Dynein在α-synuclein自噬性清除中的作用
發(fā)布時間：2018-08-06 21:07
【摘要】： 第一部分MPP+通過干擾動力蛋白活性阻礙α-synuclein的自噬性降解 目的本研究的目的是探討是否MPP+通過干擾動力蛋白(dynein)活性導致α-突觸核蛋白(α-synuclein)的自噬性清除障礙及其異常聚集。 方法取PC12細胞及我們成功構建的表達含人突變型A30Pα-synuclein的PC12細胞,按實驗要求把細胞分為PC12細胞組、MPP+處理PC12細胞組、表達含人突變型A30Pα-synuclein的PC12細胞組和MPP+處理表達含人突變型A30Pα-synuclein的PC12細胞組。在MPP+處理24h后,對細胞采用流式細胞術和MTT法檢測細胞活力,半定量RT-PCR檢測α-synuclein、LC3基因在mRNA水平表達的變化,Western Blot檢測α-synuclein、LC3-Ⅱ、dynein在蛋白水平表達的變化,單丹磺酰戊二胺(monodansylcadaverin,MDC)染色觀察自噬囊泡,免疫熒光雙標觀察自噬水平及α-synuclein、dynein、LC3和LAMP1在胞質(zhì)內(nèi)的共定位情況。 結果1)PC12細胞和表達含人突變型A30Pα-synuclein的PC12細胞經(jīng)MPP+處理后細胞活力明顯下降(與正常組比較分別為P0.001和P=0.002),同時,細胞凋亡率明顯增加,尤以MPP+處理表達含人突變型A30Pα-synuclein的PC12細胞組最顯著;2)半定量RT-PCR的結果顯示:各組細胞α-synuclein和LC3 mRNA基因表達水平較MPP+處理前均明顯上升;3)Western Blot顯示:MPP+處理后PC12細胞和表達含人突變型A30Pα-synuclein的PC12細胞dynein的蛋白表達水平均較處理前明顯下降(t=2.8414,P=0.0234;t=2.2671,P=0.043),而α-synuclein低聚體表達增加,LC3-Ⅱ水平升高(P均0.05);4)MDC染色顯示:MPP+處理后,PC12細胞和表達含人突變型A30Pα-synuclein的PC12細胞的自噬泡數(shù)目明顯增加;5)免疫熒光結果表明:MPP+處理后,LC3陽性的自噬體數(shù)目增加,并且在胞內(nèi)可見較大的自噬體,與α-synuclein的共定位也增加;α-synuclein明顯聚集,并散布于胞質(zhì)內(nèi),而dynein失去負端運動趨勢,聚集于胞質(zhì)外周,并且與α-synuclein失去共定位,同時LAMP1標記的溶酶體也聚集于胞質(zhì)外周,與LC3標記的自噬體及dynein失去共定位。 結論MPP+處理后,雖然自噬體的數(shù)目增加,但MPP+明顯影響了dynein的負端運動趨勢,使其聚集于胞質(zhì)外周,阻礙了其運輸功能,從而導致自噬體與溶酶體融合程度的下降,致使α-synuclein聚集體的自噬性清除障礙,因此,動力蛋白活性在PD的發(fā)病機制中起重要作用,是治療帕金森病的一個重要潛在靶點。 第二部分動力蛋白活性在α-synuclein的自噬性降解中的作用 目的通過給予PC12細胞ATP和EHNA影響動力蛋白活性,以及動力蛋白過表達來研究其在α-突觸核蛋白(α-synuclein)自噬性清除中的作用。 方法MTT法檢測不同濃度EHNA、ATP處理對PC12細胞活力的影響;Western Blot檢測正常PC12細胞、EHNA處理、MPP+處理、MPP+處理后再予ATP處理后細胞的LC3-Ⅱ、α-synuclein、dynein表達變化;激光共聚焦顯微鏡下觀察PC12細胞在不同藥物處理前后的α-synuclein、LC3和動力蛋白在細胞內(nèi)的共定位;透射電鏡觀察各株PC12細胞處理24小時后的細胞超微結構改變。構建pDsRED2-N1-DYNEIN重組質(zhì)粒并轉染細胞。 結果1)PC12細胞加入EHNA或MPP+后,LC3-II水平均明顯增加(Q值分別為18.7787和25.0773,P均0.01)及Dynein表達的下降(Q值分別為17.7923和17.8722,P均0.01),而加入ATP后可明顯降低LC3-II水平、升高了dynein水平(與EHNA和MPP+處理組比較P均0.01)。另外EHNA、MPP+處理均導致α-synuclein表達的升高,加入ATP后可明顯降低了α-synuclein的水平。2)激光共聚集顯微鏡顯示結果和WesternBlot結果基本一致,MPP+、EHNA處理后均導致α-synuclein和LC3累積光密度的升高、dynein光密度的下降,加入ATP后可明顯升高了dynein累積光密度從而降低了由MPP+引起的α-synuclein和LC3累積光密度的升高。3)電鏡結果表明:與未經(jīng)藥物處理對照組相比,PC12細胞MPP+、EHNA處理后,細胞核膜皺折,核染色質(zhì)散聚、邊集,見雙層膜的自噬囊泡形成,部分細胞有凋亡傾向;而ATP處理24h后PC12細胞,細胞膜、核膜完整,染色質(zhì)結構正常,胞質(zhì)中線粒體形態(tài)分布及數(shù)目正常,并可見自噬溶酶體形成,促進了自噬溶酶體的融合。4)動力蛋白過表達后α-synuclein蛋白水平下降。 結論MPP+和EHNA抑制了動力蛋白的活性,阻礙了其運輸功能,從而導致自噬體與溶酶體融合程度的下降,導致了自噬應激,致使α-synuclein的自噬性清除障礙,而加入ATP后可逆轉這一病理變化,通過提高動力蛋白的活性,增加了α-synuclein的自噬性清除,動力蛋白過表達有助于α-synuclein的清除。因此,動力蛋白的活性可能關系到細胞內(nèi)聚集體有關的疾病(如PD),從提高動力蛋白活性的角度來治療帕金森病是一個重要潛在靶點。
[Abstract]:Part one MPP+ interferes with the autophagic degradation of alpha -synuclein by interfering with the activity of dynein.
The purpose of this study was to investigate whether MPP+ can induce autophagic scavenging disorders and abnormal aggregation of alpha synuclein (alpha -synuclein) by interfering with dynein activity.
