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In JoVE (1)
Other Publications (19)
- Neurobiology of Disease
- The Biochemical Journal
- The Journal of General Virology
- Journal of Neurochemistry
- Biochemical and Biophysical Research Communications
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Proceedings of the National Academy of Sciences of the United States of America
- International Journal of Clinical and Experimental Pathology
- The FEBS Journal
- The FEBS Journal
- Biology of the Cell / Under the Auspices of the European Cell Biology Organization
- Biochimica Et Biophysica Acta
- PLoS Genetics
- Frontiers in Psychiatry / Frontiers Research Foundation
- PloS One
- Nature Communications
- Brain Research Bulletin
- Science Signaling
- PloS One
Articles by Patrick A. Lewis in JoVE
JoVE General
Assaying the Kinase Activity of LRRK2 in vitro
Department of Molecular Neuroscience, UCL Institute of Neurology
Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.
Other articles by Patrick A. Lewis on PubMed
A Presenilin 1 Mutation Associated with Familial Frontotemporal Dementia Inhibits Gamma-secretase Cleavage of APP and Notch
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
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 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
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
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
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
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
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: Tangles, Lewy Bodies and LRRK2
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
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
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
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
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
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
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
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?
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
[This corrects the article on p. e22489 in vol. 6.].
