397 results on '"Mark R Cookson"'
Search Results
2. Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.
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Adamantios Mamais, Jillian H Kluss, Luis Bonet-Ponce, Natalie Landeck, Rebekah G Langston, Nathan Smith, Alexandra Beilina, Alice Kaganovich, Manik C Ghosh, Laura Pellegrini, Ravindran Kumaran, Ioannis Papazoglou, George R Heaton, Rina Bandopadhyay, Nunziata Maio, Changyoun Kim, Matthew J LaVoie, David C Gershlick, and Mark R Cookson
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Biology (General) ,QH301-705.5 - Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.
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- 2021
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3. LAG3 is not expressed in human and murine neurons and does not modulate α‐synucleinopathies
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Marc Emmenegger, Elena De Cecco, Marian Hruska‐Plochan, Timo Eninger, Matthias M Schneider, Melanie Barth, Elena Tantardini, Pierre de Rossi, Mehtap Bacioglu, Rebekah G Langston, Alice Kaganovich, Nora Bengoa‐Vergniory, Andrès Gonzalez‐Guerra, Merve Avar, Daniel Heinzer, Regina Reimann, Lisa M Häsler, Therese W Herling, Naunehal S Matharu, Natalie Landeck, Kelvin Luk, Ronald Melki, Philipp J Kahle, Simone Hornemann, Tuomas P J Knowles, Mark R Cookson, Magdalini Polymenidou, Mathias Jucker, and Adriano Aguzzi
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LAG3 ,neurodegeneration ,prionoids ,α‐synuclein ,Medicine (General) ,R5-920 ,Genetics ,QH426-470 - Abstract
Abstract While the initial pathology of Parkinson’s disease and other α‐synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α‐synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α‐synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α‐synuclein pathology ex vivo. The overall survival of A53T α‐synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α‐synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α‐synucleinopathies is not universally valid.
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- 2021
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4. Post-translational decrease in respiratory chain proteins in the Polg mutator mouse brain.
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David N Hauser, Allissa A Dillman, Jinhui Ding, Yan Li, and Mark R Cookson
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Medicine ,Science - Abstract
Mitochondrial DNA damage is thought to be a causal contributor to aging as mice with inactivating mutations in polymerase gamma (Polg) develop a progeroid phenotype. To further understand the molecular mechanisms underlying this phenotype, we used iTRAQ and RNA-Seq to determine differences in protein and mRNA abundance respectively in the brains of one year old Polg mutator mice compared to control animals. We found that mitochondrial respiratory chain proteins are specifically decreased in abundance in the brains of the mutator mice, including several nuclear encoded mitochondrial components. However, we found no evidence that the changes we observed in protein levels were the result of decreases in mRNA expression. These results show that there are post-translational effects associated with mutations in Polg.
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- 2014
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5. LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes
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Fumitaka Kawakami, Motoki Imai, Yuki Isaka, Mark R. Cookson, Hiroko Maruyama, Makoto Kubo, Matthew J. Farrer, Makoto Kanzaki, Rei Kawashima, Tatsunori Maekawa, Shun Tamaki, Yoshifumi Kurosaki, Fumiaki Kojima, Kenichi Ohba, and Takafumi Ichikawa
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adipocyte ,glucose tolerance ,GLUT4 ,LRRK2 ,Rab GTPase ,Biology (General) ,QH301-705.5 - Abstract
Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine‐rich repeat kinase 2 (LRRK2)‐associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high‐fat diet (HFD)‐induced glucose intolerance using Lrrk2‐knockout (KO) mice. We found for the first time that HFD‐fed KO mice display improved glucose tolerance compared with their wild‐type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin‐related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin‐dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3‐L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8‐ and Rab10‐mediated GLUT4 membrane translocation.
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- 2023
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6. Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.
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Laura Civiero, Renée Vancraenenbroeck, Elisa Belluzzi, Alexandra Beilina, Evy Lobbestael, Lauran Reyniers, Fangye Gao, Ivan Micetic, Marc De Maeyer, Luigi Bubacco, Veerle Baekelandt, Mark R Cookson, Elisa Greggio, and Jean-Marc Taymans
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Medicine ,Science - Abstract
Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.
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- 2012
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7. Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.
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Tohru Matsuki, Mariam Zaka, Rita Guerreiro, Marcel P van der Brug, Jonathan A Cooper, Mark R Cookson, John A Hardy, and Brian W Howell
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Medicine ,Science - Abstract
Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.
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- 2012
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8. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.
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Jean-Marc Taymans, Renée Vancraenenbroeck, Petri Ollikainen, Alexandra Beilina, Evy Lobbestael, Marc De Maeyer, Veerle Baekelandt, and Mark R Cookson
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Medicine ,Science - Abstract
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
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- 2011
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9. Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue.
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Michael J Devine, Alice Kaganovich, Mina Ryten, Adamantios Mamais, Daniah Trabzuni, Claudia Manzoni, Philip McGoldrick, Diane Chan, Allissa Dillman, Julia Zerle, Susannah Horan, Jan-Willem Taanman, John Hardy, Jose-Felix Marti-Masso, Daniel Healy, Anthony H Schapira, Benjamin Wolozin, Rina Bandopadhyay, Mark R Cookson, Marcel P van der Brug, and Patrick A Lewis
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Medicine ,Science - Abstract
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.
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- 2011
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10. Abundant quantitative trait loci exist for DNA methylation and gene expression in human brain.
