MicroRNA-218-5p-Ddx41 axis restrains microglia-mediated neuroinflammation through downregulating type I interferon response in a mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been largely considered one of main factors to the PD pathology. MicroRNA-218-5p (miR-218-5p) is a microRNA that plays a role in neurodevelopment and function, while its potential function in PD and neuroinflammation remains unclear. METHODS: We explore the involvement of miR-218-5p in the PD in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model. The miR-218-5p agomir used for overexpression was delivered into the substantia nigra (SN) by bilateral stereotaxic infusions. The loss of dopaminergic (DA) neurons and microglial inflammation in the SN was determined using Western blotting and immunofluorescence. Motor function was assessed using the rotarod test. RNA sequencing (RNA-seq) was performed to explore the pathways regulated by miR-218-5p. The target genes of miR-218-5p were predicted using TargetScan and confirmed using dual luciferase reporter assays. The effects of miR-218-5p on microglial inflammation and related pathways were verified in murine microglia-like BV2 cells. To stimulate BV2 cells, SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP+) and the conditioned media (CM) were collected. RESULTS: MiR-218-5p expression was reduced in both the SN of MPTP-induced mice and MPP+-treated BV2 cells. MiR-218-5p overexpression significantly alleviated MPTP-induced microglial inflammation, loss of DA neurons, and motor dysfunction. RNA sequence and gene set enrichment analysis showed that type I interferon (IFN-I) pathways were upregulated in MPTP-induced mice, while this upregulation was reversed by miR-218-5p overexpression. A luciferase reporter assay verified that Ddx41 was a target gene of miR-218-5p. In vitro, miR-218-5p overexpression or Ddx41 knockdown inhibited the IFN-I response and expression of inflammatory cytokines in BV2 cells stimulated with MPP+-CM. CONCLUSIONS: MiR-218-5p suppresses microglia-mediated neuroinflammation and preserves DA neurons via Ddx41/IFN-I. Hence, miR-218-5p-Ddx41 is a promising therapeutic target for PD.

Biglycan regulated colorectal cancer progress by modulating enteric neuron-derived IL-10 and abundance of Bacteroides thetaiotaomicron.

Biglycan (BGN) is a proteoglycan with branch chains and highly expressed in enteric neurons in the tumor tissue of colorectal cancer (CRC), which is negatively associated with survival rates in patients with CRC. However, how the proteoglycan promotes the progress of CRC through interacting with bacteria and regulating the immune response of enteric neurons remains largely unknown. In the present study, we found that biglycan deficiency changed tumor distribution in a colitis-associated colon cancer model. Furthermore, we revealed that BGN deficiency inhibits tumor growth in an allograft tumor model and the migration of cancer cell by upregulating interleukin-10 expression in enteric neurons. Significantly, we demonstrated that biglycan deficiency enriched the abundance of Bacteroides thetaiotaomicron through competing with it for chondroitin sulfate to inhibit CRC progress. Our work provided new insights into the interaction between host proteoglycan and gut microbiota as well as the role of enteric neurons in the tumor microenvironment.

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 µM BaA and the developmental retardations induced by 15 µM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

The influence of alkyl substitution on the in vitro metabolism and mutagenicity of benzo[a]pyrene.

In recent years concerns over consumer exposure to mineral oil aromatic hydrocarbons (MOAH), especially those containing alkylated polycyclic aromatic hydrocarbons (PAHs), have emerged. This is especially due to the fact that some PAHs are known to be genotoxic and carcinogenic upon metabolic activation. However, available toxicological data on PAHs mainly relate to non-substituted PAHs with limited data on alkyl substituted PAHs. Therefore, the aim of the present study was to characterize in more detail the effect of alkyl substitution on the metabolism and mutagenicity of benzo[a]pyrene (B[a]P), a PAH known to be genotoxic and carcinogenic. To this end, the oxidative metabolism and mutagenicity of B[a]P and a series of its alkyl substituted analogues were quantified using in vitro microsomal incubations and the Ames test. The results obtained reveal that upon alkylation the metabolic oxidation shifts to the aliphatic side chain at the expense of aromatic ring oxidation. The overall metabolism, including metabolism via aromatic ring oxidation resulting potentially in bioactivation, was substantially reduced with elongation of the alkyl side chain, with metabolism of B[a]P with an alkyl substituent of >6 C atoms being seriously hampered. In the Ames test upon metabolic activation, the methyl substitution of B[a]P resulted in an increase or decrease of the mutagenic potency depending on the substitution position. The relevant pathways for mutagenicity of the selected monomethyl substituted B[a]P may involve the formation of a 7,8-dihydrodiol-9,10-epoxide, a 4,5-oxide and/or a benzylic alcohol as an oxidative side chain metabolite which subsequently may give rise to an unstable and reactive sulfate ester conjugate. It is concluded that alkylation of B[a]P does not systematically reduce its mutagenicity in spite of the metabolic shift from aromatic to side chain oxidation.

