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.

Blunt dopamine transmission due to decreased GDNF in the PFC evokes cognitive impairment in 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.

Association between bisphenol A exposure and body mass index in Chinese school children: a cross-sectional study.

BACKGROUND: There is increasing evidence suggesting that Bisphenol A (BPA), one of the highest volume chemicals produced worldwide, can interfere with the body's natural weight control mechanisms to promote obesity. However, epidemiological studies for this are limited, especially for children. METHODS: A cross-sectional study was conducted to investigate the association between BPA exposure and body mass index (BMI) in school children. Three primary and three middle schools were randomly selected from 26 primary and 30 middle candidate schools in Changning District of Shanghai City in China. According to the BMI-based criteria by age and sex for screening of overweight or obese children, we randomly chose 20 obese, 10 overweight, and 30 normal weight children aged 8-15 years of age from each selected school. First morning urine was collected and total urine BPA concentrations were determined by ultra-performance liquid chromatography tandem mass spectrometry. Multiple linear regression analysis was conducted to examine the association of urine BPA concentrations and daily intake estimates with BMI. RESULTS: BPA was detected in 84.9% of urine samples with a geometric mean of 0.45 ng/mL. The daily intake estimates ranged from 0.03 µg/day to 1.96 µg/day with a geometric mean of 0.37 µg/day. The average urine BPA concentrations and daily intake estimates were similar for boys and girls, but significantly higher in older children than younger ones, and showed an increasing trend with BMI. Multiple linear regression analyses showed that urine BPA concentrations were significantly associated with increasing BMI values in all subjects after adjustment for age and sex and the results were similar before and after corrected by urine specific gravity. When stratified by age or sex, the associations remained significant in females and in those 8-11 years of age before corrected by specific gravity. Similar results were shown for the association between BMI and daily intake estimates. CONCLUSIONS: There is a possibility that BPA exposure increases BMI in school children. Given the cross-sectional nature of this study, longitudinal studies are warranted to confirm BPA exposure as a contributor to increased BMI in children.

Smarcd1 antagonizes the apoptosis of injured MES23.5 DA cells by enhancing the effect of Six2 on GDNF expression.

Glial cell line-derived neurotrophic factor (GDNF) played critical roles in the survival and repair of dopaminergic (DA) neurons. Transcription factor Six2 could repair injured DA cells by promoting the expression of GDNF, however, the underlying molecular mechanisms remain largely unknown. In this study, we screened forty-three proteins that interacted with Six2 in MES23.5 DA cells treated with 6-OHDA by liquid chromatography - electrospray - ionization tandem mass spectrometry (LC-ESI-ITMS/MS). Among these proteins, Smarcd1 is a member of SWI/SNF chromatin-remodeling complex family. Our results confirmed that Smarcd1 formed a transcription complex with Six2, and Smarcd1 mainly binded to the 2840 bp-2933 bp region of the GDNF promoter. Furthermore, knockdown of Smarcd1 inhibited the effect of Six2 on GDNF expression, and resulted in decreased cell viability and increased the apoptosis of injured DA neurons, and the result of overexpression of Smarcd1 is opposite to knockdown. Taken together, our results indicate that smarcd1 can be recruited to the promoter region of GDNF by transcription factor Six2 to promote the effect of Six2 on GDNF expression and protect injured MES23.5 DA cells, which could be useful in identifying potential drug targets for promoting endogenous GDNF expression.

Possible role of glial cell line-derived neurotrophic factor for predicting cognitive impairment in Parkinson's disease: a case-control study.

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 ß-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/ß-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 ß-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 ß-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.

Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by 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.

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50 ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons.

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.

Glucose-6-phosphatase-α participates in dopaminergic differentiation.

OBJECTIVE:  Induction of dopaminergic (DA) differentiation is a cell-based therapy for Parkinson's disease (PD). Here, we explore the key factors of DA differentiation with a focus on glucose-6-phosphatase (G6Pase), a marker enzyme for the endoplasmic reticulum (ER) associated with cell differentiation. METHODS:  We cultured SH-SY5Y human neuroblastoma cells, a model system for PD research, and added glial cell-derived neurotrophic factor (GDNF; 25, 50, or 100 ng/ml) to stimulate differentiation. Subsequently, several methods, such as microRNA/mRNA microarrays, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to detect target genes and proteins respectively. RESULTS:  Light microscopy revealed that 50 ng/ml GDNF most effectively induced DA differentiation. MicroRNA/mRNA microarrays identified that G6PC mRNA was significantly upregulated, which might be influenced by three downregulated microRNAs. Follow-up qRT-PCR results were consistent with the microarray findings, and western blots also supported the results. DISCUSSION: Taken together, our results demonstrate that G6PC, a subunit of G6Pase, participates in DA differentiation. Our findings may contribute to provide a foundation for the research on the mechanism of DA differentiation as well as cell-based therapy for PD.

Neuropilin-1 is a glial cell line-derived neurotrophic factor receptor in glioblastoma.

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.

Inverse Expression Levels of EphrinA3 and EphrinA5 Contribute to Dopaminergic Differentiation of Human SH-SY5Y Cells.

Two key principles underlying successful cellular therapies for Parkinson's disease (PD) are appropriate differentiation of dopaminergic (DA) neurons from transplanted cells and precise axon growth. EphrinAs, a subclass of ephrins, act as axon guidance molecules and are highly expressed in DA brain regions. Existing evidences indicate that they act as either repulsion or attraction signals to guide axon growth. This study investigated whether ephrinAs are involved in DA neuron differentiation. Data from miRCURY™ LNA mRNAs/microRNAs microarrays and quantitative real-time polymerase chain reaction (qRT-PCR) showed upregulated ephrinA3 mRNA (EFNA3) and downregulated ephrinA5 mRNA (EFNA5) during DA neuron differentiation. In addition, hsa-miR-4271 was downregulated, which could influence EFNA3 translation. Furthermore, immunofluorescence (IF) and western blotting confirmed the mRNA results and showed increased ephrinA3 and decreased ephrinA5 protein levels in differentiating DA neurons. Taken together, our results indicate that inverse expression levels of ephrinA3 and ephrinA5, which are possibly influenced by microRNAs, contribute to DA neuron differentiation by guiding axon growth.