Botulinum Toxin A Ameliorates Neuroinflammation in the MPTP and 6-OHDA-Induced Parkinson's Disease Models.

Recently, increasing evidence suggests that neuroinflammation may be a critical factor in the development of Parkinson's disease (PD) in addition to the ratio of acetylcholine/dopamine because dopaminergic neurons are particularly vulnerable to inflammatory attack. In this study, we investigated whether botulinum neurotoxin A (BoNT-A) was effective for the treatment of PD through its anti-neuroinflammatory effects and the modulation of acetylcholine and dopamine release. We found that BoNT-A ameliorated MPTP and 6-OHDA-induced PD progression, reduced acetylcholine release, levels of IL-1ß, IL-6 and TNF-α as well as GFAP expression, but enhanced dopamine release and tyrosine hydroxylase expression. These results indicated that BoNT-A had beneficial effects on MPTP or 6-OHDA-induced PD-like behavior impairments via its anti-neuroinflammation properties, recovering dopamine, and reducing acetylcholine release.

Loss of parkin reduces lung tumor development by blocking p21 degradation.

Several epidemiological studies have demonstrated the reciprocal relationship between the development of cancer and Parkinson's disease (PD). However, the possible mechanisms underlying this relationship remain unclear. To identify this relationship, we first compared lung tumor growth in parkin knockout (KO) mice and wild-type (WT) mice. Parkin KO mice showed decreased lung tumor growth and increased expression of p21, a cell cycle arrester, as compared with WT mice. We also found that parkin interacts with p21, resulting in its degradation; however, parkin KO, knockdown, as well as mutation (R275W or G430D) reduced the degradation of p21. We investigated whether parkin KO increases the association of p21 with proliferating cell nuclear antigen (PCNA) or CDK2 by reducing p21 degradation, and, thus, arresting the cell cycle. The interaction between p21 and PCNA or CDK2 was also enhanced by parkin knockdown, and this increased interaction induced sub G0/G1 arrest, leading to cell death. Therefore, our data indicate that parkin KO reduces the development of lung tumors via cell cycle arrest by blocking the degradation of p21. These findings suggest that PD could be associated with lower lung cancer incidence.

Chitinase-3-like-1 deficiency attenuates ethanol-induced liver injury by inhibition of sterol regulatory element binding protein 1-dependent triglyceride synthesis.

OBJECTIVE: Alcohol overconsumption and abuse lead to alcoholic liver disease (ALD), which is a major chronic liver disease worldwide. Chitinase-3-like protein 1 (CHI3L1) have an important role in the pathogenesis of inflammatory disease. However, the role of CHI3L1 in ALD has not yet been reported. In the present study, we investigated the effect of CHI3L1 on chronic plus binge ethanol-induced liver injury. METHODS: CHI3L1 knock out (KO) mice and their littermate control mice based on C57BL/6 (10-12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 10 days. And, CHI3L1 siRNA or CHI3L1 expressing vector was transfected HepG2 cells were treated with ethanol or without. RESULTS: Ethanol-induced hepatic triglyceride (TG) levels and the mRNA levels of TG synthesis-related genes such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) were decreased in the liver of CHI3L1 knock out (KO) mice and the HepG2 cells transfected with CHI3L1 siRNA. Increased mRNA level and activation of SREBP1 which is transcription factor of ACC, FAS and SCD1 by ethanol feeding were reduced in the liver of ethanol-fed CHI3L1 KO mice. Moreover, ethanol-induced SREBP1 luciferase activity and mRNA level of SREBP1, ACC, FAS and SCD1 were also decreased in the HepG2 cells transfected with CHI3L1 siRNA, while those were further increased in the HepG2 cells treated with recombinant human CHI3L1. Furthermore, oxidative stress and up-regulated pro-inflammatory cytokines by ethanol were recovered in the liver of ethanol-fed CHI3L1 KO mice. CONCLUSION: Our finding suggest that inhibition of CHI3L1 suppressed ethanol-induced liver injury through inhibition of TG synthesis, and the blocking of oxidative stress and hepatic inflammation induced SREBP1 activity could be significant.

Deficiency of parkin suppresses melanoma tumor development and metastasis through inhibition of MFN2 ubiquitination.

Parkin, a critical gene of Parkinson's disease, is involved in the development of numerous cancers. However, the effect of parkin deficiency on melanoma growth and metastasis has not been reported. We showed that the tumor size and number of surface lung metastases, and expression of tumor growth and metastasis marker proteins were significantly lower in parkin-KO mice than those observed in non-transgenic controls. In an in vitro study, we also showed that parkin siRNA inhibited cell growth and migration of B16F10 and SK-Mel-28 cells. Parkin-specific ubiquitination of mitofusin-2 (MFN2) was decreased in tumors and metastasized lung tissues of parkin-KO mice. Moreover, we showed that parkin directly binds and ubiquitinates MFN2. Knockdown of MFN2 decreased the expression of Bax and apoptotic cell death, but increased that of Bcl2 and apoptotic cancer cell death. However, these effects were reversed by knockdown of parkin. Conversely, inhibitory effects on melanoma growth and migration of parkin siRNA were reversed by MFN2 siRNA. These data indicate that melanoma development was inhibited in parkin-KO mice through maintaining of MFN2 level by inhibition of ubiquitinating ability of parkin.

