Nuclear α-Synuclein-Derived Cytotoxic Effect via Altered Ribosomal RNA Processing in Primary Mouse Embryonic Fibroblasts.

α-Synuclein (αSyn) is an important player in Parkinson's disease (PD) pathogenesis. The aggregation of αSyn is mainly formed in the cytoplasm, whereas some αSyn accumulation has also been found in the nuclei of neurons. To assess the effect of nuclear αSyn, we generated αSyn conjugated with a nuclear export signal (NES) or a nuclear localization signal (NLS), and compared them with wild-type αSyn in primary mouse embryonic fibroblasts (MEF) using DNA transfection. Overexpression of NLS-αSyn increased cytotoxicity. The levels of apoptotic markers were increased by NLS-αSyn in MEF. Interestingly, an increase in the levels of 40S ribosomal protein 15 was observed in MEF expressing NLS-αSyn. These MEF also showed a higher 28S/18S rRNA ratio. Intriguingly, the expression of NLS-αSyn in MEF enhanced segmentation of nucleolin (NCL)-positive nucleolar structures. We also observed that the downregulation of NCL, using shRNA, promoted a relatively higher 28S/18S rRNA ratio. The reduction in NCL expression accelerated the accumulation of αSyn, and NCL transfection enhanced the degradation of αSyn. These results suggest that nuclear αSyn contributes to the alteration in ribosomal RNA processing via NCL malfunction-mediated nucleolar segmentation, and that NCL is a key factor for the degradation of αSyn.

LRRK2 Inhibition Mitigates the Neuroinflammation Caused by TLR2-Specific α-Synuclein and Alleviates Neuroinflammation-Derived Dopaminergic Neuronal Loss.

Evidence suggests that crosstalk occurs between microglial leucine-rich repeat kinase 2 (LRRK2)-a regulator of neuroinflammation-and neuron-released α-synuclein (αSyn)-a promoter of microglial activation and neuroinflammatory responses-in neuroinflammation-mediated Parkinson's disease (PD) progression. Therefore, we examined whether LRRK2 inhibition reduces the responses of microglia to neuroinflammation caused by neuron-released αSyn. We examined the neuroinflammatory responses provoked by Toll-like receptor 2 (TLR2)-positive αSyn of neuronal cells using an LRRK2 inhibitor in the mouse glioma cells, rat primary microglia, and human microglia cell line; and the effects of LRRK2 inhibitor in the co-culture of ectopic αSyn-expressing human neuroblastoma cells and human microglia cells and in mouse models by injecting αSyn. We analyzed the association between LRRK2 activity and αSyn oligomer and TLR2 levels in the substantia nigra tissues of human patients with idiopathic PD (iPD). The TLR2-specific αSyn elevated LRRK2 activity and neuroinflammation, and the LRRK2 inhibitor ameliorated neuroinflammatory responses in various microglia cells, alleviated neuronal degeneration along with neuroinflammation in the co-culture, and blocked the further progression of locomotor failure and dopaminergic neuronal degeneration caused by TLR2-specific αSyn in mice. Furthermore, LRRK2 phosphorylation was increased in patients with iPD showing αSyn-specific high TLR2 level. These results suggest the application of LRRK2 inhibitors as a novel therapeutic approach against αSyn-mediated PD progression.

