KCHO-1, a Novel Antineuroinflammatory Agent, Inhibits Lipopolysaccharide-Induced Neuroinflammatory Responses through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse BV2 Microglia Cells

The brain is vulnerable to oxidative stress and inflammation that can occur as a result of aging or neurodegenerative diseases. Our work has sought to identify natural products that regulate heme oxygenase (HO)-1 and to determine their mechanism of action in neurodegenerative diseases. KCHO-1 is a novel herbal therapeutic containing 30% ethanol (EtOH) extracts from nine plants. In this study, we investigated the antineuroinflammatory effects of KCHO-1 in lipopolysaccharide- (LPS-) treated mouse BV2 microglia. KCHO-1 inhibited the protein expression of inducible nitric oxide synthase (iNOS), iNOS-derived nitric oxide (NO), cyclooxygenase- (COX-) 2, and COX-2-derived prostaglandin E2 (PGE2) in LPS-stimulated BV2 microglia. It also reduced tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 production. This effect was correlated with the suppression of inhibitor of nuclear factor kappa B-α (IκB-α) phosphorylation and degradation and nuclear factor kappa B (NF-κB) translocation and DNA binding. Additionally, KCHO-1 upregulated HO-1 expression by promoting nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in mouse BV2 microglia. Tin protoporphyrin (SnPP), an HO activity inhibitor, was used to verify the inhibitory effects of KCHO-1 on proinflammatory mediators and proteins associated with HO-1 expression. Our data suggest that KCHO-1 has therapeutic potential in neurodegenerative diseases caused by neuroinflammation

Effect of leucine-rich repeat kinase 2 (LRRK2) on protein synthesis

Mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD) in an autosomal dominant manner. Pathogenic mutations of LRRK2 such as G2019S and R1441C have been observed as common genetic causes of PD. Recently, LRRK2 has been reported to increase the reporter protein synthesis in both cap-dependent and -independent manners via phosphorylation of the ribosomal protein RPS15. In this study, we tested whether LRRK2 recombinant protein would directly increase protein synthesis using a well-defined in vitro coupled transcription/translation system. Addition of commercial full-length LRRK2 or GST-fused N-terminal-deleted LRRK2 recombinant proteins to the system showed no change of protein synthesis, as measured by luciferase reporter activity. In addition, the SUnSET assay to measure newly synthesized cellular proteins showed that G2019S overexpression had a minimal effect on the total protein amount. However, we confirmed the previous result that G2019S overexpression increased the amount of protein synthesized from an exogenous gene, Flag-VAMP2, which was transfected as a reporter, whereas there was no significant change in the amount of the Flag-VAMP2 mRNA. Inhibition of protein degradation showed that protein accumulation in the vector control was higher than that of the G2019S overexpression vector. Our results suggest that LRRK2 protein influences the amount of protein by inhibiting protein degradation rather than by directly stimulating translation

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

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

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

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

A Study on the Oral Toxicity of Mecasin in Rats

Objectives: In this study, we investigated the oral toxicity of Gami-Jakyak Gamcho buja Decoction (Mecasin) to develop safe treatments. Methods: All experiments were conducted at the Medvill, an institution authorized to perform non-clinical studies, under the Good Laboratory Practice (GLP) regulations. In order to investigate the oral toxicity of Mecasin, we administered Mecasin orally to rats. Sprague-Dawley rats were divided into four groups of five male and five female animals per group: group 1 being the control group and groups 2, 3, and 4 being the experimental groups. Doses of Mecasin, 500 mg/kg, 1,000 mg/kg and 2,000 mg/kg, were administered to the experimental groups, and a dose of normal saline solution, 10 mL/kg, was administered to the control group. We examined the survival rate, weight, clinical signs, and gross findings. This study was conducted under the approval of the Institutional Animal Ethics Committee. Results: No deaths or abnormalities occurred in any of the four groups. Although slight decreases in the weights of some female rats were noted on the third day, no significant changes in weights or gross findings between the control group and the experimental groups were observed. To check for abnormalities in organs, we used microscopy to examine representative histological sections of each specified organ; the results showed no significant differences in any of the organs. Conclusion: The results showed that administration of 500 − 2,000 mg/kg of Mecasin did not cause any changes in weight or in the results of necropsy examinations. It also did not result in any mortalities. The above findings suggest that treatment with Mecasin is relatively safe. Further studies on this subject are needed to yield more concrete evidence