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J Raphael Gibbs, Marcel P van der Brug, Dena G Hernandez, Bryan J Traynor, Michael A Nalls, Shiao-Lin Lai, Sampath Arepalli, Allissa Dillman, Ian P Rafferty, Juan Troncoso, Robert Johnson, H Ronald Zielke, Luigi Ferrucci, Dan L Longo, Mark R Cookson, and Andrew B Singleton
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Genetics ,QH426-470 - Abstract
A fundamental challenge in the post-genome era is to understand and annotate the consequences of genetic variation, particularly within the context of human tissues. We present a set of integrated experiments that investigate the effects of common genetic variability on DNA methylation and mRNA expression in four human brain regions each from 150 individuals (600 samples total). We find an abundance of genetic cis regulation of mRNA expression and show for the first time abundant quantitative trait loci for DNA CpG methylation across the genome. We show peak enrichment for cis expression QTLs to be approximately 68,000 bp away from individual transcription start sites; however, the peak enrichment for cis CpG methylation QTLs is located much closer, only 45 bp from the CpG site in question. We observe that the largest magnitude quantitative trait loci occur across distinct brain tissues. Our analyses reveal that CpG methylation quantitative trait loci are more likely to occur for CpG sites outside of islands. Lastly, we show that while we can observe individual QTLs that appear to affect both the level of a transcript and a physically close CpG methylation site, these are quite rare. We believe these data, which we have made publicly available, will provide a critical step toward understanding the biological effects of genetic variation.
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- 2010
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11. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin.
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Derek P Narendra, Seok Min Jin, Atsushi Tanaka, Der-Fen Suen, Clement A Gautier, Jie Shen, Mark R Cookson, and Richard J Youle
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Biology (General) ,QH301-705.5 - Abstract
Loss-of-function mutations in PINK1 and Parkin cause parkinsonism in humans and mitochondrial dysfunction in model organisms. Parkin is selectively recruited from the cytosol to damaged mitochondria to trigger their autophagy. How Parkin recognizes damaged mitochondria, however, is unknown. Here, we show that expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 on healthy, polarized mitochondria, while facilitating the rapid accumulation of PINK1 on mitochondria that sustain damage. PINK1 accumulation on mitochondria is both necessary and sufficient for Parkin recruitment to mitochondria, and disease-causing mutations in PINK1 and Parkin disrupt Parkin recruitment and Parkin-induced mitophagy at distinct steps. These findings provide a biochemical explanation for the genetic epistasis between PINK1 and Parkin in Drosophila melanogaster. In addition, they support a novel model for the negative selection of damaged mitochondria, in which PINK1 signals mitochondrial dysfunction to Parkin, and Parkin promotes their elimination.
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- 2010
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12. The Parkinson's disease associated LRRK2 exhibits weaker in vitro phosphorylation of 4E-BP compared to autophosphorylation.
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Azad Kumar, Elisa Greggio, Alexandra Beilina, Alice Kaganovich, Diane Chan, Jean-Marc Taymans, Benjamin Wolozin, and Mark R Cookson
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Medicine ,Science - Abstract
Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was approximately 2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38alpha and DAPK2) and found that MAPK14/p38alpha could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38alpha phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.
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- 2010
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13. Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.
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Anna Sandebring, Kelly Jean Thomas, Alexandra Beilina, Marcel van der Brug, Megan M Cleland, Rili Ahmad, David W Miller, Ibardo Zambrano, Richard F Cowburn, Homira Behbahani, Angel Cedazo-Mínguez, and Mark R Cookson
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Medicine ,Science - Abstract
PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.
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- 2009
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14. Leucine-Rich Repeat Kinase 2 Mutations and Parkinson's Disease: Three Questions
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Elisa Greggio and Mark R Cookson
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Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Mutations in the gene encoding LRRK2 (leucine-rich repeat kinase 2) were first identified in 2004 and have since been shown to be the single most common cause of inherited Parkinson's disease. The protein is a large GTP-regulated serine/threonine kinase that additionally contains several protein–protein interaction domains. In the present review, we discuss three important, but unresolved, questions concerning LRRK2. We first ask: what is the normal function of LRRK2? Related to this, we discuss the evidence of LRRK2 activity as a GTPase and as a kinase and the available data on protein–protein interactions. Next we raise the question of how mutations affect LRRK2 function, focusing on some slightly controversial results related to the kinase activity of the protein in a variety of in vitro systems. Finally, we discuss what the possible mechanisms are for LRRK2-mediated neurotoxicity, in the context of known activities of the protein.
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- 2009
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15. Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans.
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Joyce van de Leemput, Jayanth Chandran, Melanie A Knight, Lynne A Holtzclaw, Sonja Scholz, Mark R Cookson, Henry Houlden, Katrina Gwinn-Hardy, Hon-Chung Fung, Xian Lin, Dena Hernandez, Javier Simon-Sanchez, Nick W Wood, Paola Giunti, Ian Rafferty, John Hardy, Elsdon Storey, R J McKinlay Gardner, Susan M Forrest, Elizabeth M C Fisher, James T Russell, Huaibin Cai, and Andrew B Singleton
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Genetics ,QH426-470 - Abstract
We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1(Delta18/Delta18)), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5' part of the ITPR1 gene, encompassing exons 1-10, 1-40, and 1-44 in three studied families, underlies SCA15 in humans.
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- 2007
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16. Molecules that cause or prevent Parkinson's disease.
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Mark R Cookson
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Biology (General) ,QH301-705.5 - Published
- 2004
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17. Differential LRRK2 Signalling and Gene Expression in WT-LRRK2 and G2019S-LRRK2 Mouse Microglia Treated with Zymosan and MLi2
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Iqra Nazish, Adamantios Mamais, Anna Mallach, Conceicao Bettencourt, Alice Kaganovich, Thomas Warner, John Hardy, Patrick A. Lewis, Jennifer Pocock, Mark R. Cookson, and Rina Bandopadhyay
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LRRK2 ,TLR2 ,LRRK2 p.G2019S knock-in ,zymosan ,MLi.2 ,RNA-Seq ,Cytology ,QH573-671 - Abstract
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson’s disease (PD), with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggests involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4, resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-sequencing analysis. We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate genome-wide association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated, respectively, with zymosan treatment, while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed that the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.
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- 2023
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18. Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment
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Jillian H. Kluss, Luis Bonet-Ponce, Patrick A. Lewis, and Mark R. Cookson
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LRRK2 ,Parkinson's disease ,Endolysosomal membranes ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway.
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- 2022
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19. Application of Aligned-UMAP to longitudinal biomedical studies.
