GSDMA at the crossroads between pyroptosis and tumor immune evasion in glioma.

Pyroptosis, an inflammatory and programmed cell death process, has been controversial in its role in tumor immunity. However, as the first molecule in the gasdermin family, the mechanism of GSDMA in glioma growth is not well understood. We identified the differentially expressed gene GSDMA from Treg cells-related genes using the TCGA database. The biological functions of GSDMA and the relationship between GSDMA expression and tumor immune cell infiltration and cancer patient survival were investigated using open-source databases and platforms. Additionally, flow cytometry analysis was used to examine the effect of GSDMA on tumor immune cell infiltration. Our study showed that GSDMA expression played an important role in immune evasion in glioma. Patients with high GSDMA expression had a worse prognosis. In vivo studies demonstrated that GSDMA knockdown could enhance the infiltration level of CD8+ T cells. High GSDMA expression was also positively correlated with poor anti-PD-L1 treatment outcomes in GBM patients, suggesting that GSDMA may be a potential biomarker that should be considered in combination with anti-PD-L1 therapy for glioma patients. In conclusion, our study demonstrates that high GSDMA expression in gliomas is associated with immune-infiltrating cells CD8+ T cells and Treg cells, and indicates a worse prognosis in glioma. Therefore, GSDMA may serve as a therapeutic target for glioma progression and should be applied in immunotherapy for glioma patients.

Novel read-through fusion transcript Bcl2l2-Pabpn1 in glioblastoma cells.

Read-through fusion transcripts have recently been identified as chimeric RNAs and have since been linked to tumour growth in some cases. Many fusion genes generated by chromosomal rearrangements have been described in glioblastoma. However, read-through fusion transcripts between neighbouring genes in glioblastoma remain unexplored. We performed paired-end RNA-seq of rat C6 glioma cells and normal cells and discovered a read-through fusion transcript Bcl2l2-Pabpn1 in which exon 3 of Bcl-2-like protein 2 (Bcl2l2) fused to exon 2 of Polyadenylate-binding protein 1 (Pabpn1). This fusion transcript was found in both human glioblastoma and normal cells. Unlike other fusions reported in glioblastoma, Bcl2l2-Pabpn1 appeared to result from RNA processing rather than genomic rearrangement. Bcl2l2-Pabpn1 fusion transcript encoded a fusion protein with BH4, BCL and RRM domains. Functionally, Bcl2l2-Pabpn1 knockdown by targeting its fusion junction decreased its expression, and suppressed cell proliferation, migration and invasion in vitro. Mechanistically, Bcl2l2-Pabpn1 blocked Bax activity and activated PI3K/AKT pathway to promote glioblastoma progression. Together, our work characterized a glioblastoma-associated Bcl2l2-Pabpn1 fusion transcript shared by humans and rats.

Susceptibility of cytoskeletal-associated proteins for tumor progression.

The traditional functions of cytoskeletal-associated proteins (CAPs) in line with polymerization and stabilization of the cytoskeleton have evolved and are currently underrated in oncology. Although therapeutic drugs have been developed to target the cytoskeletal components directly in cancer treatment, several recently established therapeutic agents designed for new targets block the proliferation of cancer cells and suppress resistance to existing target agents. It would seem like these targets only work toward inhibiting the polymerization of cytoskeletal components or hindering mitotic spindle formation in cancer cells, but a large body of literature points to CAPs and their culpability in cell signaling, molecular conformation, organelle trafficking, cellular metabolism, and genomic modifications. Here, we review those underappreciated functions of CAPs, and we delineate the implications of cellular signaling instigated by evasive properties induced by aberrant expression of CAPs in response to stress or failure to exert normal functions. We present an analogy establishing CAPs as vulnerable targets for cancer systems and credible oncotargets. This review establishes a paradigm in which the cancer machinery may commandeer the conventional functions of CAPs for survival, drug resistance, and energy generation; an interesting feature overdue for attention.

IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis.

BACKGROUND: Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE. METHODS: In vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student's t test, as appropriate. RESULTS: Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process. CONCLUSIONS: These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.

The orphan nuclear receptor Nurr1 agonist amodiaquine mediates neuroprotective effects in 6-OHDA Parkinson's disease animal model by enhancing the phosphorylation of P38 mitogen-activated kinase but not PI3K/AKT signaling pathway.

