Neurons are long-lived cells that are not replaced during the entire lifespan of the organism, and continuous exchange of materials is essential for neuronal survival and function throughout the lifespan. In addition, neurons are the most morphologically complex cells and are highly polarized. Thus, neurons can be extremely vulnerable to disruption of intracellular trafficking, and it is no coincidence that defects in vesicle or membrane trafficking have emerged as hallmarks of most, or perhaps all, neurodegenerative diseases. The link between impaired trafficking and disease progression is more evident in Parkinson’s disease (PD). A number of PD-related genes, both familial and sporadic, such as
The role of LRRK2 in the onset and progression of PD and the molecular mechanism that leads to neurodegeneration are not fully understood; however, PD-causing mutations cluster within the ROC GTPase, COR, and the kinase domains of LRRK2. G2019S mutation in the kinase domain is by far the most prevalent, known genetic cause of neurodegeneration (16). The G2019S mutation is located in the kinase activation loop and augments the kinase activity towards generic kinase substrates (17). Importantly, neurotoxicity induced by LRRK2-G2019S in culture and
Endogenous LRRK2 is largely a cytosolic protein associated with membranous structures but is generally excluded from the nucleus. Notably, LRRK2 was particularly enriched in membrane-associated fractions (23–25). At the ultrastructural level, LRRK2-positive immunolabeling was localized to Golgi apparatus, endoplasmic reticulum, mitochondria, multivesicular bodies (MVBs), and lysosomal and endosomal vesicles (23). Expression of a GFP-reporter construct in cells also located LRRK2 to a variety of different membranous compartments, including the neck of caveolae, microvilli, MVBs and autophagic vacuoles, suggesting a possible role in endolysosomal-autophagic pathway (26). A bioinformatic approach using manually-curated protein-protein interaction datasets reiterated the role of LRRK2 and LRRK2 interactome in intracellular transport and vesicular trafficking (27).
It is not clear how LRRK2 manages to associate with many different types of membranous structures. The possibility that LRRK2 binds directly to each of the membranous structures through an intrinsic affinity for lipids has not been formally excluded. However, the lack of a known membrane-spanning or membrane-targeting signal within LRRK2 together with the localization of LRRK2 in diverse vesicular or membranous structures with different lipid compositions suggest that LRRK2 might be recruited to each target membrane by interacting with specific membrane-bound proteins. This possibility is supported by the presence of several protein-protein interaction motifs in LRRK2 and is further strengthened by the identification of a growing list of membrane-associated, LRRK2-interacting proteins.
In addition to the localization of LRRK2 to multiple membranous compartments (23), mounting evidence suggests that regulation of vesicular transport is a proximal event in LRRK2 signaling. LRRK2 has been implicated in the regulation of autophagy, lysosomal maturation and trafficking, and the endosomal pathway (28, 29).
Rab GTPases are the largest subgroup of the Ras-like GTPase superfamily and comprise more than 60 members in the human genome. Rab proteins reside in specific membranous compartments and are master regulators of vesicle trafficking, controlling virtually all aspects of vesicle or membrane traffic (30–32). By cycling between GTP-bound and GDP-bound states, Rab proteins function as molecular switches and orchestrate the formation, maturation, transport, tethering, and fusion of vesicles. Once the Rab protein is inserted into its respective target membrane, a guanine nucleotide exchange factor (GEF) mediates the conversion from the GDP- to the GTP-bound form. The active, GTP-bound Rab recruits effector proteins that specifically control certain aspects of trafficking and is hydrolyzed back to its inactive state, a process which is facilitated by a GTPase-activating protein (GAP). GDP-bound Rab becomes a substrate of GDP dissociation inhibitor (GDI), which extracts Rab in its GDP-bound state from the membrane. Rab bound to GDI can then be reinserted into a membrane to start the cycle again.