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Anant Dadu, Vipul Satone, Rachneet Kaur, Mathew J. Koretsky, Hirotaka Iwaki, Yue A. Qi, Daniel M. Ramos, Brian B. Avants, Jacob Hesterman, Roger Gunn, Mark R. Cookson, Michael E. Ward, Andrew B. Singleton, Roy H. Campbell, Mike A. Nalls, and Faraz Faghri
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- 2023
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20. Glial phagocytic clearance in Parkinson’s disease
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Marie-Eve Tremblay, Mark R. Cookson, and Laura Civiero
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Reactive astrocytes ,Reactive microglia ,Phagocytosis ,Parkinson’s disease ,Neurology. Diseases of the nervous system ,RC346-429 ,Geriatrics ,RC952-954.6 - Abstract
Abstract An emerging picture suggests that glial cells’ loss of beneficial roles or gain of toxic functions can contribute to neurodegenerative conditions. Among glial cells, microglia and astrocytes have been shown to play phagocytic roles by engulfing synapses, apoptotic cells, cell debris, and released toxic proteins. As pathogenic protein accumulation is a key feature in Parkinson’s disease (PD), compromised phagocytic clearance might participate in PD pathogenesis. In contrast, enhanced, uncontrolled and potentially toxic glial clearance capacity could contribute to synaptic degeneration. Here, we summarize the current knowledge of the molecular mechanisms underlying microglial and astrocytic phagocytosis, focusing on the possible implication of phagocytic dysfunction in neuronal degeneration. Several endo-lysosomal proteins displaying genetic variants in PD are highly expressed by microglia and astrocytes. We also present the evidence that lysosomal defects can affect phagocytic clearance and discuss the therapeutic relevance of restoring or enhancing lysosomal function in PD.
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- 2019
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21. Specific Detection of Physiological S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay
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Ryan Arlinghaus, Michiyo Iba, Eliezer Masliah, Mark R. Cookson, and Natalie Landeck
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Cellular and Molecular Neuroscience ,Neurology (clinical) - Abstract
Background: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn. Objective: To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background. Methods: We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections. Results: The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue. Conclusion: We successfully developed a novel PLA method that can, in the future, be used on in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.
- Published
- 2023
22. Hallmarks of neurodegenerative diseases
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David M. Wilson, Mark R. Cookson, Ludo Van Den Bosch, Henrik Zetterberg, David M. Holtzman, and Ilse Dewachter
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General Biochemistry, Genetics and Molecular Biology - Published
- 2023
23. Leucine-rich repeat kinase 2 controls protein kinase A activation state through phosphodiesterase 4
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Isabella Russo, Giulietta Di Benedetto, Alice Kaganovich, Jinhui Ding, Daniela Mercatelli, Michele Morari, Mark R. Cookson, Luigi Bubacco, and Elisa Greggio
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LRRK2 ,PKA ,Microglia ,Neuroinflammation ,Parkinson’s disease ,PDE4 ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia. Methods To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling. Results We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming. Conclusions Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.
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- 2018
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24. Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation
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Jesús Madero-Pérez, Elena Fdez, Belén Fernández, Antonio J. Lara Ordóñez, Marian Blanca Ramírez, Patricia Gómez-Suaga, Dieter Waschbüsch, Evy Lobbestael, Veerle Baekelandt, Angus C. Nairn, Javier Ruiz-Martínez, Ana Aiastui, Adolfo López de Munain, Pawel Lis, Thomas Comptdaer, Jean-Marc Taymans, Marie-Christine Chartier-Harlin, Alexandria Beilina, Adriano Gonnelli, Mark R. Cookson, Elisa Greggio, and Sabine Hilfiker
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Centrosome ,LRRK2 ,Parkinson’s disease ,Phosphorylation ,Rab8a ,Neurology. Diseases of the nervous system ,RC346-429 ,Geriatrics ,RC952-954.6 - Abstract
Abstract Background Mutations in LRRK2 are a common genetic cause of Parkinson’s disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. Methods Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. Results Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. Conclusions Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
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- 2018
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25. Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson’s disease at 16q11.2 and MAPT H1 loci
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Marc P M Soutar, Daniela Melandri, Benjamin O’Callaghan, Emily Annuario, Amy E Monaghan, Natalie J Welsh, Karishma D’Sa, Sebastian Guelfi, David Zhang, Alan Pittman, Daniah Trabzuni, Anouk H A Verboven, Kylie S Pan, Demis A Kia, Magda Bictash, Sonia Gandhi, Henry Houlden, Mark R Cookson, Nael Nadif Kasri, Nicholas W Wood, Andrew B Singleton, John Hardy, Paul J Whiting, Cornelis Blauwendraat, Alexander J Whitworth, Claudia Manzoni, Mina Ryten, Patrick A Lewis, and Hélène Plun-Favreau
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EXPRESSION ,Science & Technology ,KANSL1 ,IDENTIFICATION ,MUTATIONS ,Parkinson's disease ,PINK1 ,FOS: Clinical medicine ,Stem Cells ,Clinical Neurology ,Neurosciences ,Cell Biology ,UBIQUITIN ,TAU GENE ,mitophagy ,MITOCHONDRIA ,KAT8 ,GWAS ,Neurosciences & Neurology ,TRANSCRIPTION ,Neurology (clinical) ,GENOME-WIDE ASSOCIATION ,Life Sciences & Biomedicine ,METAANALYSIS - Abstract
Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration. ispartof: BRAIN vol:145 issue:12 pages:4349-4367 ispartof: location:England status: published
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- 2022
26. Inhibition of p38α MAPK restores neuronal p38γ MAPK and ameliorates synaptic degeneration in a mouse model of DLB/PD
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Michiyo Iba, Changyoun Kim, Somin Kwon, Marcell Szabo, Liam Horan-Portelance, Cody J. Peer, William D. Figg, Xylena Reed, Jinhui Ding, Seung-Jae Lee, Robert A. Rissman, Mark R. Cookson, Cassia Overk, Wolf Wrasidlo, and Eliezer Masliah
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General Medicine - Abstract
Alterations in the p38 mitogen-activated protein kinases (MAPKs) play an important role in the pathogenesis of dementia with Lewy bodies (DLB) and Parkinson’s disease (PD). Activation of the p38α MAPK isoform and mislocalization of the p38γ MAPK isoform are associated with neuroinflammation and synaptic degeneration in DLB and PD. Therefore, we hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. To test this hypothesis, we treated in vitro cellular and in vivo mouse models of DLB/PD with SKF-86002, a compound that attenuates inflammation by inhibiting p38α/β, and then investigated the effects of this compound on p38γ and neurodegenerative pathology. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-synuclein. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-synuclein in mice overexpressing human α-synuclein. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-synuclein mutation) and healthy controls. Treatment with SKF-86002 ameliorated α-synuclein–induced neurodegeneration in these neurons only when microglia were pretreated with this compound. However, direct treatment of neurons with SKF-86002 did not affect α-synuclein–induced neurotoxicity, suggesting that SKF-86002 treatment inhibits α-synuclein–induced neurotoxicity mediated by microglia. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD.