Recent studies implicate the defects or altered expression of the orphan nuclear receptor Nurr1 gene in the substantia nigra in Parkinson's disease pathogenesis. In an attempt to corroborate the treatment-modifying disease that would replicate the effect of Nurr1, it has been found that amodiaquine and Nurr1 had the same chemical scaffolding, indicating a crucial structure-activity relationship. Interestingly, amodiaquine stimulate the transcriptional function of Nurr1 by physical interaction with its ligand-binding domain (LBD). However, the signaling route by which Nurr1 is activated by amodiaquine to cause the protective effect remains to be elucidated. We first demonstrated that amodiaquine treatment ameliorated behavioural deficits in 6-OHDA Parkinson's disease mouse model, and it promoted dopaminergic neurons protection signified by Tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA; Tyrosine hydroxylase (TH) protein expression level and the immunoreactivity in the substantia nigra compacta. Subsequently, we used inhibitors to ascertain the effect of amodiaquine on Akt and P38 Mapk as crucial signaling pathways for neuroprotection. Wortmannin (Akt Inhibitor) induced a significant reduction of Akt mRNA; however, there was no statistical difference between the amodiaquine-treated group and the control group suggesting that amodiaquine may not be the active stimulant of Akt. Western blot analysis confirmed that the phosphorylated Akt decreased significantly in the amodiaquine group compared to the control group. In the same vein, we found that amodiaquine substantially increased the level of phosphorylated P38 Mapk. When P38 Mapk inhibited by SB203580 (P38-Mapk Inhibitor), the total P38 Mapk but not the phosphorylated P38 Mapk decreased significantly, while tyrosine hydroxylase significantly increased. These results collectively suggest that amodiaquine can augment tyrosine hydroxylase expression via phosphorylated P38 Mapk while negatively regulating the phosphorylated Akt in protein expression.

Glial cell line-derived neurotrophic factor alters lipid composition and protein distribution in MPP+-injured differentiated SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and striatum. Glial cell line-derived neurotrophic factor (GDNF) can effectively promote the differentiation and survival of many types of neurons, especially dopaminergic neurons, suggesting it could be a treatment for PD. Lipid rafts are highly dynamic cell membrane domains that contain numerous signal protein receptors, providing an important platform for signal transduction. Compelling evidence indicates that alterations in lipid rafts are associated with PD, and some studies have reported that GDNF can regulate the expression of caveolin-1, a lipid raft-marker protein. However, the precise effects of GDNF on lipid rafts remain unknown. We developed a cellular PD model, purified detergent-resistant membranes (membrane rafts), and performed proteomic and lipid metabolomics analyses to examine changes in lipid rafts after GDNF treatment. The results showed considerable protein and lipid alterations in response to GDNF, especially altered levels of dopamine-ß-hydroxylase, heat shock 70 kDa protein, neural cell adhesion molecule, cytoskeletal proteins, and long-chain polysaturated/unsaturated fatty acids. These findings reveal a new avenue to explore the relationships between GDNF, lipid rafts, and PD and support the hypothesis that GDNF may be a useful treatment for PD.

Identification of novel LncRNA targeting Smad2/PKCα signal pathway to negatively regulate malignant progression of glioblastoma.

Glioblastoma multiforme (GBM) is a highly proliferative cancer with generally poor prognosis and accumulating evidence has highlighted the potential of long noncoding RNAs (lncRNAs) in the biological behaviors of glioma cells. This study focused on the identification of lncRNAs to identify targets for possible GBM prognosis. Microarray expression profiling found that 1,759 lncRNAs and 3,026 messenger RNAs (mRNAs) were upregulated, and 1932s lncRNA and 2,979 mRNAs were downregulated in GBM. Bioinformatics analysis and experimental verification identified TCONS_00020456 (TCON) for further analysis. In situ hybridization, along with immunohistochemical and receiver operating characteristic analysis determined TCON (truncation value = 3.5) as highly sensitive and specific in GBM. Grade IV patients with glioma life span with different lncRNA staining scores were analyzed. TCON staining scores below 3.5 indicated poor prognosis (life span ranging from 0.25 to 7 months), even if the glioma was surgically removed. TCON decreased significantly in GBM, and showed a coexpressional relationship with Smad2 and protein kinase C α (PKCα). Overexpression of TCON reduced the proliferation on one hand and migration, invasion on the other. TCON also inhibited epithelial-mesenchymal transformation and glioma progression in vivo, based on a nude mouse tumorigenicity assay. In addition, we predicted a potential binding site and intersection that microRNAs targeting Smad2, PKCα, and TCON through RACE pretest and bioinformatics analysis. Taken together, TCON, regarded as oncosuppressor, targeting the Smad2/PKCα axis plays a novel role in inhibiting the malignant progression of glioma. Moreover, it also demonstrates that the level of TCON can be used as a prognostic and diagnostic biomarker for GBM.

Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression.

BACKGROUND: Nuclear translocation of several oncogenic proteins have previously been reported, but neither the translocation of doublecortin (DCX) nor the mechanism involved has been studied. DCX is a neuronal microtubule-associated protein (MAP) that is crucial for adult neurogenesis and neuronal migration and has been associated with poor prognosis in gliomas. METHODS: We probed DCX expression in different grades of glioma tissues and conventional cells via western blotting. Then we analyzed the expression pattern in the Oncomine cancer profiling database. Confocal Immunofluorescence was used to detect DCX expression in the cellular compartments, while subcellular fractionation was probed via western blotting. Pulse shape height analysis was utilized to verify DCX localization in a larger population of cells. Co-immunoprecipitation was used in detecting DCX-import receptors interactions. To probe for DCX functions, stable cells expressing high DCX expression or knockdown were generated using CRISPR-Cas9 viral transfection, while plasmid site-directed mutant constructs were used to validate putative nuclear localization sequence (NLS) predicted via conventional algorithms and comparison with classical NLSs. in-silico modeling was performed to validate DCX interactions with import receptors via the selected putative NLS. Effects of DCX high expression, knockdown, mutation, and/or deletion of putative NLS sites were probed via Boyden's invasion assay and wound healing migration assays, and viability was detected by CCK8 assays in-vitro, while xenograft tumor model was performed in nude mice. RESULTS: DCX undergoes nucleocytoplasmic movement via the RanGTPase signaling pathway with an NLS located on the N-terminus between serine47-tyrosine70. This translocation could be stimulated by MARK's phosphorylation of the serine 47 residue flanking the NLS due to aberrant expression of glial cell line-derived neurotrophic factor (GDNF). High expression and nuclear accumulation of DCX improve invasive glioma abilities in-vitro and in-vivo. Moreover, knocking down or blocking DCX nuclear import attenuates invasiveness and proliferation of glioma cells. CONCLUSION: Collectively, this study highlights a remarkable phenomenon in glioma, hence revealing potential glioma dependencies on DCX expression, which is amenable to targeted therapy. Video abstract.

Down-Regulated CUEDC2 Increases GDNF Expression by Stabilizing CREB Through Reducing Its Ubiquitination in Glioma.

Abnormally high expression of glial cell line-derived neurotrophic factor (GDNF) derived from glioma cells has essential impacts on gliomagenesis and development, but the molecular basis underlying increased GDNF expression in glioma cells remain unclear. This work aimed to study the molecular mechanisms that may explain the accumulation of GDNF in glioma. Firstly, we observed that cAMP response element-binding protein (CREB), known as an important transcription factor for binding of GDNF promoter region, was highly expressed with an apparent accumulation into the nucleus of glioma cells, which may contribute to the transcription of GDNF. Secondly, CUE domain-containing protein 2 (CUEDC2), a ubiquitin-regulated protein, could increase the amount of binding between the E3 ligase tripartite motif-containing 21 (TRIM21) and CREB and affect the CREB level. Like our previous study, it showed that there was a significantly down-regulation of CUEDC2 in glioma. Finally, our data suggest that GDNF expression is indirectly regulated by transcription factor ubiquitination. Indeed, down-regulation of CUEDC2, decreased the ubiquitination and degradation of CREB, which was associated to high levels of GDNF. Furthermore, abundant CREB involved in the binding to the GDNF promoter region contributes to GDNF high expression in glioma cells. Collectively, it was verified the GDNF expression was affected by CREB ubiquitination regulated by CUEDC2 level.

