽

Network Maintenance ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

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Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo

Neurobiology of Disease ◽

10.1016/j.nbd.2021.105487 ◽

2021 ◽

pp. 105487

Author(s):

Federica Albanese ◽

Daniela Mercatelli ◽

Luca Finetti ◽

Giulia Lamonaca ◽

Sara Pizzi ◽

...

Keyword(s):

Kinase Activity ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

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

Biochemical Journal ◽

10.1042/bj20070209 ◽

2007 ◽

Vol 405 (2) ◽

pp. 307-317 ◽

Cited By ~ 339

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.

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