scholarly journals A Novel LRRK2 Variant p.G2294R in the WD40 Domain Identified in Familial Parkinson’s Disease Affects LRRK2 Protein Levels

2021 ◽  
Vol 22 (7) ◽  
pp. 3708
Author(s):  
Jun Ogata ◽  
Kentaro Hirao ◽  
Kenya Nishioka ◽  
Arisa Hayashida ◽  
Yuanzhe Li ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Peripheral Blood ◽  
Kinase Activity ◽  
Late Onset ◽  
Causative Gene ◽  
Protein Levels ◽  
Wd40 Domain ◽  
Lrrk2 Protein ◽  
Familial Parkinson’S Disease

Leucine-rich repeat kinase 2 (LRRK2) is a major causative gene of late-onset familial Parkinson’s disease (PD). The suppression of kinase activity is believed to confer neuroprotection, as most pathogenic variants of LRRK2 associated with PD exhibit increased kinase activity. We herein report a novel LRRK2 variant—p.G2294R—located in the WD40 domain, detected through targeted gene-panel screening in a patient with familial PD. The proband showed late-onset Parkinsonism with dysautonomia and a good response to levodopa, without cognitive decline or psychosis. Cultured cell experiments revealed that p.G2294R is highly destabilized at the protein level. The LRRK2 p.G2294R protein expression was upregulated in the patient’s peripheral blood lymphocytes. However, macrophages differentiated from the same peripheral blood showed decreased LRRK2 protein levels. Moreover, our experiment indicated reduced phagocytic activity in the pathogenic yeasts and α-synuclein fibrils. This PD case presents an example wherein the decrease in LRRK2 activity did not act in a neuroprotective manner. Further investigations are needed in order to elucidate the relationship between LRRK2 expression in the central nervous system and the pathogenesis caused by altered LRRK2 activity.

scholarly journals Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease

2021 ◽  
Author(s):  
Ludovica Iovino ◽  
Veronica Giusti ◽  
Francesca Pischedda ◽  
Elena Giusto ◽  
Nicoletta Plotegher ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Plasma Membrane ◽  
Excitatory Amino Acid ◽  
Late Onset ◽  
Glutamate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Excitatory Amino Acid Transporter ◽  
Protein Levels ◽  
Lrrk2 G2019s

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In Lrrk2 G2019S knockin mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane, causing its intracellular relocalization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

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.

LRRK2 detection in human biofluids: potential use as a Parkinson's disease biomarker?

2017 ◽  
Vol 45 (1) ◽  
pp. 207-212 ◽  
Author(s):  
Jean-Marc Taymans ◽  
Eugénie Mutez ◽  
Matthieu Drouyer ◽  
William Sibran ◽  
Marie-Christine Chartier-Harlin
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Target Engagement ◽  
Protein Levels ◽  
Disease Biomarker ◽  
Experimental Systems ◽  
Lrrk2 Protein ◽  
Potential Use ◽  
Potential Therapeutics ◽  
Inhibitor Treatment

Leucine-rich repeat kinase 2 (LRRK2) is a complex signalling protein that is a key therapeutic target, particularly in Parkinson's disease (PD). In addition, there is now evidence showing that LRRK2 expression and phosphorylation levels have potential as markers of disease or target engagement. Indeed, reports show increases in LRRK2 protein levels in the prefrontal cortex of PD patients relative to controls, suggesting that increase in total LRRK2 protein expression is correlated with disease progression. LRRK2 phosphorylation levels are reduced in experimental systems for most disease mutants, and LRRK2 is also rapidly dephosphorylated upon LRRK2 inhibitor treatment, considered potential therapeutics. Recently, the presence of LRRK2 was confirmed in exosomes from human biofluids, including urine and cerebrospinal fluid. Moreover, phosphorylation of LRRK2 at phosphosites S910, S935, S955 and S973, as well as at the autophosphoryation site S1292, was found in urinary exosomes. In this review, we summarize knowledge on detection of LRRK2 in human biofluids and the relevance of these findings for the development of PD-related biomarkers.

scholarly journals (D620N) VPS35 causes the impairment of Wnt/β-catenin signaling cascade and mitochondrial dysfunction in a PARK17 knockin mouse model

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Ching-Chi Chiu ◽  
Yi-Hsin Weng ◽  
Ying-Zu Huang ◽  
Rou-Shayn Chen ◽  
Yu-Chuan Liu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Autosomal Dominant ◽  
Late Onset ◽  
Substantia Nigra Pars Compacta ◽  
Protein Levels ◽  
Abnormal Morphology ◽  
Vacuolar Protein ◽  
Knockin Mouse ◽  
Active Caspase