Methods the PC12 cells and the PC12 cells expressing human mutant A30P alpha -synuclein were successfully constructed, and the cells were divided into PC12 cell groups according to the experimental requirements. MPP+ was used to treat PC12 cell groups. The PC12 cell group containing human mutant A30P alpha -synuclein and MPP+ treatment of the human mutant A30P alpha cells were treated. Then, cell viability was detected by flow cytometry and MTT method. Semi quantitative RT-PCR was used to detect the changes in the expression of alpha -synuclein, LC3 gene at mRNA level. Western Blot was used to detect the changes of alpha -synuclein, LC3- II, dynein in protein level. Autophagic vesicles were observed by staining of single sulfonylglutamyl two amine (monodansylcadaverin, MDC), and double immunofluorescent labeling was observed. The autophagy level and the co localization of -synuclein, dynein, LC3 and LAMP1 in the cytoplasm were observed.
Results 1) the cell viability of PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein decreased significantly after MPP+ treatment (P0.001 and P=0.002 respectively compared with the normal group), while the apoptosis rate increased obviously, especially the PC12 cell group expressing human mutant A30P alpha -synuclein in MPP+ treatment; 2) the result of semi quantitative RT-PCR. The expression level of alpha -synuclein and LC3 mRNA genes in each group was significantly higher than that before MPP+, and 3) Western Blot showed that the protein expression level of PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein after MPP+ treatment was significantly lower than that before the treatment. The expression of polymer increased, the level of LC3- II increased (P 0.05); 4) MDC staining showed that the number of autophagic vacuoles in PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein increased significantly after MPP+ treatment; 5) the immunofluorescence results showed that the number of LC3 positive autophagosomes increased after MPP+ treatment, and the larger autophagosomes were visible in the cell, and alpha -syn. The co localization of uclein also increased; alpha -synuclein obviously aggregated and scattered in the cytoplasm, and dynein lost the negative end movement trend, aggregated in the cytoplasm and lost co location with the alpha -synuclein, while the lysosomes of the LAMP1 labeled lysosomes were also clustered in the cytoplasm, and lost co localization with the LC3 labeled autophagosomes and dynein.
Conclusion after MPP+ treatment, the number of autophagosomes increased, but MPP+ significantly affected the negative end movement of dynein, which aggregated in the cytoplasm of the cytoplasm, hindered its transport function, resulting in the decrease of the fusion degree of the autophagosomes and lysosomes, resulting in the autophagic elimination of the alpha -synuclein aggregates, thus the activity of the kinetic protein was in the hair of the PD. It plays an important role in the pathogenesis and is an important potential target for the treatment of Parkinson's disease.