LRRK2 Kinase Inhibitor Rejuvenates Oxidative Stress-Induced Cellular Senescence in Neuronal Cells.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the pathogenesis of Parkinson's disease (PD). Aging is the most critical risk factor for the progression of PD. The correlation between aging and cellular senescence has been established. Cellular senescence is correlated with the dysregulation of the proteolytic pathway and mitochondrial dysfunction, which are also associated with the aggregation of α-synuclein (α-syn). METHODS: Human dopaminergic neuron-like cells (differentiated SH-SY5Y cells) were treated with rotenone in the presence or absence of the LRRK2 kinase inhibitor GSK2578215A (GSK-KI) for 48 h. The markers of cellular senescence, including p53, p21Waf1/Cip1 (p21), ß-galactosidase (ß-gal), Rb phosphorylation, senescence-associated (SA) ß-gal activity, and lysosomal activity, were examined. The dSH cells and rat primary cortical neurons were treated with α-syn fibrils 30 min before treatment with rotenone in the presence or absence of GSK-KI for 48 h. Mice were intraperitoneally injected with rotenone and MLi-2 (LRRK2 kinase inhibitor) once every two days for two weeks. RESULTS: Rotenone upregulated LRRK2 phosphorylation and ß-gal levels through the activation of the p53-p21 signaling axis and downregulated Rb phosphorylation. Additionally, rotenone upregulated SA ß-gal activity, reactive oxygen species levels, and LRRK2 phosphorylation and inhibited lysosome activity. Rotenone-induced LRRK2 upregulation impaired the clearance of α-syn fibrils. Treatment with LRRK2 inhibitor mitigated rotenone-induced cellular senescence and α-syn accumulation. CONCLUSIONS: Rotenone-induced upregulation of LRRK2 kinase activity promoted cellular senescence, which enhanced α-syn accumulation. However, the administration of an LRRK2 kinase inhibitor rejuvenated rotenone-induced cellular senescence.

Analysis of α-synuclein levels related to LRRK2 kinase activity: from substantia nigra to urine of patients with Parkinson's disease.

Research on Parkinson's disease (PD) has been focused on the development of PD diagnostic tools as much as the development of PD therapeutics. Several genetic culprits of PD, including DJ-1, Leucine-rich repeat kinase 2 (LRRK2), and α-synuclein (α-syn), have been investigated as markers of PD in human biofluids. Unfortunately, the approaches to develop PD diagnostic tools are impractical, and there is a considerable demand for an appropriate marker of PD. The measurement of α-syn in biofluids has recently been made more accurate by examining monomers and aggregates separately using enzyme-linked immunosorbent assay (ELISA). Previously, we reported on the development of two types of sandwich ELISA for total α-syn and MJFR-14-6-4-2 antibody-specific α-syn fibrillar oligomers. The pathogenic LRRK2 G2019S mutation is related to increased α-syn secretion in the extracellular space. We tested our established ELISA using differentiated SH-SH5Y cells transfected with LRRK2 G2019S. The secretory levels of fibrillar oligomeric α-syn divided by total α-syn were significantly increased in LRRK2 G2019S-expressing cells. Additionally, substantia nigra lysates or concentrated urine from PD patients and non-PD subjects were analyzed. We observed ambiguous changes in the levels of total or fibrillar oligomeric α-syn and their ratio between PD and non-PD. Despite the insignificant increase in the relative levels of fibrillar oligomeric α-syn to total α-syn in PD, the duration of disease progression after diagnosis significantly corresponded to the relative levels of fibrillar oligomeric α-syn to total α-syn in the urine. These results might provide greater understanding for the next stage of development of α-syn ELISAs.

Expression of transduced nucleolin promotes the clearance of accumulated α-synuclein in rodent cells and animal model.

Alpha-synuclein (αSyn) is a major component of Lewy bodies, which are a known pathogenic marker of Parkinson's disease (PD). The dysfunction of protein degradation machinery causes αSyn accumulation. The reinforcement of αSyn degradation is a potential therapeutic target for PD because accumulated αSyn is responsible for the pathogenesis of PD. Nucleolin (NCL) is essential in the formation of the nucleolar structure. The function of NCL is correlated with oxidative stress-mediated cell death. A previous study demonstrated that NCL overexpression alleviated rotenone-induced neurotoxic effects, whereas knockdown of NCL had the opposite effect. These results suggest that NCL malfunction would exacerbate PD pathology. Thus, it was hypothesized that the introduction of ectopic NCL could rescue α-synucleinopathy in PD. This study investigated whether the ectopic expression of NCL facilitates αSyn clearance. Ectopic expression of NCL was accomplished via the transfection of green fluorescent protein (GFP) or GFP-NCL in mouse embryonic fibroblasts (MEF) or transduction of GFP or GFP-NCL using lentivirus in rat primary cortical neurons and mouse substantia nigra. NCL overexpression enhanced the clearance of accumulated or aggregated αSyn in MEFs and rat primary cortical neurons. The activity of the autophagy-lysosome pathway was enhanced by NCL expression. NCL transduction in the substantia nigra, which was co-injected with αSyn fibrils, rescued PD manifestation. The elevation of NCL levels may reflect a therapeutic strategy for α-synucleinopathy in PD.

