ABSTRACT
Parkinson’s disease (PD)is a complex neurodegenerative disorder by motor impairments and non-motor symptoms. While dopamine-based therapies are effective in fighting the symptoms in the early stages of the disease‚ a lack of neuroprotective drugs means that the disease continues to progress. New disease modifying therapies and novel therapeutic strategies are in high demand for PD patients. Genetic studies indicated that both rare and common genetic variants could induce the development PD. As a risk candidate gene for Parkinson’s disease‚ TMEM175 encodes a lysosomal potassium channel protein with new structures‚ and the protein plays an important role in maintaining lysosomal membrane potential and pH stability. With the in-depth understanding for its structure and function‚ TMEM175 deficiency results in decreased lysosomal catalytic activity and the pathological aggregation of α-synuclein. In view of the importance of lysosome potassium channel TMEM175‚ it could be an interesting target for the development of drugs to treat Parkinson’s disease and other neurodegenerative diseases. Herein we review the structure and function TMEM175‚ and focuses on its involvement in the occurrence and development of PD by affecting the function of lysosome as a homeostatic regulator. Future drug screenings based on lysosome TMEM175 may be carried out to maintain the active state or enhance the expression of TMEM175 to improve the condition of PD patients. Further investigations are needed to study how to maintain the balance between the open and closed state of TMEM175 channels to regulate the ion homeostasis of lysosomes. Studies of this ion channel protein will bring new strategies and ideas for the treatment of PD‚ and provide support for establishing the molecular status of TMEM175 in the diagnosis and treatment of PD.
ABSTRACT
Objective To construct prokaryotic expression vector of γ-aminobutyric acid type C receptor ρ2 (GABACR ρ2) gene,induce the expression of the recombination protein GABACR ρ2,and transfect the protein into SH-SY5Y cell line,achieve the transient expression of the recombination protein GABACR ρ2 in SH-SY5Y cell line.Methods The ρ2-Tat gene was inserted into plasmid pET30 to construct the recombinant plasmid pET-ρ2-GFP-Tat.The expression of GABACR ρ2 was detected by Western blot.The histidine-tagged recombination protein ρ2 was purified through immobilized Ni2+ absorption chromatographic column and the purity of GABACR ρ2 protein was detected by sodium dodecyl sulfate polyacrylamide gel elec trophoresis.The transduction and cellular localization of the GABAC R ρ2 was observed by fluorescence microscope.The SH-SY5Y cells were divided into normal group and hypoxia low glucose group.The cells in normal group were divided into normal control group which cultured in high glucose medium and without protein transfection and normal transfection group which cultured in high glucose medium and with protein transfection;the cells in hypoxia low glucose group were divided into treatment group which cultured under hypoxia low glucose condition and treatment transfection group which cultured under hypoxia low glucose and with protein transfection;the viability of SH-SY5Y cells in each group was evaluated by cell counting Kit-8.Results The recombinant plasmid pET30-ρ2-Tat was constructed successfully.The purified recombination protein ρ2-Tat successfully crossed the cytomembrane and transfected the SH-SY5Y cells.The viability of cells in normal control group,normal transfection group,treatment group and treatment transfection group was(100.0 ± 6.9)％,(89.3 ± 3.6)％,(51.4 ± 3.6)％and (66.1 ± 8.5) ％ respectively.There was no statistic difference in the cell viability between the normal control group and normal transfection group(P ＞0.05);the cell viability in treatment group was significantly lower than that in the normal control group (P ＜ 0.01);the cell viability in treatment transfection group was significantly higher than that in the treatment group (P ＜ 0.01).Conclusion The recombination protein ρ2 can through the membrane under the effect of Tat transduction peptide and can successfully establish a SH-SY5Y cell lines which transiently express recombinant protein ρ2,which provide a new research method for the study of ρ2 subunit function.
ABSTRACT
<p><b>OBJECTIVE</b>To construct the acid-sensitive potassium hannel-3(TASK3) eukaryotic expression plasmid and to establish a stable SH-SY5Y cell line expressing enhanced green fluorescent protein (EGFP)-tagged TASK3.</p><p><b>METHODS</b>TASK3 coding region was subcloned into pEGFP-N1 plasmid to construct a recombinant vector alled pEGFP-TASK3. The correct recombinant expressing plasmid was transfected with X-feet transfection reagent to SH-SY5Y cells. The cell line stably expressiing EGFP tagged-TASK3 gene was established by screening with antibiotic G418 and fluorescence microscope. The expression and localization of the EGFP tagged-TASK3 fusion protein was detected by Western blot and confocal microscope. Exposure of the SH-SY5Y cell line expressing stably TASK3-eGFP fusion proteins was exposed to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability was assessed with cell counting Kit-8 (CCK-8).</p><p><b>RESULTS</b>All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pEGFP-TASK3 was constructed correctly. The stable SH-SY5Y cell line expressing EGFP tagged-TASK3 fusion protein was successfully established. Exposure of the wild type SH-SY5Y cells and the stable SH-SY5Y-GFP tag-TASK3 cell line to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability of two group cells significantly reduced with pH declining, and the difference was statistically significant (P < 0.05). Compared with wild type SH-SY5Y cells, the cell viability of stable SH-SYSY-GFP tag-TASK3 cell line increased significantly with the same pH media, and the difference was statistically significant (P < 0.05).</p><p><b>CONCLUSION</b>The eukaryotic expression vector pEGFP-TASK3 is successfully constructed and the cell line stably expressing TASK3-eGFP fusion is established which is important for their fundamental research and potential applications.</p>
Subject(s)
Humans , Blotting, Western , Cell Line , Gene Expression , Genetic Vectors , Green Fluorescent Proteins , Genetics , Plasmids , Polymerase Chain Reaction , Potassium Channels, Tandem Pore Domain , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To observe the neurological protective effects of progesterone (PROG) on focal cerebral ischemia/reperfusion injury in rats and to explore its possible mechanism.</p><p><b>METHODS</b>One handred and twenty male SD rats were divided into three groups randomly: sham-operated group, middle cerebral artery occlusion ( MCAO ) group and PROG + MCAO group( n = 40). The right temporary MCAO model was established by the line-embolism method. The PROG + MCAO group rats were according to 8 mg/kg intraperitoneal injection PROG, after that 30 min, the rats were suffered ischemia/reperfusion. After rats were suffered ischemia for 2 h and reperfusion 0, 24, 48, 72 h stress, the nervous functional defect degree were evaluated by longe scoring, and the expression of two-pore domain K channel 3 (TASK3) mRNA in brain tissue were detected by the real-time PCR.</p><p><b>RESULTS</b>PROG (8 mg/kg) could significantly reduced the nervous functional defect degree in rats after ischemia/reperfusion 24, 48, 72 h (P < 0.05). The results of real-time PCR showed that the TASK3 mRNA expression in the brain tissue at all time points significantly decreased in MCAO group compared with sham-operated group (P < 0.05). However, compared with MCAO group, the expression of TASK3 mRNA in brain tissue at all time points dramatically increased in PROG + MCAO group (P < 0.05).</p><p><b>CONCLUSION</b>PROG can improve the nervous functional defect degree after focal cerebral ischemia/reperfusion injury in rats, and the mechanism might be associated with up-regulating the expression of TASK3 mRNA in brain tissue.</p>