Many lines of evidence have suggested physical, genetic and functional interactions between LRRK2 and several members of the Rab GTPases (29, 33–36). In yeast-two-hybrid screening, LRRK2 has been shown to interact with Rab5, Rab32 and Rab38 (34, 37). The
Recently, we and others have identified a subset of Rab GTPases as authentic substrates of LRRK2 (38–40). Steger
Among LRRK2 substrates,
The first gene identified in familial PD was
Neurons undergo high rates of synaptic transmission, which requires efficient membrane trafficking at the presynaptic axon terminal to recover and recycle membranes that have fused with the plasma membrane during neurotransmitter release. Postmortem studies of PD brains support the notion that synaptic dysfunction is an early event in PD (1), and increasing lines of evidence suggest that LRRK2 plays a role in clathrin-dependent endocytosis and recycling of synaptic vesicles (Fig. 2). Clathrin-dependent synaptic vesicle endocytosis (SVE) is initiated by recruiting clathrin at the plasma membrane via adaptor protein 2 (AP-2). Clathrin-coated vesicle (CCV) then invaginates from the plasma membrane by the actions of proteins that sense and induce membrane curvature, such as endophilin. CCV then physically separates from the plasma membrane by the action of the GTPase dynamin. Shedding of the adaptors depends on phosphatidyl inositol hydrolysis by synaptojanin, and the ATPase Hsp70 and its cofactor auxilin remove the clathrin lattice after fission, and thereby, produce synaptic vesicles that can be reused (50). LRRK2 has been suggested to play a role in synaptic vesicle recycling via phosphorylation of endophilin A1, an essential molecule involved in clathrin-mediated SVE (51). Endophilin contains an N-terminal amphipathic α-helices followed by the Bin/amphiphysin/Rvs (BAR) domain and a C-terminal SH3 domain. Endophilin promotes early steps of SVE by generating membrane curvature at the plasma membrane and facilitates later steps via clathrin-uncoating (52–55). Membrane curvature can be generated by (i) the BAR domain, which forms a crescent-shaped dimer that interacts with and forces membranes to conform to their own intrinsic protein shape (scaffolding mechanism) and (ii) the amphipathic α-helices, which embed in the lipid bilayer and thereby generate a wedging force (wedging mechanism) (56). It has been suggested that the generation of vesicles and tubes requires different structures and mechanisms of endophilin A1 and the transition between the two membranous structures can be regulated via LRRK2-mediated phosphorylation (56). The LRRK2 phosphorylation site S75 is shallowly inserted on vesicles but is deeply embedded upon tubulation. In contrast to wild-type and the nonphosphorylatable mutant (S75A) of endophilin A1 that increased tubulation, the phosphomimetic mutant (S75D) destabilized tubulation and favored vesiculation by preventing deep insertion of the helical wedges (56). Therefore, it is possible that LRRK2-mediated phosphorylation of endophilin A1 regulates how endophilin structurally interacts with and curves the membrane. This function might play a role at the endocytic neck, a structure which is transiently generated and rapidly destabilized. It remains to be determined whether pathogenic LRRK2 mutants via its aberrant kinase activity disrupt the balance of different types of membrane curvature.
In addition to generating membrane curvature at early stages of SVE, endophilin recruits dynamin and synaptojanin (57, 58). Synaptojanin-1 has been identified as a substrate of LRRK2 via phosphoproteomic analysis of
After fission, auxilin induces clathrin uncoating of the CCVs by recruiting the ATPase Hsc70. The pathological linkage between
In addition to SVE, LRRK2 is involved in synaptic vesicle fusion by regulating the SNARE complex dissociation via phosphorylation of NSF (63). Vesicular SNARE (v-SNARE) located on the synaptic vesicle membrane and the target SNARE (t-SNARE) present on the plasma membrane assemble into a highly stable alpha-helical SNARE complex that juxtaposes the two membranes together to catalyze membrane fusion. After synaptic vesicle fusion, the SNARE complex is dissociated for reuse. At the transition period of exocytosis and endocytosis, the disassembly of SNARE complex is initiated by the formation of SNARE-SNAP-NSF complex, followed by NSF-catalyzed ATP hydrolysis and release of individual SNAREs (64). LRRK2 phosphorylates NSF at T645 located within the D2 domain of NSF. T645 is predicted to be a key residue for protein oligomerization, which regulates the ability of the D1 domain to hydrolyze ATP (65). LRRK2 phosphorylation of NSF enhanced the ATPase activity of NSF and increased the rate of SNARE complex disassembly
Snapin, known as SNAP-25 (synaptosomal-associated protein 25) binding protein, is another LRRK2 kinase substrate (66). SNAP-25 is a core component of the SNARE complex. Inhibition of the interaction between snapin and SNAP-25 blocks the association of SNARE complex with synaptotagmin. Snapin interacts with multiple vesicle trafficking-related proteins, and calcium-dependent exocytosis is impaired in snapin null mice (67). It has been shown that LRRK2 phosphorylates snapin at T117. The phosphomimetic snapin-T117D decreases its interaction with SNAP-25 and prevents the association of synaptotagmin with SNARE complex and reduces the number of readily releasable pool of synaptic vesicles. These results suggest that LRRK2 may be involved in neurotransmitter release.
Analysis of postmortem human brain tissue from PD brain donors using correlative light and electron microscopy (CLEM), serial block-free scanning electron microscopy (SEFSEM), and stimulated emission depletion (STED)-based super resolution microscopy showed that α-synuclein immunopositive inclusions were crowded with fragmented membranes, organelles and vesicular structures (68), supporting the notion that defects in membrane trafficking are a potential driver of pathogenesis of PD. Here we have discussed that intracellular localization of LRRK2, the effects of
This work was supported by the Research Resettlement Fund for the new faculty of Seoul National University (550-20180037 to EMH), the Research Institute for Veterinary Science, Seoul National University and the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016M3C7A1905386, 2017M3C7A1043848).
The authors have no conflicting interests.
Overview of LRRK2 and its substrates in vesicle or membrane trafficking. (
LRRK2 substrates involved in vesicle trafficking and synaptic transmission. LRRK2 phosphorylates a subset of Rab GTPases that control the endolysosomal-autophagy pathway and key molecules involved in synaptic vesicle endocytosis and recycling. Proteins that are suggested as direct substrates of LRRK2 are depicted in blue.