Detection and Assessment of α-Synuclein Oligomers in the Urine of Parkinson's Disease Patients.

BACKGROUND: α-Synuclein (α-syn) is a major component of Lewy bodies, a pathologic marker of Parkinson's disease (PD) in post-mortem studies. The use of α-syn as a practical PD biomarker has been investigated by numerous researchers. However, reports of differences in α-syn levels in biofluids, such as cerebrospinal fluid, plasma, and saliva, between PD patients and controls are inconsistent. Recently, the measurement of α-syn oligomer levels has emerged as a novel approach to diagnose PD. OBJECTIVE: Lysates and culture media from two different types of dopaminergic neuronal cells or urine samples from 11 non-PD and 21 PD patients were collected and analyzed. METHODS: We developed and performed an enzyme-linked immuno-absorbent assay (ELISA) to detect various oligomeric α-syn using distinct pairs of antibodies. RESULTS: We validated our ELISA using rotenone-induced alterations of α-syn levels in human dopaminergic neurons. Total urinary α-syn levels, measured using our ELISA method, showed no difference between PD and non-PD individuals, but a higher level of α-syn oligomer recognized by MJFR-14-6-5-2 in PD urine samples was observed. Levels of distinct oligomeric α-syn detected by ASyO5 were lower in PD urine samples. Three different α-syn ELISA results were analyzed with respect to the severity of PD, but only the correlation between total α-syn levels and PD index was significant. CONCLUSION: Our findings suggest that detection of distinct oligomeric formations of α-syn and measurement of their levels in urine might be feasible for use in PD diagnostics.

G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses.

Leucine rich-repeat kinase 2 (LRRK2) is involved in the pathogenesis of Parkinson's disease (PD). LRRK2 has kinase and GTPase activities, and mediates several cell functions, including vesicle trafficking, apoptosis, autophagy, mitochondrial dynamics, and neuroinflammation. G2019S (GS) is the most prevalent mutation of LRRK2. The mutation increases kinase activity, suggesting that this activity is crucial for PD pathogenesis. The activation and inhibition of LRRK2 kinase increases and reduces the levels of proinflammatory cytokines, respectively suggesting that the role of LRRK2 in neuroinflammation is critical for the pathology of PD. Previously, we demonstrated that microglial activation by lipopolysaccharide (LPS) increases mitochondrial fission via the activation of LRRK2 kinase, while LRRK2 kinase inhibition diminishes the fission morphology and release of tumor necrosis factor-alpha (TNFα) in BV2 or rat primary microglia and the brains of GS transgenic mice. In this study, the ectopic expression of GS LRRK2 in BV2 cells significantly elevated the expression of Drp1 along the fragmented mitochondria and decreased mitochondria size compared with controls. GS LRRK2-transfected BV2 cells displayed significantly increased TNFα release and neuronal death. Inhibition of LRRK2 kinase alleviated these features. TNFα levels in brains of GS mice were significantly increased compared to those in their littermates. These data further support our previous findings concerning LPS-induced neuroinflammation and mitochondrial fission in microglia via LRRK2 kinase activation.

Upregulation of the p53-p21 pathway by G2019S LRRK2 contributes to the cellular senescence and accumulation of α-synuclein.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (LB) in neurons. α-Synuclein (αSyn) is a major component of LB and promote the PD pathogenesis via its accumulation by the impaired proteasomal or autophagic clearance. Numerous studies have revealed that the reduction of proteasome activity and autophagy is accelerated by cellular senescence. Leucine-rich repeat kinase 2 (LRRK2) contributes to PD progression and its most prevalent mutation, G2019S LRRK2, increases its activity. Our previous report has shown that the G2019S LRRK2 mutant promoted p53-induced p21 expression and neuronal cytotoxicity. The p53-p21 pathway plays a role in cellular senescence. We hypothesized that the loss of dopaminergic neurons by the stimulated p53-p21 pathway via the G2019S LRRK2 mutation might be associated with cellular senescence, thereby promoting the accumulation of αSyn. We confirmed that the ectopic expression of the phosphomimetic p53 mutant, p21, or G2019 in differentiated SH-SY5Y cells increased the following: 1) the expression of ß-galactosidase, a marker of cellular senescence, and the activity of senescence-associated ß-galactosidase, 2) endogenous αSyn protein level, but not its mRNA level, and 3) αSyn fibril accumulation in dSH-SY5Y via low proteasome and cathepsin D activities. Elevated oligomeric αSyn and the increase in ß-galactosidase with induced p21 were observed in brain lysates of G2019S transgenic mice. Our results suggest that cellular senescence is promoted via the p53-p21 pathway due to the G2019S LRRK2 mutation. Eventually, decreased protein degradation by G2019S-mediated senescence could accelerate αSyn aggregate formation.

