ABSTRACT
Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.
Subject(s)
Animals , Mice , DNA-Binding Proteins/genetics , Cell Differentiation , Neural Stem Cells , Translocation, Genetic , Ubiquitins/genetics , Ligases/geneticsABSTRACT
Objective To investigate the influence of vitamin B supplementation on the plasma total homocysteine (Hcy), serum folate, serum vitamin B12, serum vitamin B6, and clinical state of senile epilepsy.Methods A total of 132 senile epilepsy patients with hyperhomocysteinemia, who visited the Second Affiliated Hospital of Soochow University from January 2013 to July 2015, were enrolled into this study.Eighty-three patients who accepted the therapy of vitamin B supplementation (folate 2.5 mg/d, vitamin B6 10.0 mg/d, vitamin B12 1.5 mg/d) were selected as treatment group, and 49 patients with no vitamin B supplementation were selected as control group.All patients were followed-up for one year.The differences of serum Hcy, folate, vitamin B12, vitamin B6 and seizure frequency, MMSE scores, Hamilton Depression Rating Scale (HDMA) scores between the treatment and the control groups were compared.According to the concentration of serum Hcy, 132 patients were divided into two groups: 75 patients (46 patients in the treatment group and 29 patients in the control group) with mild hyperhomocysteinemia (Hcy: 15.0-29.9 μmol/L), 57 patients (37 patients in the treatment group and 20 patients in the control group) with moderate-severe hyperhomocysteinemia (Hcy≥30.0 μmol/L).The differences of serum Hcy, folate, vitamin B12, vitamin B6 and seizure frequency, MMSE scores, HDMA scores between the treatment and the control group in the mild hyperhomocysteinemia group and in the moderate-severe hyperhomocysteinemia group were compared.And the influence factors of Hcy were analyzed.Results Correlation analysis revealed that Hcy was positively correlated with age (r=0.269, P=0.002), and negatively correlated with folate (r=-0.222, P=0.010).Hcy was associated with smoking (χ2=7.363,P=0.007), hypertension (χ2=6.187,P=0.013), and two or more antiepileptic drugs polytherapy (χ2=4.708,P=0.030).After vitamin B supplementation in the group of moderate-severe hyperhomocysteinemia, serum Hcy concentration ((15.68±4.85) μmol/L in the treatment group vs (31.14±11.18) μmol/L in the control group, t=5.890, P0.05).Conclusions It is necessary to monitor serum Hcy for senile epilepsy.Vitamin B supplementation is a safe and inexpensive way to reduce the concentration of Hcy, assist to control seizures and improve the clinical symptom of depression and cognitive impairment for senile epilepsy with moderate-severe hyperhomocysteinemia.
ABSTRACT
Objective To investigate the location of receptor interacting protein 3( RIP3) in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 ( RIP1 ) and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days,then pre-treated with zVAD-fmk(20μ,mol/L) for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time ,then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research,the neurons were treated with Nec-1 and shRlPl ,then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and a-chieved the peak in 12 hours of TNF treatment ( Cytoplasmic 0. 45 ± 0. 03 ,0. 41 ± 0. 02,0. 73 ± 0. 03 ,0. 90 ± 0.01,1.15 ±0.04,1.30 ±0.02,0.99 ±0.03,0.63 ±0. 03;Nucleus 0. 07 ±0.02,0. 26 ±0.02,0. 57 ±0. 02,0. 68 ± 0.02,0. 80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0. 87 ± 0.02) (P < 0.01). ②Blocking the relationship between RIP1 and RIP3 with necrostatin-1 and shRIPl , nuclear translocation of RIP3 decreased and caused a great increase in cell viability( 1.00 ±0.05,0.39 ±0.03,0.50 ±0. 03) (P<0. 01). Conclusion RIP3 mainly locates in cy-tolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.
ABSTRACT
Objective To investigated the role of the autophagy lysosomal pathway in PD cells and the possible molecular mechanisms. Methods A dopaminergic neuronal injury model was induced by 6-OHDA in PC12 cells . Autophagosomes in PC12 cells were examined by transmission electronmicro-scopy( TEM ). The expression of LC3- Ⅱ , Cathepsin B were assayed by western blot analysis. Results TEM revealed that the autophagosomes were increased in PC12 cells after 6-OHDA treatment and appeared apoptosis. The LC3-Ⅱ (2h:52.57 ±2.27,4h:56.83 ±3.51,6h:73.43 ±5.41,12h:103.90 ±2.57,24h: 100.40 ±3.91 )and Cathepsin B expression ( model group: 113.80 ± 4.46; normal group 35.89 ± 3.40) were increased after 6-OH DA treatments (P ＜ 0.05 or P ＜ 0.01 ). Conclusion The results indicate that autophagy lysosome pathway is involved in 6-OHDA-induced cell death in PC12 cells.