Other articles by Patrick A. Lewis on PubMed

• GTP Binding and Intramolecular Regulation by the ROC Domain of Death Associated Protein Kinase 1 Scientific Reports.   |   Pubmed ID: 23019516 The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases.
• α-Synuclein Mutations Cluster Around a Putative Protein Loop Neuroscience Letters.   |   Pubmed ID: 23669636 With the recent identification of two new pathogenic mutations in α-synuclein, we map the five known pathogenic mutations onto the best available models of the protein structure. We show that four of the five mutations map to a potential fold in the protein with the exception being the A30P mutation in which the substitution would be expected to have a profound effect on protein structure. We discuss this localisation in terms of the proposed mechanisms for mutation pathogenicity.
• Dysfunction of the Autophagy/lysosomal Degradation Pathway is a Shared Feature of the Genetic Synucleinopathies FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology.   |   Pubmed ID: 23682122 The past decade has witnessed huge advances in our understanding of the genetics underlying Parkinson's disease. Identifying commonalities in the biological function of genes linked to Parkinson's provides an opportunity to elucidate pathways that lead to neuronal degeneration and eventually to disease. We propose that the genetic forms of Parkinson's disease largely associated with α-synuclein-positive neuropathology (SNCA, LRRK2, and GBA) are brought together by involvement in the autophagy/lysosomal pathway and that this represents a unifying pathway to disease in these cases.
• LRRK2: Cause, Risk, and Mechanism Journal of Parkinson's Disease.   |   Pubmed ID: 23938341 In 2004 it was first shown that mutations in LRRK2 can cause Parkinson's disease. This initial discovery was quickly followed by the observation that a single particular mutation is a relatively common cause of Parkinson's disease across varied populations. Further genetic investigation has revealed a variety of genetic ties to Parkinson's disease across this gene. These include common alleles with quite broad effects on risk, likely through both alterations at the protein sequence level, and in the context of expression. A great deal of functional characterization of LRRK2 and disease-causing mutations in this protein has occurred over the last 9 years, and considerable progress has been made. Particular attention has been paid to the kinase activity of LRRK2 as a therapeutic target, and while it is no means certain that this is viable target it is likely that this hypothesis will be tested in clinical trials sooner rather than later. We believe that the future goals for LRRK2 research are, while challenging, relatively clear and that the next 10 years of research promises to be perhaps more exciting than the last.
• Rare Variants in LRRK1 and Parkinson's Disease Neurogenetics.   |   Pubmed ID: 24241507 Approximately 20 % of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset PD to identify 15 potentially causal variants. Segregation analysis and frequency assessment in 862 PD cases and 1,014 ethnically matched controls highlighted variants in EEF1D and LRRK1 as the best candidates. Mutation screening of the coding regions of these genes in 862 cases and 1,014 controls revealed several novel non-synonymous variants in both genes in cases and controls. An in silico multi-model bioinformatics analysis was used to prioritize identified variants in LRRK1 for functional follow-up. However, protein expression, subcellular localization, and cell viability were not affected by the identified variants. Although it has yet to be proven conclusively that variants in LRRK1 are indeed causative of PD, our data strengthen a possible role for LRRK1 in addition to LRRK2 in the genetic underpinnings of PD but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.
• Genetic, Structural, and Molecular Insights into the Function of Ras of Complex Proteins Domains Chemistry & Biology.   |   Pubmed ID: 24981771 Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson's disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural information available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.
• Computational Analysis of the LRRK2 Interactome PeerJ.   |   Pubmed ID: 25737818 LRRK2 was identified in 2004 as the causative protein product of the Parkinson's disease locus designated PARK8. In the decade since then, genetic studies have revealed at least 6 dominant mutations in LRRK2 linked to Parkinson's disease, alongside one associated with cancer. It is now well established that coding changes in LRRK2 are one of the most common causes of Parkinson's. Genome-wide association studies (GWAs) have, more recently, reported single nucleotide polymorphisms (SNPs) around the LRRK2 locus to be associated with risk of developing sporadic Parkinson's disease and inflammatory bowel disorder. The functional research that has followed these genetic breakthroughs has generated an extensive literature regarding LRRK2 pathophysiology; however, there is still no consensus as to the biological function of LRRK2. To provide insight into the aspects of cell biology that are consistently related to LRRK2 activity, we analysed the plethora of candidate LRRK2 interactors available through the BioGRID and IntAct data repositories. We then performed GO terms enrichment for the LRRK2 interactome. We found that, in two different enrichment portals, the LRRK2 interactome was associated with terms referring to transport, cellular organization, vesicles and the cytoskeleton. We also verified that 21 of the LRRK2 interactors are genetically linked to risk for Parkinson's disease or inflammatory bowel disorder. The implications of these findings are discussed, with particular regard to potential novel areas of investigation.
