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Title: Slit2排斥神经细胞迁移的胞内机制研究
Author: 徐华泰
Degree Level: 博士
Issued Date: 2004
Degree Grantor: 中国科学院神经科学研究所
Place of Degree Grantor: 中国科学院神经科学研究所
Supervisor: 冯林音
Abstract: 神经系统发育过程中,大量有导向作用的胞外因子能够引导未成熟的神经元,从它们的出生地到达它们要发挥功能的所在地,但它们对神经元迁移导向的胞内信号机制却不明朗。通过观察来自小脑的颗粒细胞沿着共培养的Bergmann胶质细胞的长纤维迁移,我们发现在体内可以排斥神经元迁移的Slit2的稳定梯度不影响颗粒细胞的迁移速度,但可以排斥颗粒细胞迁移。对迁移的神经元进行钙成像分析,观察到在迁移细胞胞体中有一个稳定的前低后高的内钙梯度。另外,Slit2梯度可以通过细胞钙库上的IP3受体引起胞内钙离子浓度的升高,进而逆转了先前存在的前低后高的内钙不对称分布,变成前高后低的内钙分布。这个内钙不对称分布的翻转与细胞迁移方向的改变密切相关。我们进一步发现,那些能够引起内钙升高的其它物质,如Rynodine及乙酞胆碱也能够排斥神经元迁移。基于这些结果,我们认为迁移细胞中的不对称分布内钙对神经元迁移的方向既是充分又是必须的。我们的结果表明钙信号在介导Slit2排斥神经元迁移中有重要作用,内钙的极性分布决定迁移方向也可能是迁移细胞的普遍机制。虽然大量的工作表明钙信号参与了神经元迁移,但内钙的下游机制并不清楚。通过对单独的颗粒细胞培养的观察,我们发现在没有Begmann胶质细胞时斗itZ同样也可以排斥颗粒细胞迁移。在感受到Slit2梯度后·颗粒细胞的生长锥首先塌缩,尔后缩回导向突起,接着在细胞的后端以前是尾随突起的地方长出一个新的生长锥和导向突起。利用这个颗粒细胞单独培养的系统,我们发现了微丝及钙的相关蛋白-Cofilin与颗粒细胞迁移有关,并且在细胞解读SlitZ信号的排斥作用时有重要作用。Cofilin的活性主要由它的第三位丝氨酸磷酸化与否决定,非磷酸化是它的活性形式。在外源转入了野生型及模拟部分磷酸化的S3E突变型Cofilin后,神经元的迁移速度比仅转入绿色荧光蛋白的神经元的速度要慢,但仍保留了能被Slit2排斥的性质,而那些转入了模拟去磷酸化的S3A Cofilin的神经元,迁移速度比对照组要快,而且丧失了对Slit2的排斥反应。进一步我们观察到Slit2可以降低迁移细胞的生长锥中的Cofilin的磷酸化。另外,Calcineurin抑制剂会阻断Slit2引起的排斥反应,而Calcineurin介导了由于高浓度钙离子引起的pCofilin的去磷酸化。细胞免疫组化的结果也证实了在迁移细胞中colcineur如与Cofil加有共同定位。这些结果表明由于高浓度的钙离子及钙激活的磷酸酶Calcineurin引起的pCofilin的去磷酸化在传导Slit2对迁移细胞的排斥作用时起着重要作用。
English Abstract: Migration of neuronal precursor cells in the developing brain is guided by extracellular cues, but intracellular signaling processes underlying the guidance of neuronal migration are largely unknown. By examining the migration of cerebellar granule neurons along the surface of cocultured astroglial cells, we found that an extracellular gradient of Slit2, a chemorepellant for neuronal migration in vivo, caused a reversal in the direction of migration without affecting the migration speed. Through loading calcium dye into the migrating neurons, we found that there is a stable front-low-rear-high calcium gradient in migrating neurons and Slit2 gradient elevated the intracellular concentration of Ca2+, probably due to calcium release from the internal store through IP3R, led to a reversal of the preexisting asymmetric intracellular Ca2+ distribution in the soma of migrating neurons, and this reversal was closely related with its action of reversing the migrating direction. Stimulated by other substance that can elevate intracellular calcium, such as ryanodine and acetylcholine, also repel neuronal migration. Thus we proposed that asymmetric Ca2+ distribution in the soma was both necessary and sufficient for directing neuronal migration. These results have demonstrated an important role for Ca2t in mediating neuronal responses to Slit2 and suggest a general mechanism for neuronal guidance. There is evidence that cytosolic Ca2+ signaling is involved in regulating neuronal migration, but the underlying mechanism remains unclear. In the assay of isolated granule cell culture, we found that Slit2 still repels neuronal migration. Stimulated by Slit2, migrating neurons would retract their leading process and form a new leading process at the point of previous trailing process. In this assay, we investigated the role of Cofilin, a Ca'2+ effector protein, in regulating the migration of single cerebellar granule cells in culture and in the repulsive action of Slit2 on these neurons. The activity of Cofilin is modulated mainly by phosphorylation on Ser-3. Neurons transfected with wild type or a mutant form of Cofilin (S3E) that partially simulates the phosphorylated state of the Cofilin, migrated slower than the control neurons and retained the repulsive response to Slit2, while those transfected with a mutated form (S3A) that simulates the dephosphorylated Cofilin migrated faster than control neurons and lose the Slit2-induced repulsive response. Furthermore, we found that Cofilin phosphorylation in the growth cone was decreased by Slit2, and Calcineurin inhibitors, which block the dephosphorylation of Cofilin induced by high concentrations of Ca2+, abolished Slit2-induced repulsion. Immunostaining further confimed the co-localization of Calcineurin and Cofilin in migratory neurons. Taken together, these results suggest that dephos-phorylation of Cofilin by Calcineurin, which is activated by elevated Ca2+ may be a pivotal step in the repulsive action of S1112 on neuronal migration.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2250
Appears in Collections:神经所(总)_学位论文

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Recommended Citation:
Slit2排斥神经细胞迁移的胞内机制研究.徐华泰[d].中国科学院神经科学研究所,2004.20-25
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