Expression of G2019S LRRK2 in Rat Primary Astrocytes Mediates Neurotoxicity and Alters the Dopamine Synthesis Pathway in N27 Cells via Astrocytic Proinflammatory Cytokines and Neurotrophic Factors.

Astrocytes in the brain contribute to various essential functions, including maintenance of the neuronal framework, survival, communication, metabolic processes, and neurotransmitter levels. Leucine-rich repeat kinase 2 (LRRK2) is associated with the pathogenesis of Parkinson's disease (PD). LRRK2 is expressed in neurons, microglia, and astrocytes and plays diverse roles in these cell types. We aimed to determine the effects of mutant human G2019S-LRRK2 (GS-hLRRK2) in rat primary astrocytes (rASTROs). Transfection with GS-hLRRK2 significantly decreased cell viability compared to transfection with the vector and wild-type human LRRK2 (WT-hLRRK2). GS-hLRRK2 expression significantly reduced the levels of nerve growth factor and increased the levels of proinflammatory cytokines (interleukin-1ß and tumor necrosis factor α) compared to the vector and WT-hLRRK2 expression. Furthermore, GS-hLRRK2 expression in rASTROs promoted astrogliosis, which was characterized by increased expression of glial fibrillary acidic protein and vimentin. Treatment with the conditioned medium of G2019S LRRK2-expressing rASTROs decreased N27 cell viability compared to treatment with that of WT-hLRRK2-expressing rASTROs. Consequently, the regulation of the dopamine synthesis pathway was affected in N27 cells, thereby leading to altered levels of tyrosine hydroxylase, dopamine transporter, Nurr1, and dopamine release. Overall, the G2019S LRRK2 mutation disrupted astrocyte function, thereby aggravating PD progression.

Efficacy of mecasin for treatment of amyotrophic lateral sclerosis: A phase IIa multicenter randomized double-blinded placebo-controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder characterized by progressive paralysis of voluntary muscles. Mecasin, the extract of modified jakyakgamchobuja-tang-a herbal preparation comprising of Radix Paeoniae Alba, Radix Glycyrrhizae, Radix Aconiti Lateralis Preparata, Radix Salviae Miltiorrhizae, Rhizoma Gastrodiae, Radix Polygalae, Curcuma Root, Fructus Chaenomelis, and Rhizoma Atractylodis Japonicae-shows neuroprotective and anti-neuroinflammatory effects and alleviates the symptoms in patients with ALS. AIM OF THE STUDY: This trial aimed to evaluate the efficacy and safety of mecasin in these patients. MATERIAL AND METHODS: Patients were randomized to receive mecasin 1.6 g daily, mecasin 2.4 g daily, or placebo for 12 weeks. The primary endpoint was the Korean version of ALS Functional Rating Scale-Revised (K-ALSFRS-R) score. The secondary endpoints were muscular atrophy measurements, pulmonary function test results, creatine kinase levels, body weight, safety, and scores of the Medical Research Council (MRC) scale for muscle strength; Visual Analog Scale for pain (VAS pain); Hamilton Rating Scale for Depression; and Fatigue Severity Scale. RESULTS: Among the 30 patients randomized, 24 completed the follow-up. Significant between-group differences were detected in the primary endpoint using the omnibus F-test. The changes in the K-ALSFRS-R score between 12 weeks and baseline were -0·25, -1·32, and -2·78 in the mecasin 1.6 g, mecasin 2.4 g, and placebo groups, respectively. The difference in the K-ALSFRS-R score between the mecasin 1.6 g and placebo groups was 2·53 points (95% confidence interval [CI]: 0·61-4·45), and that between the 2.4 g and placebo groups was 1·46 points (95% CI: 0·48-3·40). However, no significant differences were detected in the secondary endpoints (MRC: dyspnea, p = 0·139; VAS pain, p = 0·916; forced vital capacity, p = 0·373). The incidence of adverse events was similar and low in all groups. CONCLUSIONS: Mecasin may retard symptomatic progression without major adverse effects. A phase IIb study to evaluate its long-term effects in ALS is ongoing.