• Distinct Clinical and Neuropathological Features of G51D SNCA Mutation Cases Compared with SNCA Duplication and H50Q Mutation Molecular Neurodegeneration.   |   Pubmed ID: 26306801 We and others have described the neurodegenerative disorder caused by G51D SNCA mutation which shares characteristics of Parkinson's disease (PD) and multiple system atrophy (MSA). The objective of this investigation was to extend the description of the clinical and neuropathological hallmarks of G51D mutant SNCA-associated disease by the study of two additional cases from a further G51D SNCA kindred and to compare the features of this group with a SNCA duplication case and a H50Q SNCA mutation case.
• Parkinson's Disease: From Human Genetics to Clinical Trials Science Translational Medicine.   |   Pubmed ID: 26378242 Combining genetic insights into the pathogenesis of Parkinson's disease (PD) with findings from animal and cellular models of this disorder has advanced our understanding of the pathways that lead to the characteristic degeneration of dopaminergic neurons in the brain's nigrostriatal pathway. This has fueled an increase in candidate compounds designed to modulate these pathways and to alter the processes underlying neuronal death in this disorder. Using mitochondrial quality control and the macroautophagy/lysosomal pathways as examples, we discuss the pipeline from a comprehensive genetic architecture for PD through to clinical trials for drugs targeting pathways linked to neurodegeneration in PD. We also identify opportunities and pitfalls on the road to a clinically effective disease-modifying treatment for this disease.
• Genetic and Phenotypic Characterization of Complex Hereditary Spastic Paraplegia Brain : a Journal of Neurology.   |   Pubmed ID: 27217339 The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease-causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15 Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson's disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.
• MTOR Independent Regulation of Macroautophagy by Leucine Rich Repeat Kinase 2 Via Beclin-1 Scientific Reports.   |   Pubmed ID: 27731364 Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson's disease, Crohn's disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase.
• Genome, Transcriptome and Proteome: the Rise of Omics Data and Their Integration in Biomedical Sciences Briefings in Bioinformatics.   |   Pubmed ID: 27881428 Advances in the technologies and informatics used to generate and process large biological data sets (omics data) are promoting a critical shift in the study of biomedical sciences. While genomics, transcriptomics and proteinomics, coupled with bioinformatics and biostatistics, are gaining momentum, they are still, for the most part, assessed individually with distinct approaches generating monothematic rather than integrated knowledge. As other areas of biomedical sciences, including metabolomics, epigenomics and pharmacogenomics, are moving towards the omics scale, we are witnessing the rise of inter-disciplinary data integration strategies to support a better understanding of biological systems and eventually the development of successful precision medicine. This review cuts across the boundaries between genomics, transcriptomics and proteomics, summarizing how omics data are generated, analysed and shared, and provides an overview of the current strengths and weaknesses of this global approach. This work intends to target students and researchers seeking knowledge outside of their field of expertise and fosters a leap from the reductionist to the global-integrative analytical approach in research.
• Weighted Protein Interaction Network Analysis of Frontotemporal Dementia Journal of Proteome Research.   |   Pubmed ID: 28004582 The genetic analysis of complex disorders has undoubtedly led to the identification of a wealth of associations between genes and specific traits. However, moving from genetics to biochemistry one gene at a time has, to date, rather proved inefficient and under-powered to comprehensively explain the molecular basis of phenotypes. Here we present a novel approach, weighted protein-protein interaction network analysis (W-PPI-NA), to highlight key functional players within relevant biological processes associated with a given trait. This is exemplified in the current study by applying W-PPI-NA to frontotemporal dementia (FTD): We first built the state of the art FTD protein network (FTD-PN) and then analyzed both its topological and functional features. The FTD-PN resulted from the sum of the individual interactomes built around FTD-spectrum genes, leading to a total of 4198 nodes. Twenty nine of 4198 nodes, called inter-interactome hubs (IIHs), represented those interactors able to bridge over 60% of the individual interactomes. Functional annotation analysis not only reiterated and reinforced previous findings from single genes and gene-coexpression analyses but also indicated a number of novel potential disease related mechanisms, including DNA damage response, gene expression regulation, and cell waste disposal and potential biomarkers or therapeutic targets including EP300. These processes and targets likely represent the functional core impacted in FTD, reflecting the underlying genetic architecture contributing to disease. The approach presented in this study can be applied to other complex traits for which risk-causative genes are known as it provides a promising tool for setting the foundations for collating genomics and wet laboratory data in a bidirectional manner. This is and will be critical to accelerate molecular target prioritization and drug discovery.
