LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 is a large protein containing a small GTPase domain and a kinase domain, but its physiological role is unknown. To identify the normal function of LRRK2 in vivo, we generated two independent lines of germ-line deletion mice. The dopaminergic system of LRRK2 −/− mice appears normal, and numbers of dopaminergic neurons and levels of striatal dopamine are unchanged. However, LRRK2 −/− kidneys, which suffer the greatest loss of LRRK compared with other organs, develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins at 20 months of age. The autophagy-lysosomal pathway is also impaired in the absence of LRRK2, as indicated by accumulation of lipofuscin granules as well as altered levels of LC3-II and p62. Furthermore, loss of LRRK2 dramatically increases apoptotic cell death, inflammatory responses, and oxidative damage. Collectively, our findings show that LRRK2 plays an essential and unexpected role in the regulation of protein homeostasis during aging, and suggest that LRRK2 mutations may cause Parkinson's disease and cell death via impairment of protein degradation pathways, leading to α-synuclein accumulation and aggregation over time.Parkinson's disease | knockout | autophagy | ubiquitin-proteasome system | aging P arkinson's disease (PD) is the most common movement disorder. The neuropathological hallmarks of PD are progressive degeneration of dopaminergic (DA) neurons and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies, of which α-synuclein is a major constituent (1). Dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial PD (2, 3), highlighting the importance of LRRK2 in PD pathogenesis; however, the normal physiological role of LRRK2 is unknown. LRRK2 is a large protein containing a Ras-like small GTPase domain and a MAPKKK-like kinase domain, and has a functional homolog LRRK1, which shares similar domain structures (4). Crystal structural and biochemical studies showed that the GTPase domain forms a dimer; the pathogenic mutations destabilize the dimer and reduce GTPase activity (5-7). A recent in vitro study suggested that LRRK2 and LRRK1 can interact with each other and form a heterodimer (8). Although no physiological substrate of the LRRK2 kinase activity has been reported, studies in cultured cells have suggested that some pathogenic mutations in LRRK2 cause increases in LRRK2 kinase activity (9, 10).Protein aggregation is thought to play a major role in neurodegeneration and PD pathogenesis (11). The strongest evidence came from studies of α-synuclein. Gene multiplication and missense mutations in α-synuclein have been identified in early-onset familial PD with dominant inheritance (12). α-Synuclein is a major constituent of Lewy bodies (1). Overexpression of either WT or mutant α-synuclein in transgenic mice causes age-related neurodegeneration (13-15). Although patients carryi...

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.