Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

Glioma stem cells (GSC) were important for Glioblastoma (GBM) initiation and chemotherapy resistance. Centrosomal protein of 55 kDa (CEP55) was a biomarker for multiple cancers. However, roles and mechanism of CEP55 in glioma tumorigenesis and stemness maintains of stem like cells was still unclear. U251 cells which stable overexpression or downregulation of CEP55 was obtained by lentivirus mediated transduction. Roles and mechanism of CEP55 in stemness maintains of stem like cells and tumorigenesis was investigated. Our results implied that knockdown the expression of CEP55 inhibited the invasion and migration of U251 cells, while overexpression of CEP55 displayed opposite results. Moreover, overexpression of CEP55 promoted neurosphere formation of glioma stem-like cells, while CEP55 knockdown decreased the number and size of neurosphere. Mechanistically, overexpression of CEP55 enhanced the expression of Forkhead box protein M1 (FOXM1), Matrix metalloproteinases (MMPs) and activated the NF-κB pathway, while knockdown CEP55 displayed opposite results. In conclusion, our results indicated that CEP55 played an important role in promoting the invasion and migration of U251 cell and self-renewal of glioma stem like cells which might be a new therapeutic target for glioma.

Parkinson's disease (PD) is a neurodegenerative disease characterized by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). We recently reported that Six2 could reverse the degeneration of DA neurons in a dephosphorylation state. Here we further identified that Eya1 was the phosphatase of Six2 that could dephosphorylate the tyrosine 129 (Y129) site by forming a complex with Six2 in damaged DA cells. Dephosphorylated Six2 then translocates from the cytoplasm to the nucleus. Using ChIP-qPCR and dual luciferase assay, we found that dephosphorylated Six2 down-regulates TEA domain1 (Tead1) expression, thus inhibiting 6-hydroxydopamine (6-OHDA)-induced apoptosis in DA cells. Furthermore, we showed Six2Y129F/Tead1 signaling could protect against the loss of SNpc tyrosine hydroxylase-positive (TH+) cells and improve motor function in PD model rats. Our results demonstrate a dephosphorylation-dependent mechanism of Six2 that restores the degeneration of DA neurons, which could represent a potential therapeutic target for PD.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the substantia nigra (SN) dopamine (DA) neurons. The transmembrane signaling of GDNF is mediated by a unique receptor system, including the ligand binding receptor GDNF family receptor alpha (GFRalpha) and the transmembrane signaling receptor Ret or neural cell adhesion molecule-140 (NCAM-140). Here, we found that another transmembrane cell adhesion molecule, integrin, a heterodimer consisting of alpha and beta subunits, also mediates the transmembrane signaling of GDNF. The results showed that the level of phosphorylated Src homology 2 domain containing (Shc), which was associated with the cytoplasmic domain of integrin beta1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin beta1 could form a complex with GFRalphal. The simulation of molecular modeling showed that four H-bonds were formed between integrin beta1 and GFRalpha. These data indicate that integrin beta1 is involved in the transmembrane signaling of GDNF and suggest that integrin beta1 may be an alternative signaling receptor for GDNF.

Gliomas are the most common malignant tumors of the brain and are characteristic of severe migration and invasion. Glial cell line-derived neurotrophic factor (GDNF) promotes glioma development process. However, the regulatory mechanisms of promoting occurrence and development of glioma have not yet been clearly elucidated. In the present study, the mechanism by which GDNF promotes glioma cell migration and invasion through regulating the dispersion and location of the Golgi apparatus (GA) is described. Following GDNF treatment, a change in the volume and position of GA was observed. The stack area of the GA was enlarged and it was more concentrated near the nucleus. Golgin-160 and Golgi microtubule-associated protein 210 (GMAP210) were identified as target molecules regulating GA positioning. In the absence of either golgin-160 or GMAP210 using lentivirus, the migration and invasion of U251 cells were decreased, while it was increased following GDNF. It was also found that the GA was decreased in size and dispersed following golgin-160 or GMAP210 knockdown, as determined by GA green fluorescence assay. Once GDNF was added, the above phenomenon would be twisted, and the concentrated location and volume of the GA was restored. In combination, the present data suggested that the regulation of the position and size of the GA by golgin-160 and GMAP210 play an important role in U251 cell migration and invasion.

