Lrrk2, a gene linked to Parkinson’s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson’s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

It is now well established that chronic inflammation is a prominent feature of several neurodegenerative disorders including Parkinson’s disease (PD). Growing evidence indicates that neuroinflammation can contribute greatly to dopaminergic neuron degeneration and progression of the disease. Recent literature highlights that leucine-rich repeat kinase 2 (LRRK2), a kinase mutated in both autosomal-dominantly inherited and sporadic PD cases, modulates inflammation in response to different pathological stimuli. In this review, we outline the state of the art of LRRK2 functions in microglia cells and in neuroinflammation. Furthermore, we discuss the potential role of LRRK2 in cytoskeleton remodeling and vesicle trafficking in microglia cells under physiological and pathological conditions. We also hypothesize that LRRK2 mutations might sensitize microglia cells toward a pro-inflammatory state, which in turn results in exacerbated inflammation with consequent neurodegeneration.

Over-activated microglia and chronic neuroinflammation contribute to dopaminergic neuron degeneration and progression of Parkinson’s disease (PD). Leucine-rich repeat kinase 2 (LRRK2), a kinase mutated in autosomal dominantly inherited and sporadic PD cases, is highly expressed in immune cells, in which it regulates inflammation through a yet unclear mechanism. Here, using pharmacological inhibition and cultured Lrrk2 −/− primary microglia cells, we validated LRRK2 as a positive modulator of inflammation and we investigated its specific function in microglia cells. Inhibition or genetic deletion of LRRK2 causes reduction of interleukin-1β and cyclooxygenase-2 expression upon lipopolysaccharide-mediated inflammation. LRRK2 also takes part of the signaling trigged by α-synuclein fibrils, which culminates in induction of inflammatory mediators. At the molecular level, loss of LRRK2 or inhibition of its kinase activity results in increased phosphorylation of nuclear factor kappa-B (NF-κB) inhibitory subunit p50 at S337, a protein kinase A (PKA)-specific phosphorylation site, with consequent accumulation of p50 in the nucleus. Taken together, these findings point to a role of LRRK2 in microglia activation and sustainment of neuroinflammation and in controlling of NF-κB p50 inhibitory signaling. Understanding the molecular pathways coordinated by LRRK2 in activated microglia cells after pathological stimuli such us fibrillar α-synuclein holds the potential to provide novel targets for PD therapeutics.

Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.

One of the most promising therapeutic targets for potential diseasemodifying treatment of Parkinson’s disease (PD) is leucine-rich repeat kinase 2 (LRRK2). Specifically, targeting LRRK2’s kinase function has generated a lot of interest from both industry and academia. This work has yielded several published studies showing the feasibility of developing potent, selective and brain permeable LRRK2 kinase inhibitors. The availability of these experimental drugs is contributing to filling in the gaps in our knowledge on the safety and efficacy of LRRK2 kinase inhibition. Recent studies of LRRK2 kinase inhibition in preclinical models point to potential undesired effects in peripheral tissues such as lung and kidney. Also, while strategies are now emerging to measure target engagement of LRRK2 inhibitors, there remains an important need to expand efficacy studies in preclinical models of progressive PD. Future work in the LRRK2 inhibition field must therefore be directed towards developing molecules and treatment regimens which demonstrate efficacy in mammalian models of disease in conditions where safety liabilities are reduced to a minimum.