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.

A case study on the application evaluation of integrative medical service model for the improvement of quality of life for dementia patients and caregivers.

BACKGROUND: Since 4 major diseases and geriatric diseases require consistent management, individuals with any of these diseases cannot live alone and need caregivers' assistance. Given these characteristics, an integrative medical service model for 4 major diseases and geriatric diseases was developed in Korea, currently. Dementia, one of the typical geriatric diseases, requires caregivers' assistance from the beginning because of its enormous burden. Thus, it is necessary to provide an integrative medical service that can improve the quality of life (QoL) for both patients and caregivers. Therefore, this study aims to collect various feedback by applying an integrative medical service, which was developed to improve the QoL in patients with dementia and their caregivers, to a single case, and to modify and improve the integrative medical service model based on the results. METHOD/DESIGN: The integrative medical service program, which was developed to improve the QoL in patients with dementia and their caregivers in Korea, will be used for a patient-caregiver pair. This is an observational study with quantitative and qualitative feedback from various viewpoints. The program will be conducted in 8 sessions (twice a week, within 120 minutes). The patient will receive both Western and Korean medicine, and an integrative service will be provided to improve cognitive rehabilitation and QoL. Feedback collected at each session will be reflected on the program of the subsequent session. RESULTS: This study will then modify and improve the program with feedback and provide integrative medical services to a patient with dementia and caregiver. DISCUSSION: Patients with dementia need a program that would help them maintain cognitive function, and caregivers need a program that would improve their QoL by reducing the caregiving burden. This study is unique because the developed program is performed after modification based on feedback from the previous session. Accordingly, the patient and caregiver can check which program is the most satisfactory and helpful in improving their QoL. We expect that this study can modify the integrative medical service model to the optimized patient-based model. This study can also be used as basic data for a clinical pathway development study that applies the modified model to medical institutes.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of familial Parkinson's disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S+/- mice. However, a significant increase in ciliogenesis was observed in G2019S+/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

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.

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.

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.

Urinary Biomarkers for Neurodegenerative Diseases.

Global incidence of neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) is rapidly increasing, but the diagnosis of these diseases at their early stage is challenging. Therefore, the availability of reproducible and reliable biomarkers to diagnose such diseases is more critical than ever. In addition, biomarkers could be used not only to diagnose diseases but also to monitor the development of disease therapeutics. Urine is an excellent biofluid that can be utilized as a source of biomarker to diagnose not only several renal diseases but also other diseases because of its abundance in invasive sampling. However, urine was conventionally regarded as inappropriate as a source of biomarker for neurodegenerative diseases because it is anatomically distant from the central nervous system (CNS), a major pathologic site of NDD, in comparison to other biofluids such as cerebrospinal fluid (CSF) and plasma. However, recent studies have suggested that urine could be utilized as a source of NDD biomarker if an appropriate marker is predetermined by metabolomic and proteomic approaches in urine and other samples. In this review, we summarize such studies related to NDD.

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.

26-Week Repeated Dose Oral Toxicity Study of KCHO-1 in Sprague-Dawley Rats.

OBJECTIVES: KCHO-1(Mecasin), also called Gamijakyakgamchobuja-tang originally, is a combination of some traditional herbal medicines in East Asia. This medicine has been used mainly for alleviating neuropathic pains for centuries in Korean traditional medicine. KCHO-1 was developed to treat pain, joint contracture and muscular weakness in patients with amyotrophic lateral sclerosis. This study was carried out to investigate the chronic toxicity of KCHO-1 oral administration in rats for 26 weeks. METHODS: Sprague-Dawely rats were divided into four groups and 10 rats were placed in the control group and the high-dose group, respectively. Group 1 was the control group and the remaining groups were the experimental groups. In the oral toxicity study, 500 mg/kg, 1,000 mg/kg, and 2,000 mg/kg of KCHO-1 were administered to the experimental group, and 10 ml/kg of sterile distilled water was administered to the control group. Survival rate, body weight, feed intake, clinical signs, and visual findings were examined. Urinalysis, ophthalmologic examination, necropsy, organ weight, hematologic examination, blood chemical examination and histopathologic examination were performed. RESULTS: Mortality and toxicological lesions associated with the administration of test substance were not observed in all groups. CONCLUSION: NOAEL(No observed adverse effect level) of KCHO-1 is higher than 2000 mg/kg/day. And, the above findings suggest that treatment with KCHO-1 is relatively safe.

