Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson’s disease and Crohn’s disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis. Significance LRRK2, a protein kinase related to Parkinson’s disease, is implicated in the maintenance of lysosomes, and a subset of Rab GTPases has been identified as bona fide substrates of LRRK2. Here, we reveal a key stress-responsive pathway composed of Rab7L1, LRRK2, and phosphorylated Rab8/10 involved in lysosomal homeostasis. Lysosomal overload stress induces translocation of Rab7L1 and LRRK2 to lysosomes, where LRRK2 is activated, and stabilizes Rab8 and Rab10 through phosphorylation. The activation of this machinery protects against lysosomal enlargement and upregulates lysosomal secretion through Rab effectors, EHBP1 and EHBP1L1. These findings elucidate a novel regulatory mechanism of Rab GTPases by phosphorylation by LRRK2 in stressed lysosomes, which may also be involved in the pathomechanism of LRRK2-related disorders.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines ofCaenorhabditis elegansexpressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 inC. elegans, reduced survival associated with mitochondrial dysfunction.C. elegansexpressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers inC. elegansexpressing G2019S LRRK2 than inC. elegansexpressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

Mutations in α-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type α-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating α-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with α-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human α-synuclein in dopamine neurons. The TG worms exhibit accumulation of α-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant α-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant α-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of β-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of α-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

Mutations or multiplications in α-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type α-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating α-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of α-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C . elegans (Tg worms) that overexpress human α-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed α-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in α-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7 ) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing α-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. α-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of α-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2 , an AP-2 subunit, caused an accumulation of phosphorylated α-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and α-synuclein-induced neurotoxicity in synucleinopathy.