AbstractPatients with familial type 17 of Parkinson’s disease (PARK17) manifest autosomal dominant pattern and late-onset parkinsonian syndromes. Heterozygous (D620N) mutation of vacuolar protein sorting 35 (VPS35) is genetic cause of PARK17. We prepared heterozygous VPS35D620N/+ knockin mouse, which is an ideal animal model of (D620N) VPS35-induced autosomal dominant PARK17. Late-onset loss of substantia nigra pars compacta (SNpc) dopaminergic (DAergic) neurons and motor deficits of Parkinson’s disease were found in 16-month-old VPS35D620N/+ mice. Normal function of VPS35-containing retromer is needed for activity of Wnt/β-catenin cascade, which participates in protection and survival of SNpc DAergic neurons. It was hypothesized that (D620N) VPS35 mutation causes the malfunction of VPS35 and resulting impaired activity of Wnt/β-catenin pathway. Protein levels of Wnt1 and nuclear β-catenin were reduced in SN of 16-month-old VPS35D620N/+ knockin mice. Downregulated protein expression of survivin, which is a target gene of nuclear β-catenin, and upregulated protein levels of active caspase-8 and active caspase-9 were observed in SN of VPS35D620N/+ mice at age of 16 months. VPS35 is involved in controlling morphology and function of mitochondria. Impaired function of VPS35 caused by (D620N) mutation could lead to abnormal morphology and malfunction of mitochondria. A significant decrease in mitochondrial size and resulting mitochondrial fragmentation was found in tyrosine hydroxylase-positive and neuromelanin-positive SNpc DAergic neurons of 16-month-old VPS35D620N/+ mice. Mitochondrial complex I activity or complex IV activity was reduced in SN of 16-month-old VPS35D620N/+ mice. Increased level of mitochondrial ROS and oxidative stress were found in SN of 16-month-old VPS35D620N/+ mice. Levels of cytosolic cytochrome c and active caspase-3 were increased in SN of VPS35D620N/+ mice aged 16 months. Our results suggest that PARK17 mutant (D620N) VPS35 impairs activity of Wnt/β-catenin signaling pathway and causes abnormal morphology and dysfunction of mitochondria, which could lead to neurodegeneration of SNpc DAergic cells.

scholarly journals Functional analyses of two novel LRRK2 pathogenic variants in familial Parkinson’s disease

2021 ◽  
Author(s):  
I Coku ◽  
E Mutez ◽  
S Eddarkaoui ◽  
S Carrier ◽  
A Marchand ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Functional Testing ◽  
Pathogenic Variants ◽  
Lrrk2 Kinase ◽  
Functional Analyses ◽  
Familial Parkinson’S Disease ◽  
Inhibitor Treatment

ABSTRACTBackgroundPathogenic variants in the LRRK2 gene are a common monogenic cause of Parkinson’s disease. However, only seven variants have been confirmed to be pathogenic.ObjectivesWe identified two novel LRRK2 variants (H230R and A1440P) and performed functional testing.MethodsWe transiently expressed wildtype, the two new variants, or two known pathogenic mutants (G2019S and R1441G), in HEK-293T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins.ResultsThe two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity.ConclusionsWe identified two novel LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinson’s disease.

scholarly journals Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

2020 ◽  
Author(s):  
Yuriko Sobu ◽  
Paulina S. Wawro ◽  
Herschel S. Dhekne ◽  
Suzanne R. Pfeffer
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Cell Types ◽  
Cell Physiology ◽  
Rab Gtpases ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Lrrk2 Protein

ABSTRACTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. We have shown previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1 and together, these proteins block cilia formation in a variety of cell types including patient derived iPS cells. We have used live cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and loss of cilia seen upon serum re-addition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation. Kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss, but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis. In both cases, failure of CP110 uncapping was due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, recruitment of EHD1, another step important for ciliogenesis, appears unaltered. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation, and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.SIGNIFICANCE STATEMENTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, in particular Rab8 and Rab10. Phosphorylated Rabs bind preferentially to a distinct set of effectors and block in primary ciliation in multiple cell types. We show here that the cilia blockade is upstream of the recruitment of TTBK2 kinase to the mother centriole, a step required for the release of CP110 and subsequent cilia formation. This study provides fundamental information related to how pathogenic LRRK2 interferes with normal cell physiology.

scholarly journals Specific immune status in Parkinson’s disease at different ages of onset

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Jun Tian ◽  
Shao-Bing Dai ◽  
Si-Si Jiang ◽  
Wen-Yi Yang ◽  
Yi-Qun Yan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
T Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Rating Scale ◽  
Immune Status ◽  
Immune Cell ◽  
Late Onset ◽  
Different Ages

AbstractRecent evidence suggests that innate and adaptive immunity play a crucial role in Parkinson’s disease (PD). However, studies regarding specific immune cell classification in the peripheral blood in PD remain lacking. Therefore, we aimed to explore the different immune status in patients with PD at different ages of onset. We included 22 patients; among them were 10 who had early-onset PD (EOPD) and 12 had late-onset PD (LOPD) and 10 young healthy controls (YHCs) and 8 elder HCs (EHCs). Mass cytometry staining technology was used to perform accurate immunotyping of cell populations in the peripheral blood. Motor symptoms and cognitive function were assessed using the Unified Parkinson’s Disease Rating Scale (UPDRS) III score and Mini-mental State Examination (MMSE) score, respectively. T test and ANOVA statistical analysis were performed on the frequency of annotated cell population. Linear regression model was used to analyze the correlation between clusters and clinical symptoms. We characterized 60 cell clusters and discovered that the immune signature of PD consists of cluster changes, including decreased effector CD8+ T cells, lower cytotoxicity natural killer (NK) cells and increased activated monocytes in PD patients. In summary, we found that CD8+ T cells, NK cells, and monocytes were associated with PD. Furthermore, there may be some differences in the immune status of patients with EOPD and LOPD, suggesting differences in the pathogenesis between these groups.

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