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- 2023
27. Hexokinases link DJ-1 to the PINK1/parkin pathway
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David N. Hauser, Adamantios Mamais, Melissa M. Conti, Christopher T. Primiani, Ravindran Kumaran, Allissa A. Dillman, Rebekah G. Langston, Alexandra Beilina, Joseph H. Garcia, Alberto Diaz-Ruiz, Michel Bernier, Fabienne C. Fiesel, Xu Hou, Wolfdieter Springer, Yan Li, Rafael de Cabo, and Mark R. Cookson
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Proteomics ,RNA-Seq ,Metabolomics ,Systems biology ,Parkinson’s disease ,Mitophagy ,Neurology. Diseases of the nervous system ,RC346-429 ,Geriatrics ,RC952-954.6 - Abstract
Abstract Background Early onset Parkinson’s disease is caused by variants in PINK1, parkin, and DJ-1. PINK1 and parkin operate in pathways that preserve mitochondrial integrity, but the function of DJ-1 and how it relates to PINK1 and parkin is poorly understood. Methods A series of unbiased high-content screens were used to analyze changes at the protein, RNA, and metabolite level in rodent brains lacking DJ-1. Results were validated using targeted approaches, and cellular assays were performed to probe the mechanisms involved. Results We find that in both rat and mouse brains, DJ-1 knockout results in an age-dependent accumulation of hexokinase 1 in the cytosol, away from its usual location at the mitochondria, with subsequent activation of the polyol pathway of glucose metabolism in vivo. Both in the brain and in cultured cells, DJ-1 deficiency is associated with accumulation of the phosphatase PTEN that antagonizes the kinase AKT. In cells, addition of an inhibitor of AKT (MK2206) or addition of a peptide to dissociate association of hexokinases from mitochondria both inhibit the PINK1/parkin pathway, which works to maintain mitochondrial integrity. Conclusion Hexokinases are an important link between three major genetic causes of early onset Parkinson’s disease. Because aging is associated with deregulated nutrient sensing, these results help explain why DJ-1 is associated with age-dependent disease.
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- 2017
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28. Divergent effects of aging across human brain regions at single cell resolution
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Megan Duffy, Jinhui Ding, Rebekah Langston, D. Thad Whitaker, J. Raphael Gibbs, and Mark R. Cookson
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Single nuclear RNA-sequencing was performed on human post-mortem brain tissue from four distinct nuclei that have implications for neurodegenerative diseases, including Alzheimer's and Parkinson’s diseases. Younger, aged 20s to 30s, and middle-aged and older, aged 60s to 80s, adults were used to identify changes in cell populations and transcriptomes during aging. Files included: aging.glmmtmb_age_diffs.csv.gz - All results modeled using glmmTMB aging.glmmtmb_age_diffs_fdr.csv.gz - glmmTMB results with a statistically significant result Column headers for the glmmtmb results are: feature - gene name intercept - regression intercept estimate - regression coefficient std.error - standard error statistic - regression test statistic p.value - regression p-value tissue - the brain region or cell-type (depending on 'type' column) type - type for 'tissue' column, brain_region, specific or broad cell-type fdr_bh - B&H FDR multiple-test corrected p-value aging.diffxpy_age_diffs.csv.gz - All results using diffxpy differential expression aging.diffxpy_age_diffs_fdr.csv.gz - diffxpy results with a statistically significant result Column headers for the diffxpy results are: gene - gene name pval - p-value for differential expression qval - q-value for differential expression; multiple-test corrected p-value log2fc - log base 2 fold change in expression mean - zero_mean - zero_variance - tissue - the brain region or cell-type (depending on 'type' column) type - type for 'tissue' column, brain_region, specific or broad cell-type fdr_bh - B&H FDR cmultiple-test orrected p-value  
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- 2023
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29. A Fully Automated FAIMS-DIA Mass Spectrometry-Based Proteomic Pipeline
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Luke Reilly, Erika Lara, Daniel M. Ramos, Ziyi Li, Caroline B. Pantazis, Julia Stadler, Marianita Santiana, Jessica Roberts, Faraz Faghri, Mike A. Nalls, Priyanka Narayan, Yansheng Liu, Andrew B. Singleton, Mark R. Cookson, Michael E. Ward, and Yue Qi
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- 2023
30. Transcriptome analysis of collagen VI‐related muscular dystrophy muscle biopsies
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Carsten G. Bönnemann, Mark R. Cookson, Payam Mohassel, Eleonora Guadagnin, Lin Yang, Prech Uapinyoying, Mariarita Santi, Jahannaz Dastgir, Allissa Dillmann, Ying Hu, Kory R. Johnson, and A. Reghan Foley
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Muscle tissue ,Pathology ,medicine.medical_specialty ,Muscle biopsy ,medicine.diagnostic_test ,business.industry ,General Neuroscience ,Extracellular matrix component ,Skeletal muscle ,Neurosciences. Biological psychiatry. Neuropsychiatry ,medicine.disease ,Transcriptome ,Extracellular matrix ,medicine.anatomical_structure ,Collagen VI ,Medicine ,Neurology. Diseases of the nervous system ,Neurology (clinical) ,Muscular dystrophy ,RC346-429 ,business ,Research Articles ,RC321-571 ,Research Article - Abstract
Objective To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI‐related muscular dystrophy (COL6‐RD). Methods COL6‐RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA‐Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age‐ and sex‐matched controls. Results COL6‐RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion‐specific genes. Upregulation of the TGFβ pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6‐RD histological severity. Interpretation Overall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFβ signaling, and its downstream cellular pathways at the transcriptomic level in COL6‐RD muscle.