The second part is the role of dynein activity in the autophagic degradation of alpha -synuclein.
Objective to study the role of PC12 cells ATP and EHNA in the autophagic clearance of alpha synuclein (alpha -synuclein) by affecting the activity of the protein and the overexpression of the protein.
Methods MTT was used to detect the effects of different concentrations of EHNA and ATP on the activity of PC12 cells. Western Blot was used to detect normal PC12 cells, EHNA treatment, MPP+ treatment, MPP+ processing and ATP treated cells. The co localization of the PC12 cells was observed in the cells, and the ultrastructural changes of the cells after 24 hours treatment were observed by transmission electron microscopy. The recombinant plasmid of pDsRED2-N1-DYNEIN was constructed and transfected into cells.
Results 1) after PC12 cells were added to EHNA or MPP+, the level of LC3-II increased significantly (Q value was 18.7787 and 25.0773, P 0.01) and Dynein expression decreased (Q value was 17.7923 and 17.8722, P 0.01 respectively), and LC3-II level was significantly reduced after ATP, and dynein water level was increased (0.01). The increase of the expression of alpha -synuclein, after adding ATP, can obviously reduce the level of the level of alpha -synuclein.2). The results of the laser conclustered microscope show the same results as WesternBlot. MPP+, EHNA treatment all lead to the increase of the cumulative light density of alpha -synuclein and LC3, the decrease of dynein optical density, and the dynein accumulation can obviously increase the dynein accumulation after adding to ATP. The light density thus reduced the increase of the cumulative light density of alpha -synuclein and LC3 caused by MPP+.3. The results of electron microscopy showed that, compared with the control group without drug treatment, PC12 cells MPP+, EHNA treatment, cell nuclear membrane fold, nuclear chromatin dispersion, edge set, the formation of autophagic vesicles in the double layer membrane, and the tendency of apoptosis in some cells; ATP treated 24h after 24h PC12. Cell, cell membrane, nuclear membrane are complete, chromatin structure is normal, mitochondria form distribution and number in cytoplasm are normal, and autophagosomes are formed, and autophagosome fusion.4 is promoted. The level of alpha -synuclein protein decreases after the expression of the autophagy lysosome.
Conclusion MPP+ and EHNA inhibit the activity of the protein, which hinders its transport function, resulting in the decrease of the fusion degree of the autophagosome and lysosome, resulting in autophagic stress and the autophagic removal of the alpha -synuclein, which can reverse the pathological changes after the addition of ATP, and increase the autophagy activity and increase the autophagy of the alpha -synuclein. The elimination of phagocytosis and the overexpression of the proteins contribute to the clearance of alpha -synuclein. Therefore, the activity of the protein may be related to the disease of cell aggregation (such as PD), which is an important potential target for the treatment of Parkinson's disease from the point of increasing the activity of the protein.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329
本文編號：2168991
[Abstract]:Part one MPP+ interferes with the autophagic degradation of alpha -synuclein by interfering with the activity of dynein.
The purpose of this study was to investigate whether MPP+ can induce autophagic scavenging disorders and abnormal aggregation of alpha synuclein (alpha -synuclein) by interfering with dynein activity.
Methods the PC12 cells and the PC12 cells expressing human mutant A30P alpha -synuclein were successfully constructed, and the cells were divided into PC12 cell groups according to the experimental requirements. MPP+ was used to treat PC12 cell groups. The PC12 cell group containing human mutant A30P alpha -synuclein and MPP+ treatment of the human mutant A30P alpha cells were treated. Then, cell viability was detected by flow cytometry and MTT method. Semi quantitative RT-PCR was used to detect the changes in the expression of alpha -synuclein, LC3 gene at mRNA level. Western Blot was used to detect the changes of alpha -synuclein, LC3- II, dynein in protein level. Autophagic vesicles were observed by staining of single sulfonylglutamyl two amine (monodansylcadaverin, MDC), and double immunofluorescent labeling was observed. The autophagy level and the co localization of -synuclein, dynein, LC3 and LAMP1 in the cytoplasm were observed.