Rab GTPases as Physiological Substrates of LRRK2 Kinase.

LRRK2 (Leucine-Rich Repeat Kinase 2) is a gene whose specific mutations cause Parkinson's disease (PD), the most common neurodegenerative movement disorder. LRRK2 harbors GTPase and kinase activities, two enzyme activities that play critical roles in the regulation of cellular signal transduction. Among the several LRRK2 pathogenic mutations, the most prevalent G2019S mutation increases its kinase activity when compared with the wild-type (WT), suggesting that LRRK2 kinase substrates are potential culprits of PD pathogenesis. Although there were several studies to identify LRRK2 kinase substrates, most of them mainly employed in vitro kinase assays. Therefore, it remains uncertain whether the identified substrates were real physiological substrates. However, efforts to determine physiological LRRK2 kinase substrates have recently identified several members of the Rab GTPase family as physiological LRRK2 kinase substrates. A conserved threonine or serine in the switch II domain of certain Rab GTPase family members (Rab3A/B/C/D, Rab5A/B, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) has been pinpointed to be phosphorylated by LRRK2 in cells using sophisticated phosphoproteomics technology in combination with LRRK2-specific kinase inhibitors. The Rab GTPases regulate vesicle trafficking, suggesting that LRRK2 may be a regulator of such vesicle trafficking, confirming previously suggested LRRK2 functions. However, how the consequence of the LRRK2-mediated Rab phosphorylation is related to PD pathogenesis is not clear. This review briefly summarizes the recent results about LRRK2-mediated Rab phosphorylation studies.

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.

Characterization of Parkinson's disease-related pathogenic TMEM230 mutants.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Although most PD cases are sporadic, 5-10% of them are hereditary and several pathogenic mutations in related genes have been identified. Mutations in TMEM230 were recently identified as a cause of autosomal dominant PD. However, the basic properties of the mutant proteins are not yet known. We examined stability and neurotoxicity, important characteristics of PD pathogenesis-related proteins, of WT TMEM230 and two pathogenic mutants, R78L and PG5ext, in a dopaminergic neuronal cell line. Our study showed that amount of protein expressed in the same vector backbone was R78L > WT > PG5ext. The stabilities of the mutant proteins were similar to each other, but lower than that of the WT. In addition, overexpression of mutants and WT TMEM230 caused similar levels of neurotoxicity upon MPP+ treatment when compared to the cells transfected with an empty vector. Because the proteins encoded by two PD-causing genes, TMEM230 and LRRK2, function in vesicle trafficking, we tested whether they interact. LRRK2 neither interacts with, nor phosphorylates TMEM230. We also investigated the levels of several Rab proteins (Rab1A, 5, 7, 8A and 11) involved in vesicle trafficking after TMEM230 overexpression. However, there was no clear difference of any Rab proteins among cells transfected with an empty vector, TMEM230 WT and mutants-expressing cells, suggesting that TMEM230 does not directly regulate these Rab proteins. Thus, these TMEM230 PG5ext and R78L mutant proteins are not distinctly different from the WT proteins except for their stability. Abbreviations: LRRK2: Leucine-rich repeat kinase 2; PD: Parkinson's disease; AD: Alzheimer's disease; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FACS: fluorescence-activated cell sorting; PBS: phosphate buffered saline; FBS: fetal bovine serum; PI: propidium iodide.

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 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.

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.