title: G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons.
abstract: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions.
  
  
  
  α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294, volpicel@uab.edu.
authors.forename: Laura A
authors.identifier: 0000-0001-8934-4018
authors.initials: LA
authors.lastname: Volpicelli-Daley
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Hisham
authors.identifier: 0000-0002-0884-0843
authors.initials: H
authors.lastname: Abdelmotilib
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Stoyka
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: João Paulo Lima
authors.initials: JP
authors.lastname: Daher
authors.affiliation: Centre for Applied Neurogenetics, Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada.
authors.forename: Austen J
authors.identifier: 0000-0002-0056-1778
authors.initials: AJ
authors.lastname: Milnerwood
authors.affiliation: Jungers Center for Neurosciences Research and Parkinson Center of Oregon, Department of Neurology, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Unni
authors.affiliation: Pfizer Neuroscience and Pain Research Unit, Cambridge, Massachusetts 02139.
authors.forename: Warren D
authors.initials: WD
authors.lastname: Hirst
authors.affiliation: Departments of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yue
authors.affiliation: Ionis Pharmaceuticals, Carlsbad, California 92010, and.
authors.forename: Hien T
authors.initials: HT
authors.lastname: Zhao
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Kyle
authors.identifier: 0000-0002-6052-4469
authors.initials: K
authors.lastname: Fraser
authors.affiliation: Comprehensive Center for Healthy Aging and Division of Gerontology, Geriatrics, and Palliative Care, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Richard E
authors.identifier: 0000-0003-4638-126X
authors.initials: RE
authors.lastname: Kennedy
authors.affiliation: Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: West
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000473:pathology*; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology; D038761:Photobleaching; D051381:Rats; D051843:Synucleins / Q000378:metabolism; D057900:Transcytosis / Q000235:genetics / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000235:genetics / Q000378:metabolism; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D020320:Oligoribonucleotides, Antisense; D051843:Synucleins; D014404:Tubulin; D050994:Voltage-Dependent Anion Channels; D051844:alpha-Synuclein; C462941:beta3 tubulin, mouse; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse
keywords: LRRK2; Lewy body; Lewy neurite; Parkinson's; synuclein