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.