• LRRK2 and Autophagy Advances in Neurobiology.   |   Pubmed ID: 28353280 Leucine-rich repeat kinase 2 (LRRK2) has been implicated in a wide range of cellular processes, including the catabolic pathways collectively described as autophagy. In this chapter, the evidence linking LRRK2 to autophagy will be examined, along with how regulation of autophagy and lysosomal pathways may provide a nexus between the physiological function of this protein and the different diseases with which it has been associated. Data from cellular and animal models for LRRK2 function and dysfunction support a role in the regulation and control of autophagic pathways in the cell, although the extant results do not provide a clear indication as to whether LRRK2 is a positive or negative regulator of these pathways, and there are conflicting data as to the impact of mutations in LRRK2 causative for Parkinson's disease. Given that LRRK2 is a priority drug target for Parkinson's, the evidence suggesting that knockout or inhibition of LRRK2 can result in deregulation of autophagy may have important implications and is discussed in the context of our wider understanding of LRRK2.
• Increased Brain Expression of GPNMB is Associated with Genome Wide Significant Risk for Parkinson's Disease on Chromosome 7p15.3 Neurogenetics.   |   Pubmed ID: 28391543 Genome wide association studies (GWAS) for Parkinson's disease (PD) have previously revealed a significant association with a locus on chromosome 7p15.3, initially designated as the glycoprotein non-metastatic melanoma protein B (GPNMB) locus. In this study, the functional consequences of this association on expression were explored in depth by integrating different expression quantitative trait locus (eQTL) datasets (Braineac, CAGEseq, GTEx, and Phenotype-Genotype Integrator (PheGenI)). Top risk SNP rs199347 eQTLs demonstrated increased expressions of GPNMB, KLHL7, and NUPL2 with the major allele (AA) in brain, with most significant eQTLs in cortical regions, followed by putamen. In addition, decreased expression of the antisense RNA KLHL7-AS1 was observed in GTEx. Furthermore, rs199347 is an eQTL with long non-coding RNA (AC005082.12) in human tissues other than brain. Interestingly, transcript-specific eQTLs in immune-related tissues (spleen and lymphoblastoid cells) for NUPL2 and KLHL7-AS1 were observed, which suggests a complex functional role of this eQTL in specific tissues, cell types at specific time points. Significantly increased expression of GPNMB linked to rs199347 was consistent across all datasets, and taken in combination with the risk SNP being located within the GPNMB gene, these results suggest that increased expression of GPNMB is the causative link explaining the association of this locus with PD. However, other transcript eQTLs and subsequent functional roles cannot be excluded. This highlights the importance of further investigations to understand the functional interactions between the coding genes, antisense, and non-coding RNA species considering the tissue and cell-type specificity to understand the underlying biological mechanisms in PD.
• Leucine Rich Repeat Kinase 2: Beyond Parkinson's and Beyond Kinase Inhibitors Expert Opinion on Therapeutic Targets.   |   Pubmed ID: 28609155
• Estimating the Causal Influence of Body Mass Index on Risk of Parkinson Disease: A Mendelian Randomisation Study PLoS Medicine.   |   Pubmed ID: 28609445 Both positive and negative associations between higher body mass index (BMI) and Parkinson disease (PD) have been reported in observational studies, but it has been difficult to establish causality because of the possibility of residual confounding or reverse causation. To our knowledge, Mendelian randomisation (MR)-the use of genetic instrumental variables (IVs) to explore causal effects-has not previously been used to test the effect of BMI on PD.
• The LRRK2 Signalling System Cell and Tissue Research.   |   Pubmed ID: 29308544 The LRRK2 gene is a major contributor to genetic risk for Parkinson's disease and understanding the biology of the leucine-rich repeat kinase 2 (LRRK2, the protein product of this gene) is an important goal in Parkinson's research. LRRK2 is a multi-domain, multi-activity enzyme and has been implicated in a wide range of signalling events within the cell. Because of the complexities of the signal transduction pathways in which LRRK2 is involved, it has been challenging to generate a clear idea as to how mutations and disease associated variants in this gene are altered in disease. Understanding the events in which LRRK2 is involved at a systems level is therefore critical to fully understand the biology and pathobiology of this protein and is the subject of this review.
• PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2 Frontiers in Molecular Neuroscience.   |   Pubmed ID: 29311810 Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.
• Comparative Protein Interaction Network Analysis Identifies Shared and Distinct Functions for the Human ROCO Proteins Proteomics.   |   Pubmed ID: 29513927 Signal transduction cascades governed by kinases and GTPases are a critical component of the command and control of cellular processes, with the precise outcome partly determined by direct protein-protein interactions (PPIs). Here, we use the human ROCO proteins as a model for investigating PPI signaling events-taking advantage of the unique dual kinase/GTPase activities and scaffolding properties of these multidomain proteins. PPI networks are reported that encompass the human ROCO proteins, developed using two complementary approaches. First, using the recently developed weighted PPI network analysis (WPPINA) pipeline, a confidence-weighted overview of validated ROCO protein interactors is obtained from peer-reviewed literature. Second, novel ROCO PPIs are assessed experimentally via protein microarray screens. The networks derived from these orthologous approaches are compared to identify common elements within the ROCO protein interactome; functional enrichment analysis of this common core of the network identified stress response and cell projection organization as shared functions within this protein family. Despite the presence of these commonalities, the results suggest that many unique interactors and therefore some specialized cellular roles have evolved for different members of the ROCO proteins. Overall, this multi-approach strategy to increase the resolution of protein interaction networks represents a prototype for the utility of PPI data integration in understanding signaling biology.
• MTOR Independent Alteration in ULK1 Ser758 Phosphorylation Following Chronic LRRK2 Kinase Inhibition Bioscience Reports.   |   Pubmed ID: 29563162 Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In the present study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein leucine rich repeat kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity.
• Mutations in LRRK2 Amplify Cell-to-cell Protein Aggregate Propagation: a Hypothesis Acta Neuropathologica.   |   Pubmed ID: 29600398
• LRRK2 is a Negative Regulator of Phagosome Maturation in Macrophages The EMBO Journal.   |   Pubmed ID: 29789389 Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced phagosome maturation and mycobacterial control independently of autophagy. , LRRK2 deficiency in mice resulted in a significant decrease in burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls replication by regulating phagosome maturation.
• Stratification of Candidate Genes for Parkinson's Disease Using Weighted Protein-protein Interaction Network Analysis BMC Genomics.   |   Pubmed ID: 29898659 Genome wide association studies (GWAS) have helped identify large numbers of genetic loci that significantly associate with increased risk of developing diseases. However, translating genetic knowledge into understanding of the molecular mechanisms underpinning disease (i.e. disease-specific impacted biological processes) has to date proved to be a major challenge. This is primarily due to difficulties in confidently defining candidate genes at GWAS-risk loci. The goal of this study was to better characterize candidate genes within GWAS loci using a protein interactome based approach and with Parkinson's disease (PD) data as a test case.
• Analysis of Macroautophagy Related Proteins in G2019S LRRK2 Parkinson's Disease Brains with Lewy Body Pathology Brain Research.   |   Pubmed ID: 30055128 LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.
• A Presenilin 1 Mutation Associated with Familial Frontotemporal Dementia Inhibits Gamma-secretase Cleavage of APP and Notch Neurobiology of Disease. Mar, 2002  |   Pubmed ID: 11895378 A novel presenilin 1 mutation, insR352, associated with a frontal temporal dementia phenotype has been identified (E. A. Rogaeva et al., 2001, Neurology 57, 621-625). This mutation does not increase Abeta42 levels, but instead acts as dominant negative presenilin, decreasing amyloid beta protein (Abeta) production by inhibiting gamma-secretase cleavage of the Abeta precursor. The distinct clinical phenotype associated with this mutation suggests that chronic partial inhibition of gamma-secretase activity may result in neurodegeneration.
• Removal of the Glycosylphosphatidylinositol Anchor from PrP(Sc) by Cathepsin D Does Not Reduce Prion Infectivity The Biochemical Journal. Apr, 2006  |   Pubmed ID: 16441239 According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.
• Codon 129 Polymorphism of the Human Prion Protein Influences the Kinetics of Amyloid Formation The Journal of General Virology. Aug, 2006  |   Pubmed ID: 16847141 The human prion protein (PrP) has a common polymorphism at residue 129, which can be valine or methionine. This polymorphism has a strong influence on susceptibility to prion diseases and on prion-strain properties. Previous work has shown that this amino acid variation has no measurable effect on the native structure of cellular PrP (PrPC). Here, it is shown that the polymorphism does not change the efficiency of conversion to the beta-PrP conformation or affect the binding of copper(II) ions. However, in a partially denatured conformation, the polymorphic variation has a profound influence on the ability of the protein to form amyloid fibrils spontaneously.
