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Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

ABSTRACT: The pathogenic mechanism(s) contributing to loss of dopamine neurons in Parkinson's disease (PD) remain obscure. Leucine-rich repeat kinase 2 (LRRK2) mutations are linked, as a causative gene, to PD. LRRK2 mutations are estimated to account for 10% of familial and between 1 % and 3 % of sporadic PD. LRRK2 proximate single nucleotide polymorphisms have also been significantly associated with idiopathic/sporadic PD by genome-wide association studies. LRRK2 is a multidomain-containing protein and belongs to the protein kinase super-family. We constructed two inducible dopaminergic cell lines expressing either human-LRRK2-wild-type or human-LRRK2-mutant (G2019S). Phenotypes of these LRRK2 cell lines were examined with respect to cell viability, morphology, and protein function with or without induction of LRRK2 gene expression. The overexpression of G2019S gene promoted (1) low cellular metabolic activity without affecting cell viability, (2) blunted neurite extension, and (3) increased phosphorylation at S910 and S935. Our observations are consistent with reported general phenotypes in LRRK2 cell lines by other investigators. We used these cell lines to interrogate the biological function of LRRK2, to evaluate their potential as a drug-screening tool, and to investigate screening for small hairpin RNA-mediated LRRK2 G2019S gene knockdown as a potential therapeutic strategy. A proposed LRRK2 kinase inhibitor (i.e., IN-1) decreased LRRK2 S910 and S935 phosphorylation in our MN9DLRRK2 cell lines in a dose-dependent manner. Lentivirus-mediated transfer of LRRK2 G2019S allele-specific small hairpin RNA reversed the blunting of neurite extension caused by LRRK2 G2019S overexpression. Taken together, these inducible LRRK2 cell lines are suitable reagents for LRRK2 functional studies, and the screening of potential LRRK2 therapeutics.

SUBMITTER: Huang L

PROVIDER: S-EPMC3805857 | biostudies-other | 2013 Oct

REPOSITORIES: biostudies-other

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Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

Huang Liang L Shimoji Mika M Wang Juan J Shah Salim S Kamila Sukanta S Biehl Edward R ER Lim Seung S Chang Allison A Maguire-Zeiss Kathleen A KA Su Xiaomin X Federoff Howard J HJ

Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 20131001 4

The pathogenic mechanism(s) contributing to loss of dopamine neurons in Parkinson's disease (PD) remain obscure. Leucine-rich repeat kinase 2 (LRRK2) mutations are linked, as a causative gene, to PD. LRRK2 mutations are estimated to account for 10% of familial and between 1 % and 3 % of sporadic PD. LRRK2 proximate single nucleotide polymorphisms have also been significantly associated with idiopathic/sporadic PD by genome-wide association studies. LRRK2 is a multidomain-containing protein and b ...[more]

PMID: 23963789

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Project description:Increased leucine-rich repeat kinase 2 (LRRK2) kinase activity is an established risk factor for Parkinson's disease (PD), and several LRRK2 kinase inhibitors are in clinical development as potential novel disease-modifying therapeutics. This biomarker characterization study explored within- and between-subject variability of multiple LRRK2 pathway biomarkers (total LRRK2 [tLRRK2], phosphorylation of the serine 935 (Ser935) residue on LRRK2 [pS935], phosphorylation of Rab10 [pRab10], and total Rab10 [tRab10]) in different biological sources (whole blood, peripheral blood mononuclear cells [PBMCs], neutrophils) as candidate human target engagement and pharmacodynamic biomarkers for implementation in phase I/II pharmacological studies of LRRK2 inhibitors. PD patients with a LRRK2 mutation (n = 6), idiopathic PD patients (n = 6), and healthy matched control subjects (n = 10) were recruited for repeated blood and cerebrospinal fluid (CSF) sampling split over 2 days. Within-subject variability (geometric coefficient of variation [CV], %) of these biomarkers was lowest in whole blood and neutrophils (range: 12.64%-51.32%) and considerably higher in PBMCs (range: 34.81%-273.88%). Between-subject variability displayed a similar pattern, with relatively lower variability in neutrophils (range: 61.30%-66.26%) and whole blood (range: 44.94%-123.11%), and considerably higher variability in PBMCs (range: 189.60%-415.19%). Group-level differences were observed with elevated mean pRab10 levels in neutrophils and a reduced mean pS935/tLRRK2 ratio in PBMCs in PD LRRK2-mutation carriers compared to healthy controls. These findings suggest that the evaluated biomarkers and assays could be used to verify pharmacological mechanisms of action and help explore the dose-response of LRRK2 inhibitors in early-phase clinical studies. In addition, comparable α-synuclein aggregation in CSF was observed in LRRK2-mutation carriers compared to idiopathic PD patients.

Dataset Information

Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

Publications

Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

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