scholarly journals Human loss-of-function variants suggest that partial LRRK2 inhibition is a safe therapeutic strategy for Parkinson’s disease

2019 ◽  
Author(s):  
Nicola Whiffin ◽  
Irina M. Armean ◽  
Aaron Kleinman ◽  
Jamie L. Marshall ◽  
Eric V. Minikel ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Large Scale ◽  
Therapeutic Strategy ◽  
Kinase Activity ◽  
Model Organisms ◽  
Loss Of Function ◽  
Genomic Databases ◽  
Protein Coding

AbstractHuman genetic variants causing loss-of-function (LoF) of protein-coding genes provide natural in vivo models of gene inactivation, which are powerful indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson’s disease3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. Whilst preclinical studies in model organisms have raised some on-target toxicity concerns5–8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here we systematically analyse LoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)9 and over 4 million participants in the 23andMe genotyped dataset, to assess their impact at a molecular and phenotypic level. After thorough variant curation, we identify 1,358 individuals with high-confidence predicted LoF variants in LRRK2, several with experimental validation. We show that heterozygous LoF of LRRK2 reduces LRRK2 protein level by ~50% but is not associated with reduced life expectancy, or with any specific phenotype or disease state. These data suggest that therapeutics that downregulate LRRK2 levels or kinase activity by up to 50% are unlikely to have major on-target safety liabilities. Our results demonstrate the value of large scale genomic databases and phenotyping of human LoF carriers for target validation in drug discovery.

scholarly journals Combined knockout of Lrrk2 and Rab29 does not result in behavioral abnormalities in vivo

2020 ◽  
Author(s):  
Melissa Conti Mazza ◽  
Victoria Nguyen ◽  
Alexandra Beilina ◽  
Jinhui Ding ◽  
Mark R. Cookson
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Association Studies ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Genome Wide Association Studies ◽  
Double Knockout ◽  
Knockout Mouse Model

AbstractCoding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinson’s disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, leading to the suggestion that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, here we generated a double knockout mouse model and determined whether there are any consequences on brain function with aging. From a battery of motor and non-motor behavioral tests, we noted only that 18-24 month Rab29-/- and double (Lrrk2-/-/Rab29-/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in number of substantia nigra pars compacta (SNc) dopamine neurons or in tyrosine hydroxylase levels in the SNc and striatum, which might reflect a PD-like pathology. These results suggest that depletion of both Lrrk2 and Rab29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans.Significance statementGenetic variation in LRRK2 that result in elevated kinase activity can cause Parkinson’s disease (PD), suggesting LRRK2 inhibition as a therapeutic strategy. RAB29, a substrate of LRRK2, has also been associated with increased PD risk. Evidence exists for an interactive relationship between LRRK2 and RAB29. Mouse models lacking either LRRK2 or RAB29 do not show brain pathologies. We hypothesized that the loss of both targets would result in additive effects across in vivo and post-mortem assessments in aging mice. We found that loss of both LRRK2 and RAB29 did not result in significant behavioral deficits or dopamine neuron loss. This evidence suggests that chronic inhibition of this pathway should be tolerated clinically.

scholarly journals Machine learning discriminates a movement disorder in a zebrafish model of Parkinson's disease

2020 ◽  
Vol 13 (10) ◽  
pp. dmm045815 ◽  
Author(s):  
Gideon L. Hughes ◽  
Michael A. Lones ◽  
Matthew Bedder ◽  
Peter D. Currie ◽  
Stephen L. Smith ◽  
...  
Keyword(s):  
Machine Learning ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Movement Disorders ◽  
Molecular Mechanisms ◽  
Computational Method ◽  
Metabolic Reprogramming ◽  
Loss Of Function ◽  
Zebrafish Model

