Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe  

作　　者：Timmy Le Cynthia L.Winham Fotis Andromidas Adam C.Silver Evan R.Jellison Aime A.Levesque Andrew O.Koob 

机构地区：[1]Graduate Program in Neuroscience,Biology Department,University of Hartford,West Hartford,CT,USA [2]Department of Immunology,UCONN Health Center,Farmington,CT,USA

出　　处：《Neural Regeneration Research》2020年第10期1894-1902,共9页中国神经再生研究（英文版）

基　　金：supported by grants from the Connecticut Partnership in Innovation and Education(PIE)Fellowship(to TL);the University of Hartford College of Arts and Sciences Dean’s Fund(to TL,FA,AOK)

摘　　要：Elevated levels of γ-synuclein(γ-syn)expression have been noted in the progression of glioblastomas,and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases.γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes.Internalizedγ-syn results in increased cellular proliferation,brain derived neurotrophic factor release and astroprotection.However,the function of endogenousγ-syn in primary astrocytes,and the relationship to these two opposing disease states are unknown.γ-Syn is expressed by astrocytes in the human cortex,and to gain a better understanding of the role of endogenous γ-syn,primary human cortical astrocytes were treated with chimera RNA interference(RNAi)targeting γ-syn after release from cell synchronization.Quantitative polymerase chain reaction analysis demonstrated an increase in endogenousγ-syn expression 48 hours after release from cell synchronization,while RNAi reduced γ-syn expression to control levels.Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi γ-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization.To further investigate the consequence of γ-syn knockdown on the astrocytic cell cycle,phosphorylated histone H3 pSer10(pHH3)and phosphorylated cyclin dependent kinase-2 pTyr15(pCDK2)levels were observed via western blot analysis.The results revealed an elevated expression of pHH3,but not pCDK2,indicating γ-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours.Subsequently,flow cytometry with propidium iodide determined that increases in apoptosis coincided with γ-syn knockdown.Therefore,γ-syn exerts its effect to allow normal astrocytic progression through the cell cycle,as evidenced by decreased proliferation marker expression,increased pHH3,and mitotic catastrophe after knockdown.In this study,we demonstrated that the knockdown of γ-syn within primary human cortical astrocElevated levels of γ-synuclein(γ-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized γ-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous γ-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. γ-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous γ-syn, primary human cortical astrocytes were treated with chimera RNA interference(RNAi) targeting γ-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous γ-syn expression 48 hours after release from cell synchronization, while RNAi reduced γ-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi γ-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of γ-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10(p HH3) and phosphorylated cyclin dependent kinase-2 pTyr15(pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating γ-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with γ-syn knockdown. Therefore, γ-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of γ-syn w

关 键 词：apoptosis ASTROCYTE cell cycle GLIOBLASTOMAS phospho-histone H3 proliferation SYNUCLEIN 

分 类 号：R459.9[医药卫生—治疗学] R364[医药卫生—临床医学]