• Mutations in LRRK2/dardarin Associated with Parkinson Disease Are More Toxic Than Equivalent Mutations in the Homologous Kinase LRRK1 Journal of Neurochemistry. Jul, 2007  |   Pubmed ID: 17394548 Several mutations have been found in the leucine-rich repeat kinase 2 gene (LRRK2), encoding the protein dardarin, which are associated with autosomal dominant Parkinson disease. We have previously shown that mutant LRRK2/dardarin is toxic to neurons and neuron-like cell lines in culture and that some mutations are also associated with an inclusion-body phenotype. There is a homologous kinase, LRRK1, which has a similar domain structure but is not known to carry mutations causing Parkinson disease. In the current study, we introduced mutations at equivalent residues in both LRRK2 and LRRK1 to determine their effects in cells. We show that mutations in dardarin are more prone to form inclusion bodies in transfected cells and are more toxic than equivalent mutations in LRRK1. This work suggests that dardarin/LRRK2 is inherently more damaging than LRRK1.
• The R1441C Mutation of LRRK2 Disrupts GTP Hydrolysis Biochemical and Biophysical Research Communications. Jun, 2007  |   Pubmed ID: 17442267 Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.
• The Metalloprotease Inhibitor TIMP-3 Regulates Amyloid Precursor Protein and Apolipoprotein E Receptor Proteolysis The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2007  |   Pubmed ID: 17913923 Cellular cholesterol levels alter the processing of the amyloid precursor protein (APP) to produce Abeta. Activation of liver X receptors (LXRs), one cellular mechanism to regulate cholesterol homeostasis, has been found to alter Abeta levels in vitro and in vivo. To identify genes regulated by LXR, we treated human neuroblastoma cells with an LXR agonist (TO-901317) and examined gene expression by microarray. As expected, TO-901317 upregulated several cholesterol metabolism genes, but it also decreased expression of a metalloprotease inhibitor, TIMP-3. We confirmed this finding using real-time PCR and by measuring TIMP-3 protein in glia, SY5Y cells, and COS7 cells. TIMP-3 is a member of a family of metalloproteinase inhibitors and blocks A disintegrin and metalloproteinase-10 (ADAM-10) and ADAM-17, two APP alpha-secretases. We found that TIMP-3 inhibited alpha-secretase cleavage of APP and an apolipoprotein E (apoE) receptor, ApoER2. TIMP-3 decreased surface levels of ADAM-10, APP, and ApoER2. These changes were accompanied by increased APP beta-C-terminal fragment and Abeta production. These data suggest that TIMP-3 preferentially routes APP and ApoER2 away from the cell surface and alpha-secretase cleavage and encourages endocytosis and beta-secretase cleavage. In vivo, TO-901317 decreased brain TIMP-3 levels. TIMP-3 protein levels were increased in human Alzheimer's disease (AD) brain and in APP transgenic mice, suggesting that increased levels of TIMP-3 in AD may contribute to higher levels of Abeta.
• Structure of the ROC Domain from the Parkinson's Disease-associated Leucine-rich Repeat Kinase 2 Reveals a Dimeric GTPase Proceedings of the National Academy of Sciences of the United States of America. Feb, 2008  |   Pubmed ID: 18230735 Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity. The structure of ROC and the mechanism(s) by which it regulates kinase activity are not known. Here, we report the crystal structure of the LRRK2 ROC domain in complex with GDP-Mg(2+) at 2.0-A resolution. The structure displays a dimeric fold generated by extensive domain-swapping, resulting in a pair of active sites constructed with essential functional groups contributed from both monomers. Two PD-associated pathogenic residues, R1441 and I1371, are located at the interface of two monomers and provide exquisite interactions to stabilize the ROC dimer. The structure demonstrates that loss of stabilizing forces in the ROC dimer is likely related to decreased GTPase activity resulting from mutations at these sites. Our data suggest that the ROC domain may regulate LRRK2 kinase activity as a dimer, possibly via the C-terminal of ROC (COR) domain as a molecular hinge. The structure of the LRRK2 ROC domain also represents a signature from a previously undescribed class of GTPases from complex proteins and results may provide a unique molecular target for therapeutics in PD.
