Search

Genetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to enhanced lifetime risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2-positive lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing expression of ARL8B and SKIP or by knockdown of the motor adaptor protein JIP4 blocked recruitment and phosphorylation of Rab10 by LRRK2. Conversely, overexpression of the Rab7 effector protein RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our novel findings suggest LRRK2 can be activated at multiple cellular membranes including lysosomes, and that lysosomal positioning further provides regulation of some Rab substrates likely via differential phosphatase activity in nearby cellular compartments.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson’s disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

Parkinson’s disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. While most cases of PD are sporadic in nature, several inherited monogenic syndromes exist that overlap clinically and pathologically with sporadic PD. Of these, loss of function mutations in DNAJC6 , which encodes the protein Auxilin, cause an aggressive form of juvenile onset PD. Auxilin and its homologues are known to play a role in clathrin-mediated trafficking, which is crucial for cellular function in all eukaryotes and plays a specialized role in synaptic transmission in higher organisms. Auxilin is the major neuronal uncoating protein for clathrin-coated vesicles required for delivery of cargo from the plasma membrane and trans -Golgi network to intracellular destinations. However, how mutations in Auxilin cause PD is currently not understood. To address this problem, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation. When bred to homozygosity, this mutation induced neurological phenotypes that phenocopy clinical features observed in patients, including motor impairments reminiscent of bradykinesia and gait problems. Mapping the interactome of Auxilin confirmed clathrin and synaptic clathrin adaptor protein interactions and also identified novel Golgi-resident interactors. Critically, all tested pathogenic mutations in Auxilin retained clathrin adaptor protein binding but lost interaction with clathrin itself. These observations describe a mechanism by which impaired clathrin-mediated trafficking in R857G Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide novel insights for PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin. Highlights Auxilin interacts with Golgi-resident clathrin adaptor protein GGA2 Auxilin is involved with uncoating of CCVs at the Golgi and synapse Impaired clathrin-mediated trafficking underlies PD-like phenotypes in R857G Auxilin mice