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- 2021
31. The endoplasmic reticulum contributes to lysosomal tubulation/sorting driven by LRRK2
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Luis Bonet-Ponce and Mark R. Cookson
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Protein Transport ,Dyneins ,Humans ,Parkinson Disease ,Intracellular Membranes ,Cell Biology ,Endoplasmic Reticulum ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Lysosomes ,Molecular Biology - Abstract
Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.
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- 2022
32. PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2
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Laura Civiero, Susanna Cogo, Anneleen Kiekens, Claudia Morganti, Isabella Tessari, Evy Lobbestael, Veerle Baekelandt, Jean-Marc Taymans, Marie-Christine Chartier-Harlin, Cinzia Franchin, Giorgio Arrigoni, Patrick A. Lewis, Giovanni Piccoli, Luigi Bubacco, Mark R. Cookson, Paolo Pinton, and Elisa Greggio
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PAK6 ,14-3-3 ,LRRK2 ,Parkinson's disease ,phosphorylation ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
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.
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- 2017
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33. RNA sequencing of whole blood reveals early alterations in immune cells and gene expression in Parkinson’s disease
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Nripesh Prasad, J. Raphael Gibbs, Tobias Fehlman, Alyssa Reimer, Kendall Van Keuren-Jensen, Tatiana Foroud, Arthur W. Toga, Samantha J. Hutten, Timothy G. Whitsett, Shawn Levy, Mark Frasier, Ivo Violich, Karen Crawford, Samantha Hutten, Madison Robison, Andreas Keller, Eric Alsop, Fabian Kern, Parkinson Progression Marker Initiative, Elizabeth Hutchins, Mark R. Cookson, David Craig, Seungchan Kim, and Bradford Casey
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Aging ,Parkinson's disease ,Lymphocyte ,Neuroscience (miscellaneous) ,RNA ,Disease ,Biology ,medicine.disease ,Transcriptome ,medicine.anatomical_structure ,Immune system ,Immunology ,Gene expression ,medicine ,Geriatrics and Gerontology ,Whole blood - Abstract
Changes in the blood-based RNA transcriptome have the potential to inform biomarkers of Parkinson’s disease (PD) progression. Here we sequenced a discovery set of whole-blood RNA species in 4,871 longitudinally collected samples from 1,570 clinically phenotyped individuals from the Parkinson’s Progression Marker Initiative (PPMI) cohort. Samples were sequenced to an average of 100 million read pairs to create a high-quality transcriptome. Participants with PD in the PPMI had significantly altered RNA expression (>2,000 differentially expressed genes), including an early and persistent increase in neutrophil gene expression, with a concomitant decrease in lymphocyte cell counts. This was validated in a cohort from the Parkinson’s Disease Biomarkers Program (PDBP) consisting of 1,599 participants and by alterations in immune cell subtypes. This publicly available transcriptomic dataset, coupled with available detailed clinical data, provides new insights into PD biological processes impacting whole blood and new paths for developing diagnostic and prognostic PD biomarkers. The authors report whole-blood RNA-seq for 4,871 samples from 1,570 participants in the Parkinson Progression Marker Initiative. This Resource documents blood-based transcriptomic changes associated with PD, including early increases in neutrophil gene expression with a decrease in lymphocytes.
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- 2021
34. Lysosomal positioning regulates Rab10 phosphorylation at LRRK2 + lysosomes
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Jillian H. Kluss, Alexandra Beilina, Chad D. Williamson, Patrick A. Lewis, Mark R. Cookson, and Luis Bonet-Ponce
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Multidisciplinary - Abstract
Genetic variation at the leucine-rich repeat kinase 2 ( LRRK2 ) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2 + lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.
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- 2022
35. Pathology in the Parkinson's Progression Markers Initiative; a Finale but also a start
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Richard Camicioli and Mark R. Cookson
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Neurology ,Disease Progression ,Humans ,Parkinson Disease ,Neurology (clinical) ,Geriatrics and Gerontology ,Biomarkers - Published
- 2022
36. Generation of gene-corrected isogenic controls from Parkinson's disease patient iPSC lines carrying the pathogenic SNCA p.A53T variant
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Nikita Kozhushko, Aleksandra Beilina, and Mark R. Cookson
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Cell Biology ,General Medicine ,Developmental Biology - Published
- 2023
37. LRRK2 recruitment, activity, and function in organelles
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Luis Bonet-Ponce and Mark R. Cookson
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Organelles ,Kinase ,Neurodegeneration ,Parkinson Disease ,Cell Biology ,Biology ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,medicine.disease ,Biochemistry ,Phenotype ,LRRK2 ,Article ,nervous system diseases ,Cell biology ,medicine.anatomical_structure ,Lysosome ,Mutation ,Organelle ,medicine ,Humans ,Kinase activity ,Molecular Biology ,Gene - Abstract
Protein coding mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.
