摘要
不论是对脊椎动物还是无脊椎动物来说,神经系统都堪称最复杂的系统之但目前对于神经网络的形成及其调节的具体机制还了解甚少。
目前的研究表明,一些膜蛋白特别是细胞粘附分子家族成员参与到神经系统发育以及神经网络形成的调节过程中。唐氏综合征细胞粘附分子(Down Syndrome Cell Adhesion Molecule,DSCAM)编码的跨膜蛋白是免疫球蛋白超家族成员,属于细胞粘附分子家族。DSCAM的功能被认为与神经系统的发育密切相关。果蝇中的Dscam1基因通过选择性剪切可以产生数量众多的蛋白异构体,并通过嗜同型结合产生排斥效应,参与调节神经元轴突的分枝,树突的自我回避以及胞体的“马赛克”分布。脊椎动物DSCAM除介导嗜同型排斥外,还可介导嗜同型粘附和吸引。在脊椎动物中,还发现DSCAM具有嗜异型结合的特性。在鸡胚和小鼠胚胎发育中,DSCAM能够与DCC相互作用,共同参与介导了由netrin-1引起的轴突导向及联合纤维的跨中线转向行为。但DSCAM蛋白发挥生物学功能的具体机制及其信号通路仍需要进一步研究。
在第一部分工作中,我们发现在3周龄时,与同窝野生型(Wild-type,Wt)小鼠相比,Dscamdell7突变纯合(Homozygous Dscam mutant,Hm)小鼠发育较为迟滞。2月龄时,Hm表现出行走姿势的改变。转棒实验结果显示,Hm小鼠奔跑的持续时间(n=10,28.30s±5.54)与Wt小鼠(n=6,115.70s±23.33)和Dscamdell7突变杂合(Heterozygous Dscam mutant,Het)小鼠(n=5,132.00 s±24.39)相比显著缩短。Hm小鼠掉落时转棒的速度(n=6,14.73 rpm±2.22)与Wt(n=6,33.67 rpm±2.21)和Het小鼠(n=5.35.59 rpm±2.16)掉落时转棒的转速相比也显著减慢。经过训练,Wt小鼠奔跑的持续时间显著延长(n=4,首次：84.50 s±23.29,第八次：188.80 s±29.59),而Het小鼠(n=5,首次：111.80 s±30.08,第八次：187.60 s±23.42)和Hm小鼠则无显著差异(n=3,首次：8.00 s±2.89,第八次：18.33 s±10.87)。
在第二部分工作中,我们发现在2月龄时,Hm小鼠大脑与同窝Wt小鼠和Het小鼠相比,出现皮层的塌陷。塌陷部位和程度在Hm小鼠中存在个体差异。统计结果显示,Hm小鼠脑室体积较Wt小鼠(P<0.05)和Het小鼠显著增大(P<0.05),Hm小鼠脑室容积与整个大脑体积的百分比也较Wt(P<0.05)和Het小鼠显著升高(P<0.05)。Hm小鼠脑室的扩张还严重影响了大脑皮层,胼胝体,纹状体和海马的发育。不同脑区的大脑皮层(Cg/RS:P<0.001,M:p<0.001,S:P<0.01)和胼胝体的联合纤维(Cg/RS:P<0.001,M:P<0.001,S:P<0.01)较Wt小鼠均显著变薄。
本论文通过实验发现Dscamdell7突变小鼠存在运动能力的下降,运动学习记忆能力的受损,以及脑室显著扩张,脑内结构发育缺陷等表型。初步探索了Dscam基因在小鼠神经系统发育及大脑结构发育中的作用。并提示Dscam基因可能是一个与脑积水发病机制相关的新基因,为脑积水发病机制的研究提供了新的模型小鼠,也为脑积水的研究提供了新的思路。
The nervous system is one of the most complicated systems in both vertebrates and invertebrates. The mechanisms underlying the formation of this system are still largely unknown.
Previous studies show that many membrane proteins, especially cell adhesion molecules participate in various steps of neural development and neural network formation. The transmembrane protein encoded by DSCAM is a member of Immunoglobulin superfamily and belongs to cell adhesion molecule family. DSCAM plays a crucial role in the development of nervous system. Drosophila Dscam can generate thousands of isoforms through alternative splicing from single gene, among which only the same isoforms can bind to each other. The repulsive effect resulted from homophilic binding is involved in many aspects of neural development such as axon branching, dendritic tiling and soma spacing. The vertebrate DSCAM can play similar role in neural development such as axon branching, dendritic tiling and soma spacing. Current data suggest that in chicken retina, DSCAM can also mediate axon layer-specific targeting and synaptic formation by homophilic adhesion. Furthermore, besides mediating homophilic repulsion or adhesion, vertebrate DSCAM can heterophilically bind to DCC to form a complex receptor in the absence of netrin-1, and when netrin is present, this complex begins to dissociate. Thus, DSCAM and DCC may collaborate in mediating axon turning and midline crossing of commissural axon in response to netrin-1. However, the underlying mechanism and signaling pathway are still largely unknown.
In the first part of this study, we found that, at 3 weeks old, compared with Wild-type (Wt) littermates, homozygous Dscam mutant (Hm) mice showed maldevelopment. At 2 months old, compared with Wt mice, Hm mice exhibited severe change of walking posture. A accelerating rotarod test showed that the average running time of Hm mice (n=10,28.30 s±5.54) were significantly shorter than Wt (n= 6, 115.70 s±23.33) and heterozygous Dscam mutant (Het) (n= 5,132.00 s±24.39) mice. The speed of rotarod when Hm (n= 6,14.73 rpm±2.22) mice fell off was significantly lower than Wt (n= 6,37.67 rpm±2.21) and Het (n= 5,35.59 rpm±2.16) mice. After 8 trials of training, the average running time of the Wt mice was significantly extended (n = 4, first:84.5 s±23.39, eighth:188.80 s±29.59) while that of Het (n= 5, n= 5, first: 111.80 s±30.08, eighth:187.60 s±23.42) and Hm mice (n= 3, first:8.00 s±2.89. eighth:18.33 s±10.87) had no significant difference between before and after the training sessions.
In the second part of this study, we demonstrated that, at 2 months old, dissected Hm mice brains showed collapsed cortex comparing with littermate Wt and Het mice. There were some variations among individuals of the collapsed area and degree. Quantification results suggested that ventricles of Hm mice were significantly enlarged than Wt (P< 0.05) and Het mice (P< 0.05). The ratio of the ventricular volume to the total brain volume of Hm mice was also significantly higher than Wt (P< 0.05) and Het mice (P< 0.05). In addition, there was no significant difference between Wt and Het mice. Furthermore, we also found that as consequences of ventricular dilation, the thickness of cortex (Cg/RS:P< 0.001, M:P< 0.001, S:P< 0.01) and corpus callosum (Cg/RS:P< 0.001, M:P< 0.001, S:P< 0.01) of Hm mice in different cortical regions were significantly thinner than Wt mice. The hippocampus of Hm mice also showed malformation such as shorter longitude and rough margin. Besides, in Hm mice, the internal capsule fibers of striatum were stretched.
This work demonstrated that Hm mice had decreased motor function, decreased motor learning ability and enlarged ventricles with preliminary exploring the function of Dscam during neural development and brains structural formation. Moreover, the dilated ventricles suggested that Dscam may be a new candidate gene of hydrocephalus. The Dscam dell7 mutant mouse can be used as new animal model for hydrocephalus, providing a new idea for the researches of hydrocephalus.
引文
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