The effect of alkyl substitution on the oxidative metabolism and mutagenicity of phenanthrene.

Alkyl-substituted PAHs may be present in certain petroleum-derived products and in the environment and may eventually end up in consumer products, such as foodstuffs, cosmetics and pharmaceuticals. Safety concerns over possible exposure to alkylated PAHs have emerged. Bioactivation is a prerequisite for the mutagenicity and carcinogenicity of PAHs and has been extensively studied for non-substituted PAHs, while data on the bioactivation of alkyl-substituted PAHs are scarce. The present study investigated the effect of alkyl substitution on the CYP 450-mediated metabolism of phenanthrene and eight of its alkylated congeners by quantifying metabolite formation in rat and human liver microsomal incubations. Furthermore, the mutagenicity of four selected methylated phenanthrenes was compared to that of phenanthrene using the Ames test. The obtained results support the hypothesis that alkyl substitution shifts the oxidative metabolism from the aromatic ring to the alkyl side chain. Increasing the length of the alkyl chain reduced overall metabolism with metabolic conversion for 1-n-dodecyl-phenanthrene (C12) being negligible. 1- and 9-methyl-phenanthrene, in which the methyl group generates an additional bay region-like structural motif, showed mutagenicity toward Salmonella typhimurium TA98 and TA 100, whereas phenanthrene and also 2- and 3-methyl-phenanthrene, without such an additional bay region-like structural motif, tested negative. It is concluded that the position of the alkylation affects the metabolism and resulting mutagenicity of phenanthrene with the mutagenicity increasing in cases where the alkyl substituent creates an additional bay region-like structural motif, in spite of the extra possibilities for side chain oxidation.

Upregulated hexokinase 2 expression induces the apoptosis of dopaminergic neurons by promoting lactate production in Parkinson's disease.

Parkinson's disease (PD) is characterized by impaired mitochondrial function and decreased ATP levels. Aerobic glycolysis and lactate production have been shown to be upregulated in dopaminergic neurons to sustain ATP levels, but the effect of upregulated glycolysis on dopaminergic neurons remains unknown. Since lactate promotes apoptosis and α-synuclein accumulation in neurons, we hypothesized that the lactate produced upon upregulated glycolysis is involved in the apoptosis of dopaminergic neurons in PD. In this study, we examined the expression of hexokinase 2 (HK2) and lactate dehydrogenase (LDH), the key enzymes in glycolysis, and lactate levels in the substantia nigra pars compacta (SNpc) of a MPTP-induced mouse model of PD and in MPP+-treated SH-SY5Y cells. We found that the expression of HK2 and LDHA and the lactate levels were markedly increased in the SNpc of MPTP-treated mice and in MPP+-treated SH-SY5Y cells. Exogenous lactate treatment led to the apoptosis of SH-SY5Y cells. Intriguingly, lactate production and the apoptosis of dopaminergic neurons were suppressed by the application of 3-bromopyruvic acid (3-Brpa), a HK2 inhibitor, or siRNA both in vivo and in vitro. 3-Brpa treatment markedly improved the motor behaviour of MPTP-treated mice in pole test and rotarod test. Mechanistically, lactate increases the activity of adenosine monophosphate-activated protein kinase (AMPK) and suppresses the phosphorylation of serine/threonine kinase 1 (Akt) and mammalian target of rapamycin (mTOR). Together, our data suggest that upregulated HK2 and LDHA and increased lactate levels prompt the apoptosis of dopaminergic neurons in PD. Inhibition of HK2 expression attenuated the apoptosis of dopaminergic neurons by downregulating lactate production and AMPK/Akt/mTOR pathway in PD.

A nomogram based on iron metabolism can help identify apathy in patients with Parkinson's disease.

Backgrounds: Apathy is common in Parkinson's disease (PD) but difficult to identify. Growing evidence suggests that abnormal iron metabolism is associated with apathy in PD. We aimed to investigate the clinical features and iron metabolism of apathetic patients with PD, and construct a nomogram for predicting apathy in PD. Methods: Data of 201 patients with PD were analyzed. Demographic data, Apathy Scale (AS) assessments, and serum iron metabolism parameters were obtained. Spearman correlations were used to assess relationships between AS scores and iron metabolism parameters, separately for male and female patients. Additionally, a nomograph for detecting apathetic patients with PD was built based on the results of logistic regression analysis. Results: The serum transferrin (TRF, p < 0.0024) concentration and total iron binding capacity (TIBC, p < 0.0024) were lower in the apathetic group after Bonferroni correction, and they were negatively associated with AS scores in male participants with PD (TRF, r = -0.27, p = 0.010; TIBC, r = -0.259, p = 0.014). The nomogram was developed by incorporating the following five parameters: age, sex, serum iron concentration, TIBC and Hamilton Depression Rating Scale (HAMD) scores, which showed good discrimination and calibration, with a consistency index of 0.799 (95% confidence interval = 0.732-0.865). Conclusion: Abnormal iron metabolism may contribute to apathy in PD, especially among men. TIBC levels in combination with HAMD scores can be effectively used for the prediction of apathetic patients with PD.

Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells.

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.

[mRNA expression of MAGE genes in benign and malignant pleural effusion].

BACKGROUND: Melanoma antigen genes (MAGE) are tumor specific genes. The aim of this study is to explore the feasibility of MAGE genes detection as a diagnostic method for malignant pleural effusion. METHODS: The expression of MAGE-1, -2, -3 and -4 mRNA was detected in 18 benign and 22 malignant pleural effusion samples by RT-PCR. RESULTS: No MAGE gene expressed in the 18 cases of benign pleural effusion. Out of the 22 cases of malignant pleural effusion, 8 cases were positive by cytological examination, who all showed positive expression of MAGA genes; in the other 14 patients who were positive by pleural biopsy but negative by cytological examination, 11 cases showed positive expression of MAGE genes in both the pleural effusion and pleura samples, while the other 3 cases showed negative MAGE expression in both the pleural effusion and pleura samples. CONCLUSIONS: Detection of MAGE genes in pleural effusion may be an effective method in the differential diagnosis of benign and malignant pleural effusion.

[Detection of occult micrometastasis using MAGE genes in lymph nodes of patients with non-small cell lung cancer].

BACKGROUND: Melanoma antigen genes (MAGE genes) are tumor specific genes. In this study the expressions of MAGE-1, -2, -3 and -4 genes in lymph nodes of patients with non-small cell lung cancer (NSCLC) at mRNA level were investigated and the role of MAGE genes was analyzed in the diagnosis of occult micrometastasis in lymph nodes of patients with NSCLC. METHODS: One hundred and eleven stations of lymph nodes from 53 patients with NSCLC were studied to detect mRNA for MAGE-1, -2, -3 and -4 genes by using reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: The expression rate in samples of lymph nodes using RT-PCR (41.4%, 46/111) was significantly higher than that using routine histopathological examination (27.9%, 31/111). Of the 80 stations of lymph nodes without invasion of the tumor confirmed by routine histopathological examination, at least one of these genes was expressed in (23.8%) (19/80) out of the samples. Of the 31 stations of lymph nodes with invasion of the tumor confirmed by routine histopathological examination, at least one of these MAGE genes was expressed in 87.1% (27/31) out of the samples. In the lymph nodes of the patients with non-cancerous diseases, the MAGE-1, -2, -3 and -4 were not expressed at mRNA level. CONCLUSIONS: Micrometastasis in lymph nodes of patients with NSCLC could be diagnosed by investigating the expressions of MAGE genes at mRNA level. Detection of MAGE-1, (-2,) -3 and -4 might be helpful to diagnoze micrometastasis in lymph node and to increase the accuracy of TNM stages in NSCLC.

[Lung squamous cell carcinoma-related genes cloned by using suppression subtractive hybridization].

OBJECTIVE: To identify genes differentially expressed in human lung squamous cell carcinoma (LSCC). METHODS: A subtracted cDNA library of human LSCC constructed by using suppression subtracted hybridization (SSH) method was screened. Clones representing mRNAs that were truly differentially expressed in LSCC but not in its adjacent non-cancerous tissues were identified by semi-quantitative RT-PCR in 12 patients with LSCC. Partial novel genes were detected by Northern blot. RESULTS: Ten differentially expressed gene cDNA fragments of LSSC were obtained by SSH. Among them six were known genes; two sequences were identified but their functions were unknown (hypothetical protein); two were novel (GenBank accession numbers AF363068 and AY032661, respectively). The results from semiquantitative RT-PCR showed that the transcription expression level of PPP1CB, calumenin, S100A2, HSNOV1, OCIA and AY032661 was down-regulated in some LSCC cases, while the transcription of HSP90, ferritin, gp96 and AF363068 was up-regulated in others. CONCLUSIONS: Six known genes identified by SSH technique have been implicated in the pathogenesis of lung carcinogenesis, or they are involved in immunological defense mechanism in human body. Two hypothetical proteins probably also play an important role in the pathogenesis of lung cancer. The function of the two novel genes in lung carcinogenesis are under investigation.