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- 2021
38. Association of a common genetic variant with Parkinson’s disease is mediated by microglia
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Rebekah G. Langston, Alexandra Beilina, Xylena Reed, Alice Kaganovich, Andrew B. Singleton, Cornelis Blauwendraat, J. Raphael Gibbs, and Mark R. Cookson
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Substantia Nigra ,Humans ,Parkinson Disease ,Microglia ,General Medicine ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Article ,Genome-Wide Association Study - Abstract
Studies of multiple neurodegenerative disorders have identified many genetic variants that are associated with risk of disease throughout a lifetime. For example, Parkinson’s disease (PD) risk is attributed in part to both coding mutations in the leucine-rich repeat kinase 2 (LRRK2) gene and to a common noncoding variation in the 5′ region of theLRRK2locus, as identified by genome-wide association studies (GWAS). However, the mechanisms linking GWAS variants to pathogenicity are largely unknown. Here, we found that the influence of PD-associated noncoding variation onLRRK2expression is specifically propagated through microglia and not by other cell types that expressLRRK2in the human brain. We find microglia-specific regulatory chromatin regions that modulate theLRRK2expression in human frontal cortex and substantia nigra and confirm these results in a human-induced pluripotent stem cell–derived microglia model. We showed, using a large-scale clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen, that a regulatory DNA element containing the single-nucleotide variant rs6581593 influences theLRRK2expression in microglia. Our study demonstrates that cell type should be considered when evaluating the role of noncoding variation in disease pathogenesis and sheds light on the mechanism underlying the association of the 5′ region of LRRK2 with PD risk.
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- 2022
39. DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons
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Melissa Conti Mazza, Sarah C. Shuck, Jiusheng Lin, Michael A. Moxley, John Termini, Mark R. Cookson, and Mark A. Wilson
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Neurons ,Cellular and Molecular Neuroscience ,Mice ,Oxidative Stress ,Glycosylation ,Guanine ,Protein Deglycase DJ-1 ,Animals ,Humans ,Pyruvaldehyde ,Biochemistry - Abstract
Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.
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- 2022
40. Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in striatum and biases against heavy persistent alcohol drinking
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Daniel da Silva e Silva, Aya Matsui, Erin M. Murray, Adamantios Mamais, Marlisa Shaw, Dorit Ron, Mark R. Cookson, and Veronica A. Alvarez
- Abstract
The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition is known to require long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinson’s-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiates D1R signaling at the cellular and synaptic level, enhancing alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 are more prone to heavy alcohol drinking and consumption is insensitive to punishment. These findings identify a novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.
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- 2022
41. Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity
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Belén Fernández, Vinita G. Chittoor-Vinod, Jillian H. Kluss, Kaela Kelly, Nicole Bryant, An Phu Tran Nguyen, Syed A. Bukhari, Nathan Smith, Antonio Jesús Lara Ordóñez, Elena Fdez, Marie-Christine Chartier-Harlin, Thomas J. Montine, Mark A. Wilson, Darren J. Moore, Andrew B. West, Mark R. Cookson, R. Jeremy Nichols, and Sabine Hilfiker
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Reproducibility of Results ,Parkinson Disease ,Fibroblasts ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Rats ,Cellular and Molecular Neuroscience ,Mice ,HEK293 Cells ,Leucine ,rab GTP-Binding Proteins ,Mutation ,Animals ,Humans ,Neurology (clinical) ,Phosphorylation - Abstract
Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
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- 2022
42. DJ-1 glyoxalase activity makes a modest contribution to cellular defense against methylglyoxal damage in neurons
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Melissa Conti Mazza, Sarah Shuck, Jiusheng Lin, Michael A. Moxley, John Termini, Mark R. Cookson, and Mark A. Wilson
- Abstract
Human DJ-1 is a cytoprotective protein whose absence causes Parkinson’s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.
- Published
- 2022
43. Extracellular clusterin limits the uptake of α‐synuclein fibrils by murine and human astrocytes
- Author
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Federica Bono, Chiara Fiorentini, Veronica Mutti, Gaia Faustini, Mariacristina Missale, Mark R. Cookson, Dorien A. Roosen, Arianna Bellucci, Elisa Greggio, Isabella Russo, Federica Rizzi, Megan Duffy, Natalie Landeck, Alice Kaganovich, Alice Filippini, Isabella Tessari, Ileana Ramazzina, Francesca Longhena, Massimo Gennarelli, and Luigi Bubacco
- Subjects
0301 basic medicine ,clusterin ,Parkinson's disease ,animal diseases ,Biology ,Endocytosis ,hiPSC ,Pathogenesis ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,α-synuclein ,0302 clinical medicine ,Extracellular ,Animals ,Humans ,Gene silencing ,Induced pluripotent stem cell ,Research Articles ,astrocytes ,chemistry.chemical_classification ,α‐synuclein ,Clusterin ,Parkinson Disease ,eye diseases ,nervous system diseases ,Cell biology ,030104 developmental biology ,nervous system ,Neurology ,chemistry ,Chaperone (protein) ,alpha-Synuclein ,biology.protein ,Lewy Bodies ,sense organs ,Glycoprotein ,030217 neurology & neurosurgery ,Research Article - Abstract
The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α‐synuclein. Clearance of extracellular α‐synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α‐synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α‐synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α‐synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human‐induced pluripotent stem cell (iPSC)‐derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α‐synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α‐synuclein preformed fibrils (pffs) through dynamin‐dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α‐synuclein pffs exposure. Specifically, we found that clusterin interacts with α‐synuclein pffs in the extracellular compartment and the clusterin/α‐synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock‐out primary astrocytes and clusterin knock‐down hiPSC‐derived astrocytes we observed that clusterin limits the uptake of α‐synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno‐associated virus‐ and the α‐synuclein pffs‐ injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α‐synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology., Main Points Extracellular clusterin binds α‐synuclein fibrils thus limiting their uptake by murine and human astrocytes.Reduced levels of clusterin improve α‐synuclein aggregates clearance by astrocytes.