CCR5 knockout suppresses experimental autoimmune encephalomyelitis in C57BL/6 mice.

Multiple sclerosis (MS) is an inflammatory disease in which myelin in the spinal cord is damaged. C-C chemokine receptor type 5 (CCR5) is implicated in immune cell migration and cytokine release in central nervous system (CNS). We investigated whether CCR5 plays a role in MS progression using a murine model, experimental autoimmune encephalomyelitis (EAE), in CCR5 deficient (CCR5-/-) mice. CCR5-/- and CCR5+/+ (wild-type) mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) followed by pertussis toxin, after which EAE paralysis was scored for 28 days. We found that clinical scoring and EAE neuropathology were lower in CCR5-/- mice than CCR5+/+ mice. Immune cells (CD3+, CD4+, CD8+, B cell, NK cell and macrophages) infiltration and astrocytes/microglial activation were attenuated in CCR5-/- mice. Moreover, levels of IL-1ß, TNF-α, IFN-γ and MCP-1 cytokine levels were decreased in CCR5-/- mice spinal cord. Myelin basic protein (MBP) and CNPase were increased while NG2 and O4 were decreased in CCR5-/- mice, indicating that demyelination was suppressed by CCR5 gene deletion. These findings suggest that CCR5 is likely participating in demyelination in the spinal cord the MS development, and that it could serve as an effective therapeutic target for the treatment of MS.

Inhibition of p38 pathway-dependent MPTP-induced dopaminergic neurodegeneration in estrogen receptor alpha knockout mice.

Approximately, 7-10 million people in the world suffer from Parkinson's disease (PD). Recently, increasing evidence has suggested the protective effect of estrogens against nigrostriatal dopaminergic damage in PD. In this study, we investigated whether estrogen affects 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral impairment in estrogen receptor alpha (ERα)-deficient mice. MPTP (15mg/kg, four times with 1.5-h interval)-induced dopaminergic neurodegeneration was evaluated in ERα wild-type (WT) and knockout (KO) mice. Larger dopamine depletion, behavioral impairments (Rotarod test, Pole test, and Gait test), activation of microglia and astrocytes, and neuroinflammation after MPTP injection were observed in ERα KO mice compared to those in WT mice. Immunostaining for tyrosine hydroxylase (TH) after MPTP injection showed fewer TH-positive neurons in ERα KO mice than WT mice. Levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC, metabolite of dopamine) were also lowered in ERα KO mice after MPTP injection. Interestingly, a higher immunoreactivity for monoamine oxidase (MAO) B was found in the substantia nigra and striatum of ERα KO mice after MPTP injection. We also found an increased activation of p38 kinase (which positively regulates MAO B expression) in ERα KO mice. In vitro estrogen treatment inhibited neuroinflammation in 1-methyl-4-phenyl pyridium (MPP+)-treated cultured astrocyte cells; however, these inhibitory effects were removed by p38 inhibitor. These results indicate that ERα might be important for dopaminergic neuronal survival through inhibition of p38 pathway.

Measurement of Human Cytochrome P450 Enzyme Induction Based on Mesalazine and Mosapride Citrate Treatments Using a Luminescent Assay.

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their inter-assay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

Protective effects of Vitamin E on endocrine disruptors, PCB-induced dopaminergic neurotoxicity.

UNLABELLED: The protective effect of an antioxidant, Vitamin E (dl-alpha-tocopherol, 100 mg/kg/day, 8 days p.o. in vivo and 10 and 50 microM in vitro) was tested against PCB-induced neurotoxicity. IN VIVO STUDIES: Microdialysis was used to investigate changes in the striatal extracellular dopamine level and in p-nNOS expression in PCB-treated (Aroclor 1254, 10 microg/ml, 2 microl/min, 5 h; 6 microg was infused by microdialysis probe) rats. IN VITRO STUDIES: Cell viability and levels of p-nNOS expression were observed in PCB-treated (Aroclor 1254, 5 microg/ml) immortalized dopaminergic cell line (CATH.a cells). RESULTS: Treatment with PCB: (1) decreased the extracellular dopamine level in rat striatum, (2) increased p-nNOS expression both in rat striatal tissue and in CATH.a cells, (3) reduced the cell viability of, and (4) increased LDH release by CATH.a cells. However, Vitamin E showed a protective effect against PCB-induced toxicity and downregulation of the extracellular dopamine level. These results indicate that Vitamin E may have neuroprotective effects by inhibiting PCB-induced nNOS phosphorylation.