第一部分 对含KASH和SUN结构功能域的蛋白的功能研究(从果蝇到小鼠) KASH与SUN这两类核膜蛋白通过链接不同的细胞骨架与细胞核膜在调控多种生物学过程的细胞核迁移和锚定中发挥了重要作用。近10年来，对于KASH和SUN蛋白的研究工作已经涉及从低等生物到高等生物，从体外培养的细胞学到小鼠模型的病理学等一系列研究领域，对这两类蛋白的生物学功能以及相关机理也日益深入。本文中，我利用突变型果蝇msp-300〓研究了果蝇KASH蛋白MSP-300在卵细胞发育中的作用，并且利用KASH蛋白Syne-1和Syne-2、SUN蛋白SUN1和SUN2的基因敲除小鼠以及建立的Syne-1和SUN1的条件性基因敲除小鼠，探讨了这些KASH蛋白和SUN蛋白在多个生物学过程中介导细胞核迁移和锚定的功能。 对果蝇KASH蛋白的研究集中在对MSP-300蛋白在果蝇卵细胞发育过程中的功能研究。在果蝇卵细胞发育晚期，卵泡室内的滋养细胞的细胞质被快速转运到卵细胞中；同时，滋养细胞的细胞核会被肌动蛋白锚定。本研究发现在果蝇卵细胞发育过程中尤其是滋养细胞到卵细胞的胞质转运过程中，源自母亲的MSP-300蛋白对细胞核锚定起了重要作用。我首先用抗MSP-300的抗体对果蝇卵巢进行染色，发现MSP-300蛋白分布在滋养细胞和卵细胞的细胞质内，且大量聚集在其核膜上。并且我发现转基因表达的绿色荧光蛋白和MSP-300的碳端融合蛋白也定位于卵细胞的细胞核膜上。最后，为了消除母系效应对卵细胞发育过程的影响，我通过生殖细胞有丝分裂重组和显性雌性不育技术得到了纯合的msp-300突变克隆，发现在这些msp-300纯合突变的卵泡室内滋养细胞和卵细胞的细胞核定位紊乱，并且原本分布有序而精密的肌动蛋白结构也被严重破坏。 对于小鼠KASH/SUN蛋白功能的研究主要集中在对Syne-2，SUN1和SUN2蛋白的基因敲除小鼠视网膜发育的研究。脊椎动物视网膜内所有的神经细胞均由它们的前体细胞分化而来，且其发育过程遵循非常严格的时空顺序。果蝇KASH/SUN蛋白Klarsicht/Klaroid和斑马鱼KASH蛋白syne2a均与视网膜感光细胞的细胞核定位和迁移相关。本论文研究推断在小鼠视网膜发育过程中，Syne-2、SUN1和SUN2蛋白介导了视杆细胞细胞核的定位与视锥细胞细胞核的迁移，且SUN1和SUN2蛋白可能在视锥细胞细胞核的迁移过程中功能冗余。我通过抗Syne-2、抗SUN1和抗SUN2的抗体对小鼠视网膜冰冻切片进行染色发现，Syne-2、SUN1和SUN2蛋白定位于小鼠视网膜内大部分神经细胞的细胞核膜上。在成年Syne-2〓和Sun1〓小鼠的视网膜切片中，我观察到外核层细胞数量减少、内外核层之间细胞核定位异常的表型，且Syne-2〓和Sun1〓小鼠视网膜电生理结果也表明感光细胞和部分其他细胞可能异常。然后，我对Syne-2〓和Sun1〓小鼠视网膜切片进行了免疫荧光染色，发现其视杆细胞外节明显减少，部分视杆细胞细胞核定位异常。并且，在Syne-2〓小鼠视网膜中，我发现视锥细胞外节也明显减少，但是在Sun1〓小鼠视网膜中并没有出现这个表型。视锥细胞特异性抗体染色的结果表明，Syne-2〓小鼠视网膜发育过程中的视锥细胞细胞核迁移失败，而相同的异常表型也在Sun1〓；Sun2〓；NSE-SUN1小鼠视网膜观察到。 本论文中还报导了我们成功建立的Syne-1和Sun1条件性基因敲除小鼠，利用这些小鼠在不同组织器官水平上对KASH蛋白和SUN蛋白的功能研究中将会有非常重要的意义。由于Syne-1/2基因双敲除小鼠和SUN1/2基因双敲除小鼠都存在出生后立即死亡的现象，我们通过建立Syne-1和Sun1条件性基因敲除小鼠以对其功能及相互作用深入研究。初步实验结果表明：通过条件性基因敲除得到的Syne-1〓小鼠出现了与Syne-1〓小鼠相同的骨骼肌突触下细胞核定位异常的预期表型，Syne-1〓；Syne-2〓小鼠出生后立即死亡，而Syne-1〓；Syne-2〓小鼠没有出现明显异常。通过条件性基因敲除得到的Sun1〓小鼠也出现了与Sun1〓小鼠相同的不育表型。Sun1〓；Sun2〓小鼠出生后也立即死亡，而Sun1〓；Sun2〓小鼠并没有出现明显异常。对Sun1和Sun2条件性基因双敲除小鼠的研究发现，神经组织特异性敲除了SUN1和SUN2蛋白的Sun1〓；Sun2〓；Nestin-Cre小鼠出生后并没有立即死亡，而是至少可以存活24小时，最终死亡是由于丧失吃奶的能力而营养不良致死。组织学分析结果表明，Sun1〓；Sun2〓；Nestin-Cre小鼠与Sun1〓；Sun2〓小鼠表型非常相似，其大脑皮层发育严重受损，由于神经细胞辐射方向迁移失败导致大脑皮层发育无法正常分层。 我分别在果蝇和小鼠体内对MSP-300、Syne-1/2和SUN1/2蛋白的生物学功能研究揭示了这些蛋白在多类细胞核定位和迁移过程中的重要作用，包括果蝇卵泡室内滋养细胞和卵细胞细胞核的锚定，小鼠感光细胞细胞核的迁移，以及大脑皮层神经元的辐射方向迁移。本研究结果不仅丰富了对细胞核锚定和迁移过程的分子机制的研究，而且增进了相关人类疾病发病机理的认识。 关键词： KASH结构功能域，SUN结构功能域，核膜蛋白，MSP-300蛋白，Syne-1蛋白，Syne-2蛋白，SUN1蛋白，SUN2蛋白，胞质转运，视杆细胞，视锥细胞，外节，视网膜电生理，视网膜发育，条件性基因敲除小鼠，基因双敲除 第二部分 对多聚谷氨酰胺疾病DRRLA致病基因Atrophin-1的研究 齿状核红核苍白球丘脑底核萎缩(dentatorubral-pallidoluysian atrophy， DRPLA)是一种显性遗传的神经退行性疾病，是由Atrophin-1基因中编码谷氨酰胺的CAG重复序列的扩增所造成。用转基因的方法在小鼠神经细胞内表达包含多聚谷氨酰胺扩增的人类Atrophin-1突变蛋白足以使小鼠产生神经退行性表型。但是，对于多聚谷氨酰胺扩增的Atrophin-1突变蛋白是否会和存在于病人和转基因小鼠疾病模型中的正常蛋白相互作用的问题，我们还不是很了解。因此，我们建立了Atrophin-1基因敲除小鼠。研究表明纯合基因敲除小鼠出现了生长延迟和雄性小鼠逐渐丧失繁殖能力的表型，但是并没有明显的神经退行性疾病的表型。而且小鼠Atrophin-1基因的敲除既不能增强也不能减弱在小鼠中由于过表达包含多聚谷氨酰胺扩增的人类Atrophin-1突变蛋白所产生的DRPLA表型。因此我们认为，DRPLA是由多聚谷氨酰胺在Atrophin-1蛋白中扩增所造成，而且这个过程与多聚谷氨酰胺扩增的蛋白和正常Atriphin-1蛋白之间的相互作用无关。 关键词：齿状核红核苍白球丘脑底核萎缩，多聚谷氨酰胺，转基因，基因敲除，神经退行性疾病

