SUMMARY Recent genome-wide association studies have linked common variants in the human genome to Parkinson’s disease (PD) risk. Here we show that the consequences of variants at 2 such loci, PARK16 and LRRK2, are highly interrelated, both in terms of their broad impacts on human brain transcriptomes of unaffected carriers, and in terms of their associations with PD risk. Deficiency of the PARK16 locus gene RAB7L1 in primary rodent neurons, or of a RAB7L1 orthologue in Drosophila dopamine neurons, recapitulated degeneration observed with expression of a familial PD mutant form of LRRK2, whereas RAB7L1 overexpression rescued the LRRK2 mutant phenotypes. PD-associated defects in RAB7L1 or LRRK2 led to endolysosomal and Golgi apparatus sorting defects and deficiency of the VPS35 component of the retromer complex. Expression of wild-type VPS35, but not a familial PD-associated mutant form, rescued these defects. Taken together, these studies implicate retromer and lysosomal pathway alterations in PD risk.
SignificanceLRRK2, 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 of Caenorhabditis elegans expressing 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 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing 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 in C. elegans expressing G2019S LRRK2 than in C. elegans expressing 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.
The antioxidant activity of an anti-ischemic agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was examined. The pKa value of MCI-186 is 7.0 and the rate of oxidation of MCI-186 initiated with an azo compound increased with increasing pH, suggesting that the anionic form of MCI-186 is much more reactive than the non-ionic form. The major products were 3-methyl-1-phenyl-2-pyrazolin-4,5-dione (4,5-dione) and 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB). Hydrolysis of 4,5-dione gave OPB. The minor intermediate product was 4-hydroxy-4-(3-methyl-1-phenyl-1H-pyrazolin-5-on-4-yl)-3-methyl-1-phenyl-1H-pyrazolin-5-one (BPOH). The nucleophilic attack of the anionic form of MCI-186 to 4,5-dione is likely to give BPOH. MCI-186 (50 μM) inhibited the aerobic oxidation at 37°C of 5.2 mM unilamellar soybean phosphatidylcholine (PC) liposomal membranes, initiated with a water-soluble initiator, as efficientlyas did ascorbate (100 μM). MCI-186 (50 μM) also inhibited the oxidation of the same PC liposomal membranes, this time initiated with a lipid-soluble initiator, almost as efficiently as did α-tocopherol (2 μM). Furthermore, the combination of MCI-186 with ascorbate or α-tocopherol showed almost complete inhibition of PC oxidation induced by both initiators. These data suggest that MCI-186 may work as a good antioxidant in cellular systems as well as in cell-free systems.
Mutations in Parkinson disease (PD)3 is a major neurodegenerative disease of the adulthood affecting the extrapyramidal motor system (1). The brains of patients affected with PD are characterized by a loss of neurons in the brainstem monoaminergic neurons, e.g. dopamine neurons in the substantia nigra and noradrenergic neurons in the locus caeruleus, which is accompanied by the deposition of Lewy bodies (LBs) in the cytoplasm of remaining neurons. LBs are characteristic spherical intracytoplasmic inclusions composed of filaments of ϳ7-10 nm in diameter and are pathognomonic for PD and related dementing disorder, dementia with Lewy bodies (DLB) (2). A small percentage of patients inherit PD as an autosomal dominant trait (familial PD; FPD), and missense mutations (3-5) or multiplications (6 -8) of the ␣-synuclein gene have been identified in these families. LBs in the brains of patients with sporadic PD or DLB were shown to be composed of ␣-synuclein (9, 10). Glial cytoplasmic inclusions in the brains of patients with multiple system atrophy, another major sporadic neurodegenerative disease, or dystrophic neurites in Hallervorden-Spatz disease also were shown to be composed of ␣-synuclein, and these neurodegenerative diseases characterized by deposition of ␣-synuclein are collectively designated "synucleinopathies" (2). In vitro studies suggested that ␣-synuclein aggregates and forms filaments that are similar to those seen in PD or DLB brains, and amino acid substitutions linked to familial PD (A53T, A30P, or E46K) have been shown to enhance the aggregation of ␣-synuclein possibly through conformational changes (11-14), implicating deposition of ␣-synuclein in the pathogenesis of synucleinopathies including PD. Taken together with the gene dosage effects of ␣-synuclein in a subset of FPD (i.e. duplication and triplication), transgenic overexpression of ␣-synuclein in neurons would be a rational strategy to model neurodegeneration in PD.A number of transgenic models overexpressing ␣-synuclein using heterologous organisms Drosophila (21,22), Caenorhabditis elegans (23), yeast (24)) have been reported, and some abnormal behavioral or pathological phenotypes have been documented in a subset of these animal models. However, an ideal model in which deposition of ␣-synuclein in dopamine neurons, the most vulnerable subset of neurons in PD, causes behavioral phenotypes that are inherent to the functions of dopamine neurons, has not been established yet. Here we describe a transgenic C. elegans model in which human ␣-synuclein overexpressed specifically in dopamine neurons causes an abnormal phenotype in food-sensing behavior that has been attributed to the function of C. elegans dopamine neurons (25), in a manner dependent on FPD-linked mutations, through reduction of neuronal dopamine.
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