Significance: Parkinson’s disease (PD) is the second-most common neurodegenerative disorder. Mutations leading to overactivation of LRRK2 are a leading cause of familial PD, and this protein is therefore considered as an appealing target for drug design. Here, we describe the discovery and characterization of a diverse set of LRRK2-targeting nanobodies. A subset of these nanobodies inhibit LRRK2 via a mechanism that differs from the commonly used LRRK2 kinase inhibitors. Importantly, some of these nanobodies selectively inhibit certain LRRK2 activities (Rab phosphorylation) while leaving other activities (autophosphorylation) unaffected. We anticipate that these nanobodies will find multiple applications as research tools and will open up opportunities for the development of new PD diagnostics and therapeutics in parallel to other currently pursued strategies.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson’s Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

Dysfunction of the immune system and mitochondrial metabolism has been associated with Parkinson’s disease (PD) pathology. Mutations and increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) are linked to both idiopathic and familial PD. However, the function of LRRK2 in the immune cells under inflammatory conditions is contradictory. Our results showed that lipopolysaccharide (LPS) stimulation increased the kinase activity of LRRK2 in parental RAW 264.7 (WT) cells. In addition to this, LRRK2 deletion in LRRK2 KO RAW 264.7 (KO) cells altered cell morphology following LPS stimulation compared to the WT cells, as shown by an increase in the cell impedance as observed by the xCELLigence measurements. LPS stimulation caused an increase in the cellular reactive oxygen species (ROS) levels in both WT and KO cells. However, WT cells displayed a higher ROS level compared to the KO cells. Moreover, LRRK2 deletion led to a reduction in interleukin-6 (IL-6) inflammatory cytokine and cyclooxygenase-2 (COX-2) expression and an increase in lactate production after LPS stimulation compared to the WT cells. These data illustrate that LRRK2 has an effect on inflammatory processes in RAW macrophages upon LPS stimulation.