Serum levels of glial cell line-derived neurotrophic factor and multiple neurotransmitters: In relation to cognitive performance in Parkinson's disease with mild cognitive impairment.

OBJECTIVES: Mild cognitive impairment is a common non-motor feature of Parkinson's disease, termed PD-MCI. But there is a scarcity of data on the role of glial cell line-derived neurotrophic factor (GDNF) and neurotransmitters in pathogenesis of PD-MCI. The aim of this project was to detect the serum levels of GDNF and multiple neurotranmitters and explore their relationships with cognitive performance in PD-MCI patients. METHODS: Neuropsychological testing was administered to PD patients and healthy controls to investigate different domains of cognitive function. Serum levels of GDNF and four cognition-related neurotransmitters including Dopamine metabolites Homovanillic acid (HVA), acetylcholine (Ach), γ-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) were detected by enzyme-linked immunosorbent assay and liquid chromatography-electrospray ionization tandem mass spectrometry analysis respectively. RESULTS: The more serious cognitive impairment of PD, the lower levels of GDNF, HVA and 5-HT. In PD-MCI patients, the levels of GDNF, HVA, Ach, 5-HT, and GABA had a significant positive correlation with Digit span backward test (DSB-T) scores and negative correlation with the scores of Trail Making Test A (TMT-A) and Trail Making Test B (TMT-B) respectively. Effect size analysis showed that GDNF and GDNF*Ach have a significant effect on DSB-T, TMT-A and TMT-B respectively; GDNF*HVA, GDNF*5-HT and GDNF*GABA play important part in Auditory Verbal Learning Test separately. CONCLUSIONS: Serum GDNF may be involved in the impairment of attention, memory and executive function of PD-MCI patients, by acting alone or in conjunction with neurotransmitters (HVA, 5-HT, GABA, and Ach).

The reversible effects of glial cell line-derived neurotrophic factor (GDNF) in the human brain.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor, and a member of the transforming growth factor ß (TGF-ß) superfamily acting on different neuronal activities. GDNF was originally identified as a neurotrophic factor crucially involved in the survival of dopaminergic neurons of the nigrostriatal pathway and is currently an established therapeutic target in Parkinson's disease. However, GDNF was later reported to be highly expressed in gliomas, especially in glioblastomas, and was demonstrated as a potent proliferation factor involved in the development and migration of gliomas. Here, we review our current understanding and progress made so far by researchers in our laboratories with references to relevant articles to support our discoveries. We present past and recent discoveries on the mechanisms involved in the protection of neurons by GDNF and examine its emerging roles in gliomas, as well as reasons for the abnormal expression in Glioblastoma Multiforme (GBM). Collectively, our work establishes a paradigm by which the ability of GDNF to protect dopaminergic neurons from degradation and its corresponding effects on glioma cells points to an underlying biological vulnerability in the effects of GDNF in the normal brain which can be subverted for use by cancer cells. Hence, presenting novel opportunities for intervention in glioma therapies.

MiR-409-3p and MiR-1896 co-operatively participate in IL-17-induced inflammatory cytokine production in astrocytes and pathogenesis of EAE mice via targeting SOCS3/STAT3 signaling.

Th17 cells and interleukin-17 (IL-17) have been found to play an important role in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Response to IL-17, reactive astrocytes accompany with immune cells infiltration and axonal damage in MS/EAE. However, the role and the regulatory mechanism of IL-17-activated astrocytes in inflammation and in the EAE process still remain largely unknown. Here, we elucidated that miR-409-3p and miR-1896, as co-upregulated microRNAs in activated astrocytes and in EAE mice, targeted suppressor of cytokine signaling proteins 3 (SOCS3). Overexpression of miR-409-3p or miR-1896 significantly reduced SOCS3 expression and increased phosphorylation of STAT3 as well as induced the inflammatory cytokines production (IL-1ß, IL-6, IP-10, MCP-1, and KC), CD4+ T cells migration and demyelination, in turn aggravating EAE development. Importantly, the effects of co-overexpression of miR-409-3p and miR-1896 in vitro or in vivo are strongly co-operative. In contrast, simultaneously silenced miR-409-3p and miR-1896 co-operatively ameliorates inflammation and demyelination in the central nervous system of EAE mice. Collectively, our findings highlight that miR-409-3p and miR-1896 co-ordinately promote the production of inflammatory cytokines in reactive astrocytes through the SOCS3/STAT3 pathway and enhance reactive astrocyte-directed chemotaxis of CD4+ T cells, leading to aggravate pathogenesis in EAE mice. Co-inhibition of miR-409-3p and miR-1896 may be a therapeutic target for treating MS and neuroinflammation.