- Published
- 2020
44. Large-scale pathway specific polygenic risk and transcriptomic community network analysis identifies novel functional pathways in Parkinson disease
- Author
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Anthony R. Soltis, Ziv Gan-Or, Clifton L. Dalgard, Debra Ehrlich, Leonard H, Sara Saez-Atienzar, Cornelis Blauwendraat, Ali Torkamani, J. R. Gibbs, Sonja W. Scholz, Bryan J. Traynor, Clemens R. Scherzer, Jonggeol Jeff Kim, Jinhui Ding, Mark R. Cookson, Juan A. Botía, Matt Bookman, Andrew B. Singleton, Mike A. Nalls, Sara Bandres-Ciga, Monica Diez-Fairen, Hirotaka Iwaki, Lasse Pihlstrøm, Alastair J. Noyce, Dena G. Hernandez, Mina Ryten, Mary B. Makarious, and Faraz Faghri
- Subjects
Original Paper ,Polygenic risk ,Transcriptome community maps ,Context (language use) ,Computational biology ,Disease ,Quantitative trait locus ,Biology ,Chromatin remodeling ,Pathology and Forensic Medicine ,Parkinson disease ,Transcriptome ,Cellular and Molecular Neuroscience ,Mendelian randomization ,Neurology (clinical) ,Allele ,Signal transduction ,Gene - Abstract
Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological processes underlying PD using the largest currently available cohorts of genetic and gene expression data from International Parkinson’s Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership-Parkinson’s disease initiative (AMP-PD), among other sources. We applied large-scale gene-set specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk focusing on publicly annotated gene sets representative of curated pathways. We nominated specific molecular sub-processes underlying protein misfolding and aggregation, post-translational protein modification, immune response, membrane and intracellular trafficking, lipid and vitamin metabolism, synaptic transmission, endosomal–lysosomal dysfunction, chromatin remodeling and apoptosis mediated by caspases among the main contributors to PD etiology. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data and found evidence for a burden of rare damaging alleles in a range of processes, including neuronal transmission-related pathways and immune response. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for dopaminergic neurons, serotonergic neurons, hypothalamic GABAergic neurons, and neural progenitors. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of PD patients, which revealed functional enrichment in inflammatory signaling pathways, cell death machinery related processes, and dysregulation of mitochondrial homeostasis. Our analyses highlight several specific promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done. Electronic supplementary material The online version of this article (10.1007/s00401-020-02181-3) contains supplementary material, which is available to authorized users.
- Published
- 2020
45. APOE4 is Associated with Differential Regional Vulnerability to Bioenergetic Deficits in Aged APOE Mice
- Author
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Rishi R. Agrawal, Kathleen Shannon, Ernest Fraenkel, Steven S. Gross, David N. Guilfoyle, Helen Y. Figueroa, Allissa Dillman, Karen Duff, Archana Ashok, Tal Nuriel, Estela Area-Gomez, Marta Pera, Hirra A. Arain, Qiuying Chen, Mark R. Cookson, Leila Pirhaji, and Delfina Larrea
- Subjects
Male ,Apolipoprotein E ,medicine.medical_specialty ,Bioenergetics ,Apolipoprotein E4 ,Hippocampus ,lcsh:Medicine ,Biology ,Molecular neuroscience ,Article ,Pathogenesis ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Internal medicine ,mental disorders ,medicine ,Animals ,Allele ,lcsh:Science ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,lcsh:R ,Brain ,Alzheimer's disease ,Entorhinal cortex ,Mitochondria ,Cortex (botany) ,Endocrinology ,Metabolome ,lipids (amino acids, peptides, and proteins) ,lcsh:Q ,Energy Metabolism ,Transcriptome ,030217 neurology & neurosurgery - Abstract
The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason for the association between APOE4 and AD remains unclear. While much of the research has focused on the ability of the apoE4 protein to increase the aggregation and decrease the clearance of Aβ, there is also an abundance of data showing that APOE4 negatively impacts many additional processes in the brain, including bioenergetics. In order to gain a more comprehensive understanding of APOE4′s role in AD pathogenesis, we performed a transcriptomics analysis of APOE4 vs. APOE3 expression in the entorhinal cortex (EC) and primary visual cortex (PVC) of aged APOE mice. This study revealed EC-specific upregulation of genes related to oxidative phosphorylation (OxPhos). Follow-up analysis utilizing the Seahorse platform showed decreased mitochondrial respiration with age in the hippocampus and cortex of APOE4 vs. APOE3 mice, but not in the EC of these mice. Additional studies, as well as the original transcriptomics data, suggest that multiple bioenergetic pathways are differentially regulated by APOE4 expression in the EC of aged APOE mice in order to increase the mitochondrial coupling efficiency in this region. Given the importance of the EC as one of the first regions to be affected by AD pathology in humans, the observation that the EC is susceptible to differential bioenergetic regulation in response to a metabolic stressor such as APOE4 may point to a causative factor in the pathogenesis of AD.
- Published
- 2020
46. Application of Aligned-UMAP to Longitudinal Biomedical Studies
- Author
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Anant Dadu, Vipul K. Satone, Rachneet Kaur, Mathew J. Koretsky, Hirotaka Iwaki, Yue A. Qi, Daniel M. Ramos, Brian Avants, Jacob Hesterman, Roger Gunn, Mark R. Cookson, Michael E. Ward, Andrew B Singleton, Roy H Campbell, Mike A Nalls, and Faraz Faghri
- Subjects
History ,Polymers and Plastics ,General Decision Sciences ,Business and International Management ,Industrial and Manufacturing Engineering - Abstract
Longitudinal multi-dimensional biological datasets are ubiquitous and highly abundant. These datasets are essential to understanding disease progression, identifying subtypes, and drug discovery. Discovering meaningful patterns or disease pathophysiologies in these datasets is challenging due to their high dimensionality, making it difficult to visualize hidden patterns. Several methods have been developed for dimensionality reduction, but they are limited to cross-sectional datasets. Recently proposed Aligned-UMAP, an extension of the UMAP algorithm, can visualize high-dimensional longitudinal datasets. In this work, we applied Aligned-UMAP on a broad spectrum of clinical, imaging, proteomics, and single-cell datasets. Aligned-UMAP reveals time-dependent hidden patterns when color-coded with the metadata. We found that the algorithm parameters also play a crucial role and must be tuned carefully to utilize the algorithm’s potential fully.Altogether, based on its ease of use and our evaluation of its performance on different modalities, we anticipate that Aligned-UMAP will be a valuable tool for the biomedical community. We also believe our benchmarking study becomes more important as more and more high-dimensional longitudinal data in biomedical research becomes available.Highlights-explored the utility of Aligned-UMAP in longitudinal biomedical datasets-offer insights on optimal uses for the technique-provide recommendations for best practicesIn BriefHigh-dimensional longitudinal data is prevalent yet understudied in biological literature. High-dimensional data analysis starts with projecting the data to low dimensions to visualize and understand the underlying data structure. Though few methods are available for visualizing high dimensional longitudinal data, they are not studied extensively in real-world biological datasets. A recently developed nonlinear dimensionality reduction technique, Aligned-UMAP, analyzes sequential data. Here, we give an overview of applications of Aligned-UMAP on various biomedical datasets. We further provide recommendations for best practices and offer insights on optimal uses for the technique.