KASH and SUN proteins are two kinds of nuclear envelope (NE) proteins bridging the cytoskeletons and nuclear envelope (NE), which play critical roles in nuclear migration and anchorage during multiple biological processes. In the past decade, the extensive investigation on KASH and SUN proteins has been carried out from lower to higher organisms using various systems from in vitro tissue culture cell systems to pathological analysis with mouse models. The knowledge of the biological functions and the molecular mechanisms of these two protein families are increasing very fast. Using msp-300〓 mutant fly and gene knockout mice for KASH proteins Syne-1 and Syne-2, SUN proteins SUN1 and SUN2, we have studied their essential functions in nuclear anchorage and migration during multiple biological processes from Drosophila to mouse. The investigation of Drosophila KASH proteins focused on the functional analysis of MSP-300 protein in oogenesis. During late stages of Drosophila oogenesis, the cytoplasm of nurse cells in the egg chamber is rapidly transferred ("dumped"）to oocytes, while the nurse cell nuclei are anchored by a mechanism that involves the actin cytoskeleton. This thesis reports that maternal MSP-300 plays an important role in the actin-dependent nuclear anchorage during the cytoplasmic transport. MSP-300 is distributed throughout the cytoplasm and accumulates at the nuclear envelope of nurse cells and the oocyte with an antibody against the C-terminus of MSP-300. A GFP fusion protein containing the C-terminal region of MSP-300 is sufficient to localize the protein on the nuclear envelope in oocytes. To eliminate the maternal effect during oogenesis, we generated homozygous germ-line clones of a msp-300 loss-of-function mutation in otherwise heterozygous mothers. In the mutant egg chambers that develop from such clones, cytoplasmic dumping of nurse cells is severely disturbed. The nuclei of nurse cells and the oocyte are mislocalized and the normally well-organized actin structures are severely disrupted. The investigation of mammalian KASH and SUN proteins is mainly focused on the functional analysis of Syne-2, Sun1 and Sun2 knockout mice in retinal development. Retinal progenitor cells can form all kinds of retinal neurons, and all of the proliferation and differentiation take place in a well-organized temporal sequence. Studies on Drosophila KASH/SUN proteins Klarsicht/Klaroid and zebrafish KASH protein syne2a have