Dynein在α-synuclein自噬性清除中的作用
发布时间：2018-08-06 21:07
【摘要】： 第一部分MPP+通过干扰动力蛋白活性阻碍α-synuclein的自噬性降解 目的本研究的目的是探讨是否MPP+通过干扰动力蛋白(dynein)活性导致α-突触核蛋白(α-synuclein)的自噬性清除障碍及其异常聚集。 方法取PC12细胞及我们成功构建的表达含人突变型A30Pα-synuclein的PC12细胞,按实验要求把细胞分为PC12细胞组、MPP+处理PC12细胞组、表达含人突变型A30Pα-synuclein的PC12细胞组和MPP+处理表达含人突变型A30Pα-synuclein的PC12细胞组。在MPP+处理24h后,对细胞采用流式细胞术和MTT法检测细胞活力,半定量RT-PCR检测α-synuclein、LC3基因在mRNA水平表达的变化,Western Blot检测α-synuclein、LC3-Ⅱ、dynein在蛋白水平表达的变化,单丹磺酰戊二胺(monodansylcadaverin,MDC)染色观察自噬囊泡,免疫荧光双标观察自噬水平及α-synuclein、dynein、LC3和LAMP1在胞质内的共定位情况。 结果1)PC12细胞和表达含人突变型A30Pα-synuclein的PC12细胞经MPP+处理后细胞活力明显下降(与正常组比较分别为P0.001和P=0.002),同时,细胞凋亡率明显增加,尤以MPP+处理表达含人突变型A30Pα-synuclein的PC12细胞组最显著;2)半定量RT-PCR的结果显示:各组细胞α-synuclein和LC3 mRNA基因表达水平较MPP+处理前均明显上升;3)Western Blot显示:MPP+处理后PC12细胞和表达含人突变型A30Pα-synuclein的PC12细胞dynein的蛋白表达水平均较处理前明显下降(t=2.8414,P=0.0234;t=2.2671,P=0.043),而α-synuclein低聚体表达增加,LC3-Ⅱ水平升高(P均0.05);4)MDC染色显示:MPP+处理后,PC12细胞和表达含人突变型A30Pα-synuclein的PC12细胞的自噬泡数目明显增加;5)免疫荧光结果表明:MPP+处理后,LC3阳性的自噬体数目增加,并且在胞内可见较大的自噬体,与α-synuclein的共定位也增加;α-synuclein明显聚集,并散布于胞质内,而dynein失去负端运动趋势,聚集于胞质外周,并且与α-synuclein失去共定位,同时LAMP1标记的溶酶体也聚集于胞质外周,与LC3标记的自噬体及dynein失去共定位。 结论MPP+处理后,虽然自噬体的数目增加,但MPP+明显影响了dynein的负端运动趋势,使其聚集于胞质外周,阻碍了其运输功能,从而导致自噬体与溶酶体融合程度的下降,致使α-synuclein聚集体的自噬性清除障碍,因此,动力蛋白活性在PD的发病机制中起重要作用,是治疗帕金森病的一个重要潜在靶点。 第二部分动力蛋白活性在α-synuclein的自噬性降解中的作用 目的通过给予PC12细胞ATP和EHNA影响动力蛋白活性,以及动力蛋白过表达来研究其在α-突触核蛋白(α-synuclein)自噬性清除中的作用。 方法MTT法检测不同浓度EHNA、ATP处理对PC12细胞活力的影响;Western Blot检测正常PC12细胞、EHNA处理、MPP+处理、MPP+处理后再予ATP处理后细胞的LC3-Ⅱ、α-synuclein、dynein表达变化;激光共聚焦显微镜下观察PC12细胞在不同药物处理前后的α-synuclein、LC3和动力蛋白在细胞内的共定位;透射电镜观察各株PC12细胞处理24小时后的细胞超微结构改变。构建pDsRED2-N1-DYNEIN重组质粒并转染细胞。 结果1)PC12细胞加入EHNA或MPP+后,LC3-II水平均明显增加(Q值分别为18.7787和25.0773,P均0.01)及Dynein表达的下降(Q值分别为17.7923和17.8722,P均0.01),而加入ATP后可明显降低LC3-II水平、升高了dynein水平(与EHNA和MPP+处理组比较P均0.01)。另外EHNA、MPP+处理均导致α-synuclein表达的升高,加入ATP后可明显降低了α-synuclein的水平。2)激光共聚集显微镜显示结果和WesternBlot结果基本一致,MPP+、EHNA处理后均导致α-synuclein和LC3累积光密度的升高、dynein光密度的下降,加入ATP后可明显升高了dynein累积光密度从而降低了由MPP+引起的α-synuclein和LC3累积光密度的升高。3)电镜结果表明:与未经药物处理对照组相比,PC12细胞MPP+、EHNA处理后,细胞核膜皱折,核染色质散聚、边集,见双层膜的自噬囊泡形成,部分细胞有凋亡倾向;而ATP处理24h后PC12细胞,细胞膜、核膜完整,染色质结构正常,胞质中线粒体形态分布及数目正常,并可见自噬溶酶体形成,促进了自噬溶酶体的融合。4)动力蛋白过表达后α-synuclein蛋白水平下降。 结论MPP+和EHNA抑制了动力蛋白的活性,阻碍了其运输功能,从而导致自噬体与溶酶体融合程度的下降,导致了自噬应激,致使α-synuclein的自噬性清除障碍,而加入ATP后可逆转这一病理变化,通过提高动力蛋白的活性,增加了α-synuclein的自噬性清除,动力蛋白过表达有助于α-synuclein的清除。因此,动力蛋白的活性可能关系到细胞内聚集体有关的疾病(如PD),从提高动力蛋白活性的角度来治疗帕金森病是一个重要潜在靶点。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
本文编号：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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 钱进军;程言博;刘春风;杨亚萍;刘康永;;人SNCA基因真核表达载体的构建及其在PC12细胞中的表达[J];细胞与分子免疫学杂志;2008年01期
2 ;Molecular mechanism and regulation of autophagy[J];Acta Pharmacologica Sinica;2005年12期
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