title: Phosphoproteomic evaluation of pharmacological inhibition of leucine-rich repeat kinase 2 reveals significant off-target effects of LRRK-2-IN-1.
abstract: Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH-SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition (LRRK2-IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC-MS/MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2-IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2-IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha (TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2-IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2-IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function.
authors.affiliation: Pfizer Global Research & Development, Neuroscience Research Unit, Cambridge, MA, USA.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Luerman
authors.forename: Chuong
authors.initials: C
authors.lastname: Nguyen
authors.forename: Harry
authors.initials: H
authors.lastname: Samaroo
authors.forename: Paula
authors.initials: P
authors.lastname: Loos
authors.forename: Hualin
authors.initials: H
authors.lastname: Xi
authors.forename: Andres
authors.initials: A
authors.lastname: Hurtado-Lorenzo
authors.forename: Elie
authors.initials: E
authors.lastname: Needle
authors.forename: G
authors.initials: G
authors.lastname: Stephen Noell
authors.forename: Paul
authors.initials: P
authors.lastname: Galatsis
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
authors.forename: Kieran F
authors.initials: KF
authors.lastname: Geoghegan
authors.forename: Warren D
authors.initials: WD
authors.lastname: Hirst
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008070:Lipopolysaccharides / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D061251:Primary Cell Culture; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D040901:Proteomics; D015398:Signal Transduction / Q000187:drug effects; D014025:Titanium / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D054357:Chemokine CXCL10; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; C009495:titanium dioxide; D014025:Titanium; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: Inflammation; LRRK2; LRRK2-IN-1; Neurite outgrowth; Parkinson's disease; SILAC