ABSTRACTAnimal models of human disease provide an in vivo system that can reveal molecular mechanisms by which mutations cause pathology, and, moreover, have the potential to provide a valuable tool for drug development. Here, we have developed a zebrafish model of Parkinson's disease (PD) together with a novel method to screen for movement disorders in adult fish, pioneering a more efficient drug-testing route. Mutation of the PARK7 gene (which encodes DJ-1) is known to cause monogenic autosomal recessive PD in humans, and, using CRISPR/Cas9 gene editing, we generated a Dj-1 loss-of-function zebrafish with molecular hallmarks of PD. To establish whether there is a human-relevant parkinsonian phenotype in our model, we adapted proven tools used to diagnose PD in clinics and developed a novel and unbiased computational method to classify movement disorders in adult zebrafish. Using high-resolution video capture and machine learning, we extracted novel features of movement from continuous data streams and used an evolutionary algorithm to classify parkinsonian fish. This method will be widely applicable for assessing zebrafish models of human motor diseases and provide a valuable asset for the therapeutics pipeline. In addition, interrogation of RNA-seq data indicate metabolic reprogramming of brains in the absence of Dj-1, adding to growing evidence that disruption of bioenergetics is a key feature of neurodegeneration.This article has an associated First Person interview with the first author of the paper.

scholarly journals Neuron-autonomous susceptibility to induced synuclein aggregation is exacerbated by endogenous Lrrk2 mutations and ameliorated by Lrrk2 genetic knock-out

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Sarah MacIsaac ◽  
Thaiany Quevedo Melo ◽  
Yuting Zhang ◽  
Mattia Volta ◽  
Matthew J Farrer ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Therapeutic Strategy ◽  
Germ Line ◽  
Lewy Bodies ◽  
Neuronal Cultures ◽  
Leucine Rich Repeat ◽  
Kinase Inhibition ◽  
Knock Out

Abstract Neuronal aggregates containing α-synuclein are a pathological hallmark of several degenerative diseases; including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Understanding the process of α-synuclein aggregation, and discovering means of preventing it, may help guide therapeutic strategy and drug design. Recent advances provide tools to induce α-synuclein aggregation in neuronal cultures. Application of exogenous pre-formed fibrillar α-synuclein induces pathological phosphorylation and accumulation of endogenous α-synuclein, typical of that seen in disease. Genomic variability and mutations in α-synuclein and leucine-rich repeat kinase 2 proteins are the major genetic risk factors for Parkinson’s disease. Reports demonstrate fibril-induced α-synuclein aggregation is increased in cells from leucine-rich repeat kinase 2 pathogenic mutant (G2019S) overexpressing mice, and variously decreased by leucine-rich repeat kinase 2 inhibitors. Elsewhere in vivo antisense knock-down of leucine-rich repeat kinase 2 protein has been shown to protect mice from fibril-induced α-synuclein aggregation, whereas kinase inhibition did not. To help bring clarity to this issue, we took a purely genetic approach in a standardized neuron-enriched culture, lacking glia. We compared fibril treatment of leucine-rich repeat kinase 2 germ-line knock-out, and G2019S germ-line knock-in, mouse cortical neuron cultures with those from littermates. We found leucine-rich repeat kinase 2 knock-out neurons are resistant to α-synuclein aggregation, which predominantly forms within axons, and may cause axonal fragmentation. Conversely, leucine-rich repeat kinase 2 knock-in neurons are more vulnerable to fibril-induced α-synuclein accumulation. Protection and resistance correlated with basal increases in a lysosome marker in knock-out, and an autophagy marker in knock-in cultures. The data add to a growing number of studies that argue leucine-rich repeat kinase 2 silencing, and potentially kinase inhibition, may be a useful therapeutic strategy against synucleinopathy.

scholarly journals Opposing functions of β-arrestin 1 and 2 in Parkinson’s disease via microglia inflammation and Nprl3

2021 ◽  
Author(s):  
Yinquan Fang ◽  
Qingling Jiang ◽  
Shanshan Li ◽  
Hong Zhu ◽  
Rong Xu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Inflammatory Responses ◽  
Functional Divergence ◽  
Primary Cultures ◽  
Loss Of Function ◽  
Neuron Damage ◽  
Dopaminergic Neuron Loss