Results 1) the cell viability of PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein decreased significantly after MPP+ treatment (P0.001 and P=0.002 respectively compared with the normal group), while the apoptosis rate increased obviously, especially the PC12 cell group expressing human mutant A30P alpha -synuclein in MPP+ treatment; 2) the result of semi quantitative RT-PCR. The expression level of alpha -synuclein and LC3 mRNA genes in each group was significantly higher than that before MPP+, and 3) Western Blot showed that the protein expression level of PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein after MPP+ treatment was significantly lower than that before the treatment. The expression of polymer increased, the level of LC3- II increased (P 0.05); 4) MDC staining showed that the number of autophagic vacuoles in PC12 cells and PC12 cells expressing human mutant A30P alpha -synuclein increased significantly after MPP+ treatment; 5) the immunofluorescence results showed that the number of LC3 positive autophagosomes increased after MPP+ treatment, and the larger autophagosomes were visible in the cell, and alpha -syn. The co localization of uclein also increased; alpha -synuclein obviously aggregated and scattered in the cytoplasm, and dynein lost the negative end movement trend, aggregated in the cytoplasm and lost co location with the alpha -synuclein, while the lysosomes of the LAMP1 labeled lysosomes were also clustered in the cytoplasm, and lost co localization with the LC3 labeled autophagosomes and dynein.
Conclusion after MPP+ treatment, the number of autophagosomes increased, but MPP+ significantly affected the negative end movement of dynein, which aggregated in the cytoplasm of the cytoplasm, hindered its transport function, resulting in the decrease of the fusion degree of the autophagosomes and lysosomes, resulting in the autophagic elimination of the alpha -synuclein aggregates, thus the activity of the kinetic protein was in the hair of the PD. It plays an important role in the pathogenesis and is an important potential target for the treatment of Parkinson's disease.
The second part is the role of dynein activity in the autophagic degradation of alpha -synuclein.
Objective to study the role of PC12 cells ATP and EHNA in the autophagic clearance of alpha synuclein (alpha -synuclein) by affecting the activity of the protein and the overexpression of the protein.
Methods MTT was used to detect the effects of different concentrations of EHNA and ATP on the activity of PC12 cells. Western Blot was used to detect normal PC12 cells, EHNA treatment, MPP+ treatment, MPP+ processing and ATP treated cells. The co localization of the PC12 cells was observed in the cells, and the ultrastructural changes of the cells after 24 hours treatment were observed by transmission electron microscopy. The recombinant plasmid of pDsRED2-N1-DYNEIN was constructed and transfected into cells.
Results 1) after PC12 cells were added to EHNA or MPP+, the level of LC3-II increased significantly (Q value was 18.7787 and 25.0773, P 0.01) and Dynein expression decreased (Q value was 17.7923 and 17.8722, P 0.01 respectively), and LC3-II level was significantly reduced after ATP, and dynein water level was increased (0.01). The increase of the expression of alpha -synuclein, after adding ATP, can obviously reduce the level of the level of alpha -synuclein.2). The results of the laser conclustered microscope show the same results as WesternBlot. MPP+, EHNA treatment all lead to the increase of the cumulative light density of alpha -synuclein and LC3, the decrease of dynein optical density, and the dynein accumulation can obviously increase the dynein accumulation after adding to ATP. The light density thus reduced the increase of the cumulative light density of alpha -synuclein and LC3 caused by MPP+.3. The results of electron microscopy showed that, compared with the control group without drug treatment, PC12 cells MPP+, EHNA treatment, cell nuclear membrane fold, nuclear chromatin dispersion, edge set, the formation of autophagic vesicles in the double layer membrane, and the tendency of apoptosis in some cells; ATP treated 24h after 24h PC12. Cell, cell membrane, nuclear membrane are complete, chromatin structure is normal, mitochondria form distribution and number in cytoplasm are normal, and autophagosomes are formed, and autophagosome fusion.4 is promoted. The level of alpha -synuclein protein decreases after the expression of the autophagy lysosome.
Conclusion MPP+ and EHNA inhibit the activity of the protein, which hinders its transport function, resulting in the decrease of the fusion degree of the autophagosome and lysosome, resulting in autophagic stress and the autophagic removal of the alpha -synuclein, which can reverse the pathological changes after the addition of ATP, and increase the autophagy activity and increase the autophagy of the alpha -synuclein. The elimination of phagocytosis and the overexpression of the proteins contribute to the clearance of alpha -synuclein. Therefore, the activity of the protein may be related to the disease of cell aggregation (such as PD), which is an important potential target for the treatment of Parkinson's disease from the point of increasing the activity of the protein.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329
【參考文獻】
相關期刊論文 前2條
1 錢進軍;程言博;劉春風;楊亞萍;劉康永;;人SNCA基因真核表達載體的構建及其在PC12細胞中的表達[J];細胞與分子免疫學雜志;2008年01期
2 ;Molecular mechanism and regulation of autophagy[J];Acta Pharmacologica Sinica;2005年12期
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