• Genetic Neuropathology of Parkinson's Disease International Journal of Clinical and Experimental Pathology. 2008  |   Pubmed ID: 18784814 Parkinson's disease (PD) has long been considered to be a sporadic entity, perhaps with an environmental etiology. However, recent genetic discoveries have challenged this view, as there are many families with diseases of Mendelian inheritance that clinically resemble PD. Here, we will review in detail the neuropathological data relating to familial cases of PD. We will discuss the complicated relationships between the genetically defined cases and the two key pathological events seen in PD, namely loss of dopaminergic neurons in the substantia nigra pars compacta and the formation of protein inclusions, Lewy bodies, in the neurons that survive to the end stage of the disease course. These observations will be synthesized into an overall scheme that emphasizes the two key aspects of the neuropathology as distinct events and suggest that each gene tells us something a little different about the neuropathology of PD.
• Emerging Pathways in Genetic Parkinson's Disease The FEBS Journal. Dec, 2008  |   Pubmed ID: 19021751
• Emerging Pathways in Genetic Parkinson's Disease: Tangles, Lewy Bodies and LRRK2 The FEBS Journal. Dec, 2008  |   Pubmed ID: 19021752 The last decade has seen clear links emerge between the genetic determinants and neuropathological hallmarks of parkinsonism and dementia, notably with the discovery of mutations in alpha-synuclein and tau. Following the description of mutations in LRRK2 linked to Parkinson's disease, characterized by variable pathology including either alpha-synuclein or tau deposition, it has been suggested that LRRK2 functions as an upstream regulator of Parkinson's disease pathogenesis. This minireview explores this model, in the context of our current understanding of the biochemistry of LRRK2, alpha-synuclein and tau.
• The Function of ROCO Proteins in Health and Disease Biology of the Cell / Under the Auspices of the European Cell Biology Organization. Mar, 2009  |   Pubmed ID: 19152505 The ROCO family of multidomain proteins extends across the eukaryotes, and has been implicated in numerous cellular processes. Following the description of mutations causing PD (Parkinson's disease) in a human representative of the ROCO family, LRRK2 (leucine-rich repeat kinase 2), a great deal of research has been carried out into these proteins. This review examines the published data regarding the roles the ROCO proteins are thought to play in cell processes, and how the structure and domain organization of these proteins relates to their function.
• The R1441C Mutation Alters the Folding Properties of the ROC Domain of LRRK2 Biochimica Et Biophysica Acta. Dec, 2009  |   Pubmed ID: 19781641 LRRK2 is a 250 kDa multidomain protein, mutations in which cause familial Parkinson's disease. Previously, we have demonstrated that the R1441C mutation in the ROC domain decreases GTPase activity. Here we show that the R1441C alters the folding properties of the ROC domain, lowering its thermodynamic stability. Similar to small GTPases, binding of different guanosine nucleotides alters the stability of the ROC domain, suggesting that there is an alteration in conformation dependent on GDP or GTP occupying the active site. GTP/GDP bound state also alters the self-interaction of the ROC domain, accentuating the impact of the R1441C mutation on this property. These data suggest a mechanism whereby the R1441C mutation can reduce the GTPase activity of LRRK2, and highlights the possibility of targeting the stability of the ROC domain as a therapeutic avenue in LRRK2 disease.
• Cancer and Neurodegeneration: Between the Devil and the Deep Blue Sea PLoS Genetics. 2010  |   Pubmed ID: 21203498 Cancer and neurodegeneration are often thought of as disease mechanisms at opposite ends of a spectrum; one due to enhanced resistance to cell death and the other due to premature cell death. There is now accumulating evidence to link these two disparate processes. An increasing number of genetic studies add weight to epidemiological evidence suggesting that sufferers of a neurodegenerative disorder have a reduced incidence for most cancers, but an increased risk for other cancers. Many of the genes associated with either cancer and/or neurodegeneration play a central role in cell cycle control, DNA repair, and kinase signalling. However, the links between these two families of diseases remain to be proven. In this review, we discuss recent and sometimes as yet incomplete genetic discoveries that highlight the overlap of molecular pathways implicated in cancer and neurodegeneration.