- Published
- 2022
47. Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC
- Author
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María Queralt-Martín, Mark R. Cookson, Alexandra Beilina, Amandine Rovini, William M. Rosencrans, Philip A. Gurnev, Sergey M. Bezrukov, and Tatiana K. Rostovtseva
- Subjects
Pharmacology ,0303 health sciences ,Voltage-dependent anion channel ,biology ,030302 biochemistry & molecular biology ,Chromosomal translocation ,Cell Biology ,Proximity ligation assay ,Gating ,Mitochondrion ,Neuroprotection ,Cell biology ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,chemistry ,biology.protein ,Molecular Medicine ,Olesoxime ,Bacterial outer membrane ,Molecular Biology - Abstract
An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC β-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.
- Published
- 2019
48. Controlling homology-directed repair outcomes in human stem cells with dCas9
- Author
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William C. Skarnes, Gang Ning, Sofia Giansiracusa, Alexander S. Cruz, Cornelis Blauwendraat, Brandon Saavedra, Kevin Holden, Mark R. Cookson, Michael E. Ward, and Justin A. McDonough
- Subjects
viruses - Abstract
Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive ‘dCas9’was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.
- Published
- 2021
49. A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons
- Author
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Luke Reilly, Marianita Santiana, Yue Andy Qi, Julia Stadler, Priyanka Narayan, Anant Dadu, Caroline B. Pantazis, Michael S. Fernandopulle, Michael E. Ward, Lirong Peng, Faraz Faghri, Andrew B. Singleton, James R. Iben, Erika Lara, Steven L. Coon, Mike A. Nalls, Mark R. Cookson, and Daniel Ramos
- Subjects
Computer science ,Proteomic Profiling ,Pipeline (computing) ,Proteome ,Neuron differentiation ,Data-independent acquisition ,Computational biology ,Proteomics ,Induced pluripotent stem cell ,Throughput (business) - Abstract
Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.
- Published
- 2021
50. A reference human induced pluripotent stem cell line for large-scale collaborative studies
- Author
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Caroline B. Pantazis, Andrian Yang, Erika Lara, Justin A. McDonough, Cornelis Blauwendraat, Lirong Peng, Hideyuki Oguro, Jitendra Kanaujiya, Jizhong Zou, David Sebesta, Gretchen Pratt, Erin Cross, Jeffrey Blockwick, Philip Buxton, Lauren Kinner-Bibeau, Constance Medura, Christopher Tompkins, Stephen Hughes, Marianita Santiana, Faraz Faghri, Mike A. Nalls, Daniel Vitale, Shannon Ballard, Yue A. Qi, Daniel M. Ramos, Kailyn M. Anderson, Julia Stadler, Priyanka Narayan, Jason Papademetriou, Luke Reilly, Matthew P. Nelson, Sanya Aggarwal, Leah U. Rosen, Peter Kirwan, Venkat Pisupati, Steven L. Coon, Sonja W. Scholz, Theresa Priebe, Miriam Öttl, Jian Dong, Marieke Meijer, Lara J.M. Janssen, Vanessa S. Lourenco, Rik van der Kant, Dennis Crusius, Dominik Paquet, Ana-Caroline Raulin, Guojun Bu, Aaron Held, Brian J. Wainger, Rebecca M.C. Gabriele, Jackie M. Casey, Selina Wray, Dad Abu-Bonsrah, Clare L. Parish, Melinda S. Beccari, Don W. Cleveland, Emmy Li, Indigo V.L. Rose, Martin Kampmann, Carles Calatayud Aristoy, Patrik Verstreken, Laurin Heinrich, Max Y. Chen, Birgitt Schüle, Dan Dou, Erika L.F. Holzbaur, Maria Clara Zanellati, Richa Basundra, Mohanish Deshmukh, Sarah Cohen, Richa Khanna, Malavika Raman, Zachary S. Nevin, Madeline Matia, Jonas Van Lent, Vincent Timmerman, Bruce R. Conklin, Katherine Johnson Chase, Ke Zhang, Salome Funes, Daryl A. Bosco, Lena Erlebach, Marc Welzer, Deborah Kronenberg-Versteeg, Guochang Lyu, Ernest Arenas, Elena Coccia, Lily Sarrafha, Tim Ahfeldt, John C. Marioni, William C. Skarnes, Mark R. Cookson, Michael E. Ward, Florian T. Merkle, Human genetics, Amsterdam Neuroscience - Cellular & Molecular Mechanisms, Neurology, Merkle, Florian [0000-0002-8513-2998], Apollo - University of Cambridge Repository, Functional Genomics, and Amsterdam Neuroscience - Neurodegeneration
- Subjects
Gene Editing ,p53 ,iPSC ,Induced Pluripotent Stem Cells ,Cell Differentiation ,Cell Biology ,differentiation ,single-cell ,reference ,whole-genome ,karyotype ,stem cell ,pluripotent ,ddc:570 ,CRISPR ,Genetics ,Molecular Medicine ,Humans ,Biological Assay ,Human medicine ,Biology - Abstract
Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.
- Published
- 2022
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