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.

Frequency of Loss of Function Variants in LRRK2 in Parkinson Disease

“…Recognizing that LOF variants in LRRK2 are not damaging to an individual indicates that kinase inhibitors and allele-specific oligos could be used as PD therapies. Previous studies 14,15 have shown that mutant alleles of LRRK2 can be selectively knocked down in various cell lines using allele-specific oligos. Additional studies of chronic LRRK2 kinase inhibition in mice and nonhuman primates 16,17 have shown that multiple inhibitors are effective at reducing LRRK2 phosphorylation in the brain with no adverse effects in the kidneys; however, a lysosomal phenotype has been reported in treated nonhuman primate lungs that is similar to reports from Lrrk2 knockout mice.…”

The role of the LRRK2 gene in Parkinsonism

“…Experiment conducted in LRRK2 p.G2019S cells with a doxorubicin inducible neuritic phenotype showing a reversed phenotype after adding a specific and potent kinase inhibitor, IN-1. Moreover, LRRK2 p.G2019S-mediated blunting of neurite extension is reversed by Lentivirus-mediated transfer of LRRK2 p.G2019S allele-specific small hairpin RNA [ 119 ]. Another similar study conducted in transgenic C. elegans which expressing human p.R1441C- and p.G2019S-LRRK2 showed that TTT-3002 and LRRK2-IN1 rescued the behavioral deficit characteristic of dopaminergic impairment.…”

The role of posttranslational modifications of α-synuclein and LRRK2 in Parkinson's disease: Potential contributions of environmental factors

“…Mutations in the KIN and ROCO domains alter constitutive phosphorylation of LRRK2 and are associated with PD pathogenesis [102]. The G2019S mutation of LRRK2 increased phosphorylation in dopaminergic cell lines, resulting in abnormal neurite growth and increased neuronal vulnerability [103]. Mutation in G2019S also increased LRRK2 kinase activity [104, 105], while inhibition of kinase activity reduced its constitutive phosphorylation [103].…”

“…The G2019S mutation of LRRK2 increased phosphorylation in dopaminergic cell lines, resulting in abnormal neurite growth and increased neuronal vulnerability [103]. Mutation in G2019S also increased LRRK2 kinase activity [104, 105], while inhibition of kinase activity reduced its constitutive phosphorylation [103]. This indicates that G2019 mutations increase LRRK2 constitutive phosphorylation and promote LRRK2 kinase activity.…”