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the protection of dopaminergic neurons, but there are few reports of the relationship between GDNF and its precursors (α-pro-GDNF and β-pro-GDNF) and cognitive impairment in Parkinson's disease. This study aimed to investigate the relationship between the serum levels of GDNF and its precursors and cognitive impairment in Parkinson's disease, and to assess their potential as a diagnostic marker. Fifty-three primary outpatients and hospitalized patients with Parkinson's disease (23 men and 30 women) with an average age of 66.58 years were enrolled from the Affiliated Hospital of Xuzhou Medical University of China in this case-control study. The patients were divided into the Parkinson's disease with cognitive impairment group (n = 27) and the Parkinson's disease with normal cognitive function group (n = 26) based on their Mini-Mental State Examination, Montreal Cognitive Assessment, and Clinical Dementia Rating scores. In addition, 26 age- and sex-matched healthy subjects were included as the healthy control group. Results demonstrated that serum GDNF levels were significantly higher in the Parkinson's disease with normal cognitive function group than in the other two groups. There were no significant differences in GDNF precursor levels among the three groups. Correlation analysis revealed that serum GDNF levels, GDNF/α-pro-GDNF ratios, and GDNF/β-pro-GDNF ratios were moderately or highly correlated with the Mini-Mental State Examination, Montreal Cognitive Assessment, and Clinical Dementia Rating scores. To explore the risk factors for cognitive impairment in patients with Parkinson's disease, logistic regression analysis and stepwise linear regression analysis were performed. Both GDNF levels and Hoehn-Yahr stage were risk factors for cognitive impairment in Parkinson's disease, and were the common influencing factors for cognitive scale scores. Neither α-pro-GDNF nor β-pro-GDNF was risk factors for cognitive impairment in Parkinson's disease. A receiver operating characteristic curve of GDNF was generated to predict cognitive function in Parkinson's disease (area under the curve = 0.859). This result indicates that the possibility that serum GDNF can correctly distinguish whether patients with Parkinson's disease have cognitive impairment is 0.859. Together, these results suggest that serum GDNF may be an effective diagnostic marker for cognitive impairment in Parkinson's disease. However, α-pro-GDNF and β-pro-GDNF are not useful for predicting cognitive impairment in this disease. This study was approved by Ethics Committee of the Affiliated Hospital of Xuzhou Medical University, China (approval No. XYFY2017-KL047-01) on November 30, 2017.

The aim of this study was to identify the receptor for glial cell line-derived neurotrophic factor (GDNF) in glioblastoma multiforme (GBM). After GST pull-down assays, membrane proteins purified from C6 rat glioma cells were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The differentially expressed proteins were annotated using Gene Ontology, and neuropilin-1 (NRP1) was identified as the putative GDNF receptor in glioma. NRP1 was more highly expressed in human GBM brains and C6 rat glioma cells than in normal human brains or primary rat astrocytes. Immunofluorescence staining showed that NRP1 was recruited to the membrane by GDNF, and NRP1 co-immunoprecipitated with GDNF. Using the NRP1 and GDNF protein structures to assess molecular docking in the ZDOCK server and visualization with the PyMOL Molecular Graphics System revealed 8 H-bonds and stable positive and negative electrostatic interactions between NRP1 and GDNF. RNAi knockdown of NRP1 reduced proliferation of C6 glioma cells when stimulated with GDNF. NRP1 was an independent risk factor for both survival and recurrence in GBM patients. High NRP1 mRNA expression correlated with shorter OS and DFS (OS: χ2=4.6720, P=0.0307; DFS: χ2=11.013, P=0.0009). NRP1 is thus a GDNF receptor in glioma cells and a potential therapeutic target.

Malignant astrocytoma (MA) is the most common and severe type of brain tumor. A greater understanding of the underlying mechanisms responsible for the development of MA would be beneficial for the development of targeted molecular therapies. In the present study, the upregulated differentially expressed genes (DEGs) in MA were obtained from the Gene Expression Omnibus database using R/Bioconductor software. DEGs in different World Health Organization classifications were compared using the Venny tool and 15 genes, including collagen type I α1 chain (COL1A1) and laminin subunit γ1 (LAMC1), were revealed to be involved in the malignant progression of MA. In addition, the upregulated DEGs in MA were evaluated using functional annotations of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes with the Database for Annotation, Visualization, and Integrated Discovery tool. The results indicated that invasion‑associated enrichment was observed in 'extracellular matrix' (ECM), 'cell adhesion' and 'phosphoinositide 3‑kinase‑protein kinase B signaling pathway'. Subsequently, the analysis of the protein‑protein interactions was performed using STRING and Cytoscape software, which revealed that the ECM component was the invasion‑associated module and its corresponding genes included COL1A1, LAMC1 and fibronectin 1. Finally, survival Kaplan‑Meier estimate was conducted using cBioportal online, which demonstrated that COL1A1 expression affected the survival of and recurrence in patients with MA. Moreover, the results of in vitro Transwell assay and western blot analysis revealed that the depleted levels of COL1A1 also decreased the expression of several proteins associated with cell invasion, including phosphorylated‑signal transducer and activator of transcription 3, matrix metalloproteinase (MMP)‑2, MMP‑9 and nuclear factor‑κB. On the whole, the present study identified the invasion‑related target genes and the associated potential pathways in MA. The results indicated that COL1A1 may be a candidate biomarker for the prognosis and treatment of MA.