[mRNA expressions of MAGE genes in human lung cancer].

BACKGROUND: To investigate the expressions of MAGE-1,-2,-3 and -4 in lung cancer at mRNA level. METHODS: Using a reverse transcription-polymerase chain reaction assay (RT-PCR), the expressions of MAGE-1,-2,-3 and -4 in 35 lung cancer samples and non-tumorous lung tissues were examined. RESULTS: Of the 35 tumor samples, the MAGE-1,-2,-3 and -4 were expressed at mRNA level in 22.9% (8/35), 62.9% (22/35), 37.1% (13/35) and 77.1% (27/35) respectively. At least one of these genes was expressed in 85.7% (30/35) of the samples. Two or more of these genes were expressed in 71.4% (25/35) of the samples. None of the non-tumorous lung tissue was positive for these genes. No significant difference of the frequency of MAGE gene expression was found between adenocarcinoma and squamous cell carcinoma, between different tumor stages, and between groups with or without lymph node metastasis (P > 0.05). CONCLUSIONS: MAGE-1,-2,-3 and -4 are expressed at a high percentage in lung cancer. All of these genes are negative in the adjacent non tumorous lung tissue. These results indicate the possibility of a future specific immunotherapy for lung cancer based on these MAGE antigens.

[Molecular cloning of genes differentially expressed in human lung squamous cell carcinoma using suppression subtractive hybridization].

BACKGROUND: To clone and identify genes differentially expressed in human lung squamous cell carcinoma (LSCC). METHODS: A subtracted cDNA library of human LSCC was constructed by suppression subtracted hybridization (SSH) method. After screening, the subtracted library clones representing mRNAs that were truly differentially expressed in LSCC but not in its adjacent non cancerous tissues were selected to identify by RT-PCR and DNA sequencing were performed. Nucleic acid homology searches were performed using the BLAST program. RESULTS: By this technique, 10 differentially expressed gene cDNA fragments of LSSC were obtained. Two were novel and eight were already known genes. CONCLUSIONS: SSH is a useful technique with high sensitivity for the detection of differential genes expression in LSCC and an effective method to clone novel genes.

[Expression of cancer-testis antigens in human lung cancer].

BACKGROUND: To investigate the expression of cancer-testis antigens(CTA) in human lung cancer. METHODS: Reverse-transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of the MAGE-1, -3, SSX-1, -2, -4, -5 and NY-ESO-1 genes in 35 lung cancer samples and corresponding non-tumorous lung tissues. Three samples selected randomly from each CTA PCR product were sequenced. RESULTS: In 35 tumor samples, the MAGE-1, -3, SSX-1, -2, -4, -5 and NY-ESO-1 mRNA expression rates were 34.3%(12/35), 57.1%(20/35), 17.1%(6/35), 17.1%(6/35), 20.0%(7/35), 25.7%(9/35) and 37.1%(13/35), respectively. The positive rate was 74.3%(26/35) for at least one of these genes expression, and 65.7%(23/35) for two or more genes coexpression. No non-tumorous lung tissue was positive for these genes. The DNA sequence confirmed that the RT-PCR products were truly CTA cDNA. CONCLUSIONS: The cancer-testis antigens are potential targets for antigen-special immunotherapy of lung cancer. The coexpression pattern of these antigens provides a theoretic foundation for developing a polyvalent lung cancer vaccine.

[Autologous lung cancer tissues derived gp96-peptide complex/dendritic cell vaccine induces the cytotoxic T cell response in vitro].

BACKGROUND: To investigate whether autologous lung cancer tissues derived gp96-peptide complex/dendritic cell vaccine could induce peptide specific cytotoxic T lymphocyte (CTL) response in vitro. METHODS: A patient's tumor-derived antigens including gp96-peptide complexes and tumor cell lysate were co cultured with DCs derived from the same patient's bone marrow blood mononuclear cells. The various antigen/DC vaccines were used to stimulate peripheral lymphocytes. Interferon-γ (IFN-γ) level of activated lymphocytes was detected by ELISA method and the Cr51 release test was performed to evaluate the gp96-peptide specific CTL response in three kinds of target cells including the primary cultured tumor cells, PG cells and K562 cells. RESULTS: IFN-γ could be observed from the supernate collected in all antigen groups after the cognate T lymphocytes were stimulated by various vaccines. The concentration of IFN-γ induced by gp96-peptide complexes/DC vaccine was higher than that of other groups. In addition, the killing effect of the activated T lymphocytes on patient's primary tumor cells was higher than that on PG and K562 cells. CONCLUSIONS: Autologous tumor-derived gp96-peptide complexes can induce a peptide complex specific CTL response, and the CTL response is significantly intensified after DCs are pulsed.