• A Tangled Web - Tau and Sporadic Parkinson's Disease Frontiers in Psychiatry / Frontiers Research Foundation. 2010  |   Pubmed ID: 21423457 Parkinson's disease (PD) represents a major challenge for health care systems around the world: it is the most common degenerative movement disorder of old age, affecting over 100,000 people in the UK alone (Schrag et al., 2000). Despite the remarkable success of treatments directed at potentiating or replacing dopamine within the brain, which can relieve symptoms for over a decade, PD remains an incurable and invariably fatal disorder. As such, efforts to understand the processes that lead to cell death in the brains of patients with PD are a priority for neurodegenerative researchers. A great deal of progress has been made in this regard by taking advantage of advances in genetics, initially by the identification of genes responsible for rare Mendelian forms of PD (outlined in Table 1), and more recently by applying genome wide association studies (GWAS) to the sporadic form of the disease (Hardy et al., 2009). Several such GWAS have now been carried out, with a meta-analysis currently under way. Using over 6000 cases and 10,000 controls, two of these studies have identified variation at a number of loci as being associated with an increased risk of disease (Satake et al., 2009; Simon-Sanchez et al., 2009). Three genes stand out as candidates from these studies - the SNCA gene, coding for α-synuclein, the LRRK2 gene, coding for leucine rich repeat kinase 2, and MAPT, coding for the microtubule-associated protein tau. Mutations at all three of these loci have been associated with Mendelian forms of disease presenting with the clinical syndrome of Parkinsonism, however only SNCA and LRRK2 have been previously associated with pathologically defined PD (Hardy et al., 2009). Point mutations in α-synuclein, along with gene multiplication events, result in autosomal dominant PD, often with a significant dementia component. In addition to this, α-synuclein is the principle component of the main pathological hallmark of idiopathic PD, the Lewy body, making it an unsurprising hit in the GWAS (Spillantini et al., 1997). Mutations in LRRK2 are the most common genetic cause of PD, and so again made this gene a likely candidate as a susceptibility locus for the sporadic form of disease (Kumari and Tan, 2009). More surprising, perhaps, was the identification of tau as a susceptibility factor for Parkinson's. In this review we will outline the role of tau in neurodegeneration and in different forms of Parkinsonism, and speculate as to what the functional basis of the association between MAPT and PD might be.
• Pathogenic LRRK2 Mutations Do Not Alter Gene Expression in Cell Model Systems or Human Brain Tissue PloS One. 2011  |   Pubmed ID: 21799870 Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.
• Parkinson's Disease Induced Pluripotent Stem Cells with Triplication of the α-synuclein Locus Nature Communications. 2011  |   Pubmed ID: 21863007 A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction.
• Gene Expression in the Parkinson's Disease Brain Brain Research Bulletin. Dec, 2011  |   Pubmed ID: 22173063 The study of gene expression has undergone a transformation in the past decade as the benefits of the sequencing of the human genome have made themselves felt. Increasingly, genome wide approaches are being applied to the analysis of gene expression in human disease as a route to understanding the underlying pathogenic mechanisms. In this review, we will summarise current state of gene expression studies of the brain in Parkinson's disease, and examine how these techniques can be used to gain an insight into aetiology of this devastating disorder.
• LRRK2 and Human Disease: A Complicated Question or a Question of Complexes? Science Signaling. 2012  |   Pubmed ID: 22253261 Leucine-rich repeat kinase 2 (LRRK2) is linked to various diseases, including Parkinson's disease, cancer, and leprosy. Data from LRRK2 knockout mice has highlighted a possible role for LRRK2 in regulating signaling pathways that are linked to the pathogenesis of Crohn's disease. Here, we examine how LRRK2's role as a signaling hub in the cell could lead to diverse pathologies.
• Correction: Pathogenic LRRK2 Mutations Do Not Alter Gene Expression in Cell Model Systems or Human Brain Tissue PloS One. 2012  |   Pubmed ID: 22303433 [This corrects the article on p. e22489 in vol. 6.].
• Divergent α-synuclein Solubility and Aggregation Properties in G2019S LRRK2 Parkinson's Disease Brains with Lewy Body Pathology Compared to Idiopathic Cases Neurobiology of Disease. Oct, 2013  |   Pubmed ID: 23747310 Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). The most prevalent LRRK2 mutation is the G2019S coding change, located in the kinase domain of this complex multi-domain protein. The majority of G2019S autopsy cases feature typical Lewy Body pathology with a clinical phenotype almost indistinguishable from idiopathic PD (iPD). Here we have investigated the biochemical characteristics of α-synuclein in G2019S LRRK2 PD post-mortem material, in comparison to pathology-matched iPD. Immunohistochemistry with pS129 α-synuclein antibody showed that the medulla is heavily affected with pathology in G2019S PD whilst the basal ganglia (BG), limbic and frontal cortical regions demonstrated comparable pathology scores between G2019S PD and iPD. Significantly lower levels of the highly aggregated α-synuclein species in urea-SDS fractions were observed in G2019S cases compared to iPD in the BG and limbic cortex. Our data, albeit from a small number of cases, highlight a difference in the biochemical properties of aggregated α-synuclein in G2019S linked PD compared to iPD, despite a similar histopathological presentation. This divergence in solubility is most notable in the basal ganglia, a region that is affected preclinically and is damaged before overt dopaminergic cell death.