AbstractAlthough β-arrestins (ARRBs) regulate diverse physiological and pathophysiological processes, their functions and regulation in Parkinson’s disease (PD) remain poorly defined. In this study, we show that the expression of β-arrestin 1 (ARRB1) and β-arrestin 2 (ARRB2) is reciprocally regulated in PD mouse models, particularly in microglia. ARRB1 ablation ameliorates, whereas ARRB2 knockout aggravates, the pathological features of PD, including dopaminergic neuron loss, neuroinflammation and microglia activation in vivo, and microglia-mediated neuron damage in vitro. We also demonstrate that ARRB1 and ARRB2 produce adverse effects on inflammation and activation of the inflammatory STAT1 and NF-κB pathways in primary cultures of microglia and macrophages and that two ARRBs competitively interact with the activated form of p65, a component of the NF-κB pathway. We further find that ARRB1 and ARRB2 differentially regulate the expression of nitrogen permease regulator-like 3 (Nprl3), a functionally poorly characterized protein, as revealed by RNA sequencing, and that in the gain- and loss-of-function studies, Nprl3 mediates the functions of both ARRBs in microglia inflammatory responses. Collectively, these data demonstrate that two closely related ARRBs exert opposite functions in microglia-mediated inflammation and the pathogenesis of PD which are mediated at least in part through Nprl3 and provide novel insights into the understanding of the functional divergence of ARRBs in PD.

scholarly journals LRRK2 and the Endolysosomal System in Parkinson’s Disease

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.

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 The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

2012 ◽  
Vol 446 (1) ◽  
pp. 99-111 ◽  
Author(s):  
Iakov N. Rudenko ◽  
Alice Kaganovich ◽  
David N. Hauser ◽  
Aleksandra Beylina ◽  
Ruth Chia ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Missense Mutations ◽  
Leucine Rich Repeat ◽  
Loss Of Function ◽  
Partial Loss ◽  
G2019s Mutation ◽  
Enzymatic Function ◽  
C Terminus

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2–3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.

scholarly journals Targeting mitophagy in Parkinson's disease

2020 ◽  
pp. jbc.REV120.014294
Author(s):  
Emily H Clark ◽  
Aurelio Vázquez de la Torre ◽  
Tamaki Hoshikawa ◽  
Thomas Briston
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Quality Control ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Biological Properties ◽  
Loss Of Function ◽  
Mitochondrial Quality Control

The genetics and pathophysiology of Parkinson’s disease (PD) strongly implicate mitochondria in disease aetiology. Elegant studies over the last two decades have elucidated complex molecular signalling governing the identification and removal of dysfunctional mitochondria from the cell, a process of mitochondrial quality control known as mitophagy. Mitochondrial deficits and specifically reduced mitophagy are evident in both sporadic and familial PD. Mendelian genetics attributes loss-of-function mutations in key mitophagy regulators PINK1 and Parkin to early-onset PD. Pharmacologically enhancing mitophagy and accelerating the removal of damaged mitochondria are of interest for developing a disease-modifying PD therapeutic. However, despite significant understanding of both PINK1/Parkin-dependent and -independent mitochondrial quality control pathways, the therapeutic potential of targeting mitophagy remains to be fully explored. Here, we provide a summary of the genetic evidence supporting the role for mitophagy-failure as a pathogenic mechanism in PD. We assess the tractability of mitophagy pathways and prospects for drug discovery and consider intervention points for mitophagy enhancement. We explore the numerous hit molecules beginning to emerge from high-content/high-throughput screening as well as the biochemical and phenotypic assays that enabled these screens. The chemical and biological properties of these reference compounds suggest many could be used to interrogate and perturb mitochondrial biology to validate promising drug targets. Finally, we address key considerations and challenges in achieving pre-clinical proof-of-concept, including in vivo mitophagy reporter methodologies and disease models, as well as patient stratification and biomarker development for mitochondrial forms of the disease.

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