HIV-1 Tat enhances purinergic P2Y4 receptor signaling to mediate inflammatory cytokine production and neuronal damage via PI3K/Akt and ERK MAPK pathways.

BACKGROUND: HIV-associated neurocognitive disorders (HANDs) afflict more than half of HIV-1-positive individuals. The transactivator of transcription (Tat) produced by HIV virus elicits inflammatory process and is a major neurotoxic mediator that induce neuron damage during HAND pathogenesis. Activated astrocytes are important cells involved in neuroinflammation and neuronal damage. Purinergic receptors expressed in astrocytes participate in a positive feedback loop in virus-induced neurotoxicity. Here, we investigated that whether P2Y4R, a P2Y receptor subtype, that expressed in astrocyte participates in Tat-induced neuronal death in vitro and in vivo. METHODS: Soluble Tat protein was performed to determine the expression of P2Y4R and proinflammatory cytokines in astrocytes using siRNA technique via real-time PCR, Western blot, and immunofluorescence assays. Cytometric bead array was used to measure proinflammatory cytokine release. The TUNEL staining and MTT cell viability assay were analyzed for HT22 cell apoptosis and viability, and the ApopTag® peroxidase in situ apoptosis detection kit and cresyl violet staining for apoptosis and death of hippocampal neuron in vivo. RESULTS: We found that Tat challenge increased the expression of P2Y4R in astrocytes. P2Y4R signaling in astrocytes was involved in Tat-induced inflammatory cytokine production via PI3K/Akt- and ERK1/2-dependent pathways. Knockdown of P2Y4R expression significantly reduced inflammatory cytokine production and relieved Tat-mediated neuronal apoptosis in vitro. Furthermore, in vivo challenged with Tat, P2Y4R knockdown mice showed decreased inflammation and neuronal damage, especially in hippocampal CA1 region. CONCLUSIONS: Our data provide novel insights into astrocyte-mediated neuron damage during HIV-1 infection and suggest a potential therapeutic target for HANDs.

Analysis of Long Noncoding RNA and mRNA Expression Profiles in IL-9-Activated Astrocytes and EAE Mice.

BACKGROUND/AIMS: Multiple sclerosis (MS) is an autoimmune disease in the central nervous system associated with demyelination and axonal injury. Astrocyte activation is involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This study was designed to find potential lncRNAs in EAE mice and activated astrocytes. METHODS: we performed microarray analysis of lncRNAs from the brain tissues of EAE mice and primary mouse astrocytes treated with IL-9(50 ng/ml). 12 lncRNAs were validated through real-time PCR. Gene ontology and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RESULTS: Differentially expressed 3300 lncRNAs and 3250 mRNAs were in the brain tissues of EAE mice, and 3748 lncRNAs and 3332 mRNAs were in activated astrocytes. Notably, there were 2 co-up-regulated lncRNAs and 3 co-down-regulated lncRNAs both in the brain tissues of EAE mice and in activated astrocytes, including Gm14005, Gm12478, mouselincRNA1117, AK080435, and mouselincRNA0681, which regulate the ER calcium flux kinetics, zinc finger protein and cell apoptosis. Similarly, there were 7 mRNAs co-up-regulated and 2 mRNAs co-down-regulated both in vivo and in vitro. Gene ontology and KEGG pathway analysis showed that the biological functions of differentially expressed mRNAs were associated with metabolism, development and inflammation. The results of realtime PCR validation were consistent with the data from the microarrays. CONCLUSIONS: Our data uncovered the expression profiles of lncRNAs and mRNAs in vivo and in vitro, which may help delineate the mechanisms of astrocyte activation during MS/EAE process.

Decreased intracellular chloride promotes ADP induced platelet activation through inhibition of cAMP/PKA instead of activation of Lyn/PI3K/Akt pathway.