• Inhibition of LRRK2 Kinase Activity Stimulates Macroautophagy Biochimica Et Biophysica Acta. Dec, 2013  |   Pubmed ID: 23916833 Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.
• Fine-mapping, Gene Expression and Splicing Analysis of the Disease Associated LRRK2 Locus PloS One. 2013  |   Pubmed ID: 23967090 Association studies have identified several signals at the LRRK2 locus for Parkinson's disease (PD), Crohn's disease (CD) and leprosy. However, little is known about the molecular mechanisms mediating these effects. To further characterize this locus, we fine-mapped the risk association in 5,802 PD and 5,556 controls using a dense genotyping array (ImmunoChip). Using samples from 134 post-mortem control adult human brains (UK Human Brain Expression Consortium), where up to ten brain regions were available per individual, we studied the regional variation, splicing and regulation of LRRK2. We found convincing evidence for a common variant PD association located outside of the LRRK2 protein coding region (rs117762348, A>G, P = 2.56×10(-8), case/control MAF 0.083/0.074, odds ratio 0.86 for the minor allele with 95% confidence interval [0.80-0.91]). We show that mRNA expression levels are highest in cortical regions and lowest in cerebellum. We find an exon quantitative trait locus (QTL) in brain samples that localizes to exons 32-33 and investigate the molecular basis of this eQTL using RNA-Seq data in n = 8 brain samples. The genotype underlying this eQTL is in strong linkage disequilibrium with the CD associated non-synonymous SNP rs3761863 (M2397T). We found two additional QTLs in liver and monocyte samples but none of these explained the common variant PD association at rs117762348. Our results characterize the LRRK2 locus, and highlight the importance and difficulties of fine-mapping and integration of multiple datasets to delineate pathogenic variants and thus develop an understanding of disease mechanisms.
• Pathogenic Parkinson's Disease Mutations Across the Functional Domains of LRRK2 Alter the Autophagic/lysosomal Response to Starvation Biochemical and Biophysical Research Communications. Nov, 2013  |   Pubmed ID: 24211199 LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.
• Parkinson's Disease-linked Mutations in VPS35 Induce Dopaminergic Neurodegeneration Human Molecular Genetics. Sep, 2014  |   Pubmed ID: 24740878 Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.
• Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution The Journal of Biological Chemistry. Aug, 2014  |   Pubmed ID: 24942733 Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser(910)/Ser(935) mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser(910)/Ser(935) phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser(910)/Ser(935) phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function.
• Leucine-rich Repeat Kinase 2 Interacts with P21-activated Kinase 6 to Control Neurite Complexity in Mammalian Brain Journal of Neurochemistry. Sep, 2015  |   Pubmed ID: 26375402 Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.
• Additional Rare Variant Analysis in Parkinson's Disease Cases with and Without Known Pathogenic Mutations: Evidence for Oligogenic Inheritance Human Molecular Genetics. Oct, 2016  |   Pubmed ID: 27798102 Oligogenic inheritance implies a role for several genetic factors in disease etiology. We studied oligogenic inheritance in Parkinson's (PD) by assessing the potential burden of additional rare variants in established Mendelian genes and/or GBA, in individuals with and without a primary pathogenic genetic cause in two large independent cohorts totaling 7,900 PD cases and 6,166 controls. An excess (≥30%) of cases with a recognised primary genetic cause had ≥1 additional rare variants in Mendelian PD genes, as compared with no known mutation PD cases (17%) and unaffected controls (16%), supporting our hypothesis. Carriers of additional Mendelian gene variants have younger ages at onset (AAO). The effect of additional Mendelian variants in LRRK2 G2019S mutation carriers, of which ATP13A2 variation is particularly common, may account for some of the variation in penetrance. About 10% of No Known Mutation-PD cases harbour a rare GBA variant compared to known pathogenic mutation PD cases (8%) and controls (5%), with carriers having earlier AAOs. Together, the data suggest that the oligogenic inheritance of rare Mendelian variants may be important in patient with a primary pathogenic cause, whereas GBA increases risk across all forms of PD. This study highlights the potential genetic complexity of Mendelian PD. The identification of potential modifying variants provides new insights into disease mechanisms by potentially separating relevant from benign variants and by the interaction between genes in specific pathways. In the future this may be relevant to genetic testing and counselling of patients with PD and their families.