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

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.

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor (GDNF) in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin (sh)RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 shRNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region (GDNF-luciferase) were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-shRNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-shRNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain, α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neuroprotective effects on dopamine neurons. However, the expression of the GDNF splice variants in dopaminergic neurons in the brain remains unclear. Therefore, in this study, we investigated the mRNA and protein expression of α- and β-pro-GDNF in the mouse brain by real-time quantitative polymerase chain reaction, using splice variant-specific primers, and western blot analysis. At the mRNA level, β-pro-GDNF expression was significantly greater than that of α-pro-GDNF in the mouse brain. In contrast, at the protein level, α-pro-GDNF expression was markedly greater than that of β-pro-GDNF. To clarify the mechanism underlying this inverse relationship in mRNA and protein expression levels of the GDNF splice variants, we analyzed the expression of sorting protein-related receptor with A-type repeats (SorLA) by real-time quantitative polymerase chain reaction. At the mRNA level, SorLA was positively associated with β-pro-GDNF expression, but not with α-pro-GDNF expression. This suggests that the differential expression of α- and β-pro-GDNF in the mouse brain is related to SorLA expression. As a sorting protein, SorLA could contribute to the inverse relationship among the mRNA and protein levels of the GDNF isoforms. This study was approved by the Animal Ethics Committee of Xuzhou Medical University, China on July 14, 2016.

Previous studies have found that deficiency in nuclear receptor-related factor 1 (Nurr1), which participates in the development, differentiation, survival, and degeneration of dopaminergic neurons, is associated with Parkinson's disease, but the mechanism of action is perplexing. Here, we first ascertained the repercussion of knocking down Nurr1 by performing liquid chromatography coupled with tandem mass spectrometry. We found that 231 genes were highly expressed in dopaminergic neurons with Nurr1 deficiency, 14 of which were linked to the Parkinson's disease pathway based on Kyoto Encyclopedia of Genes and Genomes analysis. To better understand how Nurr1 deficiency autonomously invokes the decline of dopaminergic neurons and elicits Parkinson's disease symptoms, we performed single-nuclei RNA sequencing in a Nurr1 LV-shRNA mouse model. The results revealed cellular heterogeneity in the substantia nigra and a number of activated genes, the preponderance of which encode components of the major histocompatibility II complex. Cd74, H2-Ab1, H2-Aa, H2-Eb1, Lyz2, Mrc1, Slc6a3, Slc47a1, Ms4a4b, and Ptprc2 were the top 10 differentially expressed genes. Immunofluorescence staining showed that, after Nurr1 knockdown, the number of CD74-immunoreactive cells in mouse brain tissue was markedly increased. In addition, Cd74 expression was increased in a mouse model of Parkinson's disease induced by treatment with 6-hydroxydopamine. Taken together, our results suggest that Nurr1 deficiency results in an increase in Cd74 expression, thereby leading to the destruction of dopaminergic neurons. These findings provide a potential therapeutic target for the treatment of Parkinson's disease.

Studies have found that the absence of glial cell line-derived neurotrophic factor may be the primary risk factor for Parkinson's disease. However, there have not been any studies conducted on the potential relationship between glial cell line-derived neurotrophic factor and cognitive performance in Parkinson's disease. We first performed a retrospective case-control study at the Affiliated Hospital of Xuzhou Medical University between September 2018 and January 2020 and found that a decreased serum level of glial cell line-derived neurotrophic factor was a risk factor for cognitive disorders in patients with Parkinson's disease. We then established a mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and analyzed the potential relationships among glial cell line-derived neurotrophic factor in the prefrontal cortex, dopamine transmission, and cognitive function. Our results showed that decreased glial cell line-derived neurotrophic factor in the prefrontal cortex weakened dopamine release and transmission by upregulating the presynaptic membrane expression of the dopamine transporter, which led to the loss and primitivization of dendritic spines of pyramidal neurons and cognitive impairment. In addition, magnetic resonance imaging data showed that the long-term lack of glial cell line-derived neurotrophic factor reduced the connectivity between the prefrontal cortex and other brain regions, and exogenous glial cell line-derived neurotrophic factor significantly improved this connectivity. These findings suggested that decreased glial cell line-derived neurotrophic factor in the prefrontal cortex leads to neuroplastic degeneration at the level of synaptic connections and circuits, which results in cognitive impairment in patients with Parkinson's disease.