Decrease of chloride concentration contributes to cardiovascular diseases, however, whether decrease of chloride concentration is involved in platelet activation remains elusive. In the present study, we found that ACI patients had lower serum chloride which would be rescued after Aspirin administration. ADP induced chloride concentration reduction in platelets. Blockade of chloride channel prevented ADP-induced platelet adhesion, activation and aggregation, however, decreasing the extracellular chloride concentration promoted ADP-induced platelet adhesion and activation. Decrease of the extracellular chloride concentration facilitated the inactivation of Src family kinase Lyn, which was not involved in PI3K/Akt phosphorylation. Nevertheless, low chloride concentration promoted the production of platelet cytosol Gαi2 subunit. This subunit prevents AC from converting ATP into cAMP, which therefore, inhibited the phosphorylation of PKA to promote platelet activation. In conclusion, decreased intracellular chloride promotes ADP induced platelet activation through the Gαi2/cAMP/PKA pathway instead of the Lyn/PI3K/Akt signal pathway.

High Concentration of Glial Cell Line-Derived Neurotrophic Factor Protects Primary Astrocytes from Apoptosis.

BACKGROUND: Studies have shown that astrocytes play an important role in a variety of biological processes, so damage to astrocytes can cause a series of related diseases. Glial cell line-derived neurotrophic factor (GDNF) has always been considered a protective factor for dopamine neurons. However, it remains unclear whether GDNF has a protective effect on glial cells, especially astrocytes. In this study, we put forward the hypothesis that a high concentration of GDNF in the microenvironment of astrocytes exerts an inhibitory effect on the apoptosis of astrocytes by DNA-damaging reagents. METHODS: We isolated, purified, and identified primary astrocytes from neonate rats. Astrocytes were exposed to mitoxantrone (MTN, a DNA-damaging compound) for 24 h. The effects of MTN on astrocytes were tested by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay. One of the concentrations of MTN was applied to construct an apoptotic model of astrocytes. The astrocytes were then treated with GDNF together with a selected concentration of MTN for 24 h. The cell viability, cell nucleus morphology, and apoptosis ratio of the cells was assessed by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay, respectively. RNA sequencing (RNA-Seq), quantitative PCR analysis, and KEGG pathway mapping were performed to examine the genes involved in the procedure. Finally, Western blot analysis was applied to confirm the expression levels of the proteins of interest. RESULTS: Hoechst 33342 staining revealed a one-tenth change in the percentage of Hoechst-positive cells after the addition of 500 ng/mL GDNF combined with 1,000 nM MTN for 24 h. The viability of the cells treated the same as described above was 1.4-fold that of the control group. Flow cytometry assays indicated that the apoptotic rates were 17.67, 8.67, and 4.34% for 0, 200, and 500 ng/mL GDNF, respectively. Birc2, Birc3, and Gadd45b were linked to the antiapoptotic process induced by GDNF in astrocytes. Western blot analysis confirmed the elevated expression of Birc2 and Gadd45b. CONCLUSIONS: Our studies revealed that GDNF has a noticeable antiapoptotic effect on gene-injured astrocytes. This may provide critical clues for the treatment of a series of diseases in which damaged astrocytes are involved.

HIV-1 Nef-induced lncRNA AK006025 regulates CXCL9/10/11 cluster gene expression in astrocytes through interaction with CBP/P300.

BACKGROUND: HIV-associated neurocognitive disorder (HAND) is a neurodegenerative disease associated with persistent neuroinflammation and subsequent neuron damage. Pro-inflammatory factors and neurotoxins from activated astrocytes by HIV-1 itself and its encoded proteins, including the negative factor (Nef), are involved in the pathogenesis of HAND. This study was designed to find potential lncRNAs that regulate astrocyte functions and inflammation process. METHODS: We performed microarray analysis of lncRNAs from primary mouse astrocytes treated with Nef protein. Top ten lncRNAs were validated through real-time PCR analysis. Gene ontology (GO) and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RIP and ChIP assays were performed to demonstrate the mechanism of lncRNA regulating gene expression. RESULTS: There were 638 co-upregulated lncRNAs and 372 co-downregulated lncRNAs in primary astrocytes treated with Nef protein for both 6 h and 12 h. GO and KEGG pathway analysis showed that the biological functions of top differential-expressed mRNAs were associated with inflammatory cytokines and chemokine. Knockdown of lncRNA AK006025, not AK138360, inhibited significantly CXCL9, CXCL10 (IP-10), and CXCL11 expression in astrocytes treated with Nef protein. Mechanism study showed that AK006025 associated with CBP/P300 was enriched in the promoter of CXCL9, CXCL10, and CXCL11 genes. CONCLUSIONS: Our findings uncovered the expression profiles of lncRNAs and mRNAs in vitro, which might help to understand the pathways that regulate astrocyte activation during the process of HAND.

Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins.

Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of α/ß hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.

MiRNAs Mediate GDNF-Induced Proliferation and Migration of Glioma Cells.

BACKGROUND/AIMS: Glial cell line-derived neurotrophic factor (GDNF) is an important factor promoting invasive glioma growth. This study was performed to reveal a unique mechanism of glioma cell proliferation and migration. METHODS: Human U251 glioma cells were used to screen the optimal GDNF concentration and treatment time to stimulate proliferation and migration. MicroRNA (MiRNA) expression profiles were detected by microarray and confirmed by real-time polymerase chain reaction (PCR). The target genes of differentially expressed miRNAs were predicted by miRWalk, and those targeted by multiple miRNAs were screened with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A regulatory miRNA network was constructed using ingenuity pathway analysis (IPA). Target gene expression of differentially expressed miRNAs was examined by real-time PCR or mRNA microarray. RESULTS: The results show that 50 ng/mL GDNF for 24 h significantly promotes U251 glioma cell proliferation and migration (P < 0.05). Seven miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-629-5p, hsa-miR-3609, hsa-miR-183-5p, and hsa-miR-487b-3p) were significantly up-regulated after GDNF treatment (P < 0.05). These miRNAs are primarily involved in signal transduction, cell adhesion and cell cycle through mitogen-activated protein kinase (MAPK) signaling, focal adhesion and glioma signal pathways. Five of these miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-183-5p, and hsa-miR-487b-3p) co-regulate TP53 and Akt. mRNA expression levels of four genes co-targeted by two or more up-regulated miRNAs were significantly decreased after GDNF treatment (P < 0.05). CONCLUSION: GDNF treatment of U251 glioma cells significantly increased the expression of seven miRNAs involved in cell adhesion and the cell cycle.

PERK and XBP1 differentially regulate CXCL10 and CCL2 production.

Inflammation plays a key role in the pathogenesis of many retinal degenerative diseases related with photoreceptor dysfunction/degeneration. However the involvement of photoreceptor cells in inflammatory reactions is largely unknown as they are not considered as inflammatory cells. In this study, we assessed whether photoreceptor cells can produce CCL2 and CXCL10, two important players in inflammation during endoplasmic reticulum (ER) stress. After photoreceptor 661 W cells were treated with ER stress inducer thapsigargin (TG), induction of ER stress increased CXCL10 and CCL2 expression at both mRNA and protein levels, which was significantly blocked by an ER stress blocker 4-phenylbutyrate. ER stress contains three pathways: PERK, ATF6 and IRE1α. Knockdown of PERK attenuated TG-induced CXCL10 and CCL2 mRNA expression, associated with significant decreases in phosphorylation of NF-κB RelA and STAT3. In contrast to PERK, knockdown of XBP1, which is activated by IRE1α-mediated splicing, robustly enhanced TG-induced CXCL10 and CCL2 expression and phosphorylation of NF-κB RelA and STAT3. Blockade of NF-κB or STAT3 markedly diminished TG-induced CXCL10 and CCL2 expression. The specific roles of PERK and XBP1 in CXCL10 and CCL2 expression were further investigated by treating photoreceptor cells with advanced glycation end products (AGE) and high glucose (HG), two of the major contributors to diabetic complications. Similarly, AGE and HG induced CXCL10 and CCL2 expression in which PERK was a positive regulator while XBP1 was a negative regulator. These studies suggest that photoreceptors may be involved in retinal inflammation by expressing chemokines CXCL10 and CCL2. PERK and IRE1α/XBP1 in the unfolded protein response differentially regulate the expression of CXCL10 and CCL2 likely through modulation of ER stress-induced NF-κB RelA and STAT3 activation.