LRRK2 impairs autophagy by mediating phosphorylation of leucyl-tRNA synthetase.

Leucine-rich repeat kinase 2 (LRRK2) is a causal gene of Parkinson disease. G2019S pathogenic mutation increases its kinase activity. LRRK2 regulates various phenotypes including autophagy, neurite outgrowth, and vesicle trafficking. Leucyl-tRNA synthetase (LRS) attaches leucine to tRNALeu and activates mTORC1. Down-regulation of LRS induces autophagy. We investigated the relationship between LRRK2 and LRS in regulating autophagy and observed interaction between endogenous LRRK2 and LRS proteins and LRS phosphorylation by LRRK2. Mutation studies implicated that T293 in the LRS editing domain was a putative phosphorylation site. Phospho-Thr in LRS was increased in cells overexpressing G2019S and dopaminergic neurons differentiated from induced pluripotent stem (iPS) cells of a G2019S carrier. It was decreased by treatment with an LRRK2 kinase inhibitor (GSK2578215A). Phosphomimetic T293D displayed lower leucine bindings than wild type (WT), suggesting its defective editing function. Cellular expression of T293D increased expression of GRP78/BiP, LC3B-II, and p62 proteins and number of LC3 puncta. Increase of GRP78 and phosphorylated LRS was diminished by treatment with GSK2578215A. Levels of LC3B, GRP78/BiP, p62, and α-synuclein proteins were also increased in G2019S transgenic (TG) mice. These data suggest that LRRK2-mediated LRS phosphorylation impairs autophagy by increasing protein misfolding and endoplasmic reticulum stress mediated by LRS editing defect. SIGNIFICANCE OF THE STUDY: Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of Parkinson disease (PD), and the most prevalent pathogenic mutation, G2019S, increases its kinase activity. In this study, we elucidated that leucyl-tRNA synthetase (LRS) was an LRRK2 kinase substrate and identified T293 as an LRRK2 phosphorylation site. LRRK2-meidated LRS phosphorylation or G2019S can lead to impairment of LRS editing, increased ER stress, and accumulation of autophagy markers. These results demonstrate that LRRK2 kinase activity can facilitate accumulation of misfolded protein, suggesting that LRRK2 kinase might be a potential PD therapeutic target along with previous studies.

Increase in anti-apoptotic molecules, nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity.

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson's disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression. Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson's disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species.

LRRK2 Kinase Activity Induces Mitochondrial Fission in Microglia via Drp1 and Modulates Neuroinflammation.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase domain and G2019S, the most prevalent LRRK2 pathogenic mutation, increases its kinase activity. LRRK2 regulates mitochondria morphology and autophagy in neurons. LPS treatment increases LRRK2 protein level and mitochondrial fission in microglia, and down-regulation of LRRK2 expression or inhibition of its kinase activity attenuates microglia activation. Here, we evaluated the direct role of LRRK2 G2019S in mitochondrial dynamics in microglia. Initial observation of microglia in G2019S transgenic mice revealed a decrease in mitochondrial area and shortage of microglial processes compared with their littermates. Next, we elucidated the molecular mechanisms of these phenotypes. Treatment of BV2 cells and primary microglia with LPS enhanced mitochondrial fission and increased Drp1, a mitochondrial fission marker, as previously reported. Importantly, both phenotypes were rescued by treatment with GSK2578215A, a LRRK2 kinase inhibitor. Finally, the protein levels of CD68, an active microglia marker, Drp1 and TNF-α were significantly higher in brain lysates of G2019S transgenic mice compared with the levels in their littermates. Taken together, our data suggest that LRRK2 could promote microglial mitochondrial alteration via Drp1 in a kinase-dependent manner, resulting in stimulation of pro-inflammatory responses. This mechanism in microglia might be a potential target to develop PD therapy since neuroinflammation by active microglia is a major symptom of PD.

Oxidized DJ-1 Levels in Urine Samples as a Putative Biomarker for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Oxidative stress is the most critical risk factor for neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). Numerous reports have demonstrated that oxidative stress aggravates cytotoxicity in dopaminergic neurons and accelerates the formation of protein inclusions. In addition, oxidative stress, such as 4-hydroxynonenal (HNE), oxidized protein, and dopamine quinone, are related to PD progression. DJ-1 is a PD-causative gene, and it plays a pivotal role as a sensor and eliminator of oxidative stress. Several studies have shown that oxidized DJ-1 (OxiDJ-1) formation is induced by oxidative stress. Hence, previous studies suggest that oxidized DJ-1 could be a biomarker for PD. We previously reported higher DJ-1 levels in Korean male PD patient urine exosomes than male non-PD controls. We speculate that OxiDJ-1 levels in PD patient urine might be higher than that in non-PD controls. In this study, we established an ELISA for OxiDJ-1 using recombinant DJ-1 treated with H2O2. Using Western blot assay and ELISA, we confirmed an increase of OxiDJ-1 from HEK293T cells treated with H2O2. Using our ELISA, we observed significantly higher, 2-fold, OxiDJ-1 levels in the urine of Korean PD patients than in non-PD controls.

Phosphorylation of p53 by LRRK2 induces microglial tumor necrosis factor α-mediated neurotoxicity.

Leucine-rich repeat kinase (LRRK2), a major causal gene of Parkinson's disease (PD), functions as a kinase. The most prevalent mutation of LRRK2 is G2019S. It exhibits increased kinase activity compared to the wildtype LRRK2. Previous studies have shown that LRRK2 can phosphorylate p53 at T304 and T377 of threonine-X-arginine (TXR) motif in neurons. Reduction of LRRK2 expression or inhibition of LRRK2 kinase activity has been shown to be able to alleviate LPS-induced neuroinflammation in microglia cells. In this study, we found that LRRK2 could also phosphorylate p53 in microglia model BV2 cells. Transfection of BV2 with phosphomimetic p53 T304/377D significantly increased the secretion of pro-inflammatory cytokine TNFα compared to BV2 transfected with p53 wild type after LPS treatment. In addition, conditioned media from these transfected cells increased the death of dopaminergic neuronal SN4741 cells. Moreover, such neurotoxic effect was rescued by co-treatment with the conditioned media and etanercept, a TNFα blocking antibody. Furthermore, TNFα secretion was significantly increased in primary microglia derived from G2019S transgenic mice treated with LPS compared to that in cells derived from their littermates. These results suggest that LRRK2 kinase activity in microglia can contribute to neuroinflammation in PD via phosphorylating p53 at T304 and T377 site.

G2385R and I2020T Mutations Increase LRRK2 GTPase Activity.

The LRRK2 mutation is a major causal mutation in familial Parkinson's disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities.

Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21(WAF1/CIP1) expression.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a gene in which a mutation causes Parkinson's disease (PD), and p53 is a prototype tumor suppressor. In addition, activation of p53 in patient with PD has been reported by several studies. Because phosphorylation of p53 is critical for regulating its activity and LRRK2 is a kinase, we tested whether p53 is phosphorylated by LRRK2. RESULTS: LRRK2 phosphorylates threonine (Thr) at TXR sites in an in vitro kinase assay, and the T304 and T377 were identified as putative phosphorylated residues. An increase of phospho-Thr in the p53 TXR motif was confirmed in the cells overexpressing G2019S, and human induced pluripotent stem (iPS) cells of a G2019S carrier. Interactions between LRRK2 and p53 were confirmed by co-immunoprecipitation of lysates of differentiated SH-SY5Y cells. LRRK2 mediated p53 phosphorylation translocalizes p53 predominantly to nucleus and increases p21(WAF1/CIP1) expression in SH-SY5Y cells based on reverse transcription-polymerase chain reaction and Western blot assay results. The luciferase assay using the p21(WAF1/CIP1) promoter-reporter also confirmed that LRRK2 kinase activity increases p21 expression. Exogenous expression of G2019S and the phosphomimetic p53 T304/377D mutants increased expression of p21(WAF1/CIP1) and cleaved PARP, and cytotoxicity in the same cells. We also observed increase of p21 expression in rat primary neuron cells after transient expression of p53 T304/377D mutants and the mid-brain lysates of the G2019S transgenic mice. CONCLUSION: p53 is a LRRK2 kinase substrate. Phosphorylation of p53 by LRRK2 induces p21(WAF1/CIP1) expression and apoptosis in differentiated SH-SY5Y cells and rat primary neurons.

An early endosome regulator, Rab5b, is an LRRK2 kinase substrate.

Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine residue was confirmed by western analysis using cells stably expressing LRRK2 G2019S. The phosphomimetic T6D mutant exhibited stronger GTPase activity than that of the wild-type Rab5b. In addition, phosphorylation of Rab5b by LRRK2 also exhibited GTPase activity stronger than that of the unphosphorylated Rab5b protein. Two assays testing Rab5's activity, neurite outgrowth analysis and epidermal growth factor receptor degradation assays, showed that Rab5b T6D exhibited phenotypes that were expected to be observed in the inactive Rab5b, including longer neurite length and less degradation of EGFR. These results suggest that LRRK2 kinase activity functions as a Rab5b GTPase activating protein and thus, negatively regulates Rab5b signalling.

Increased DJ-1 in urine exosome of Korean males with Parkinson's disease.

Parkinson's disease (PD) is a difficult disease to diagnose although it is the second most common neurodegenerative disease. Recent studies show that exosome isolated from urine contains LRRK2 or DJ-1, proteins whose mutations cause PD. To investigate a potential use for urine exosomes as a tool for PD diagnosis, we compared levels of LRRK2, α-synuclein, and DJ-1 in urine exosomes isolated from Korean PD patients and non-PD controls. LRRK2 and DJ-1, but not α-synuclein, were detected in the urine exosome samples, as reported previously. We initially could not detect any significant difference in these protein levels between the patient and the control groups. However, when age, disease duration, L-dopa daily dose, and gender were considered as analytical parameters, LRRK2 and DJ-1 protein levels showed clear gender-dependent differences. In addition, DJ-1 level was significantly higher (1.7-fold) in male patients with PD than that in male non-PD controls and increased in an age-dependent manner in male patients with PD. Our observation might provide a clue to lead to a novel biomarker for PD diagnosis, at least in males.

Dexamethasone induces the expression of LRRK2 and α-synuclein, two genes that when mutated cause Parkinson's disease in an autosomal dominant manner.

LRRK2 (leucine-rich repeat kinase 2) has been identified as a gene corresponding to PARK8, an autosomal-dominant gene for familial Parkinson's disease (PD). LRRK2 pathogenic-specific mutants induce neurotoxicity and shorten neurites. To elucidate the mechanism underlying LRRK2 expression, we constructed the LRRK2-promoter-luciferase reporter and used it for promoter analysis. We found that the glucocorticoid receptor (GR) transactivated LRRK2 in a ligand-dependent manner. Using quantitative RT-PCR and Western analysis, we further showed that treatment with dexamethasone, a synthetic GR ligand, induced LRRK2 expression at both the transcriptional and translational levels, in dopaminergic MN9D cells. Dexamethasone treatment also increased expression of α α-synuclein, another PD causative gene, and enhanced transactivation of the α-synuclein promoter-luciferase reporter. In addition, dexamethasone treatment to MN9D cells weakly induced cytotoxicity based on an LDH assay. Because glucocorticoid hormones are secreted in response to stress, our data suggest that stress might be a related factor in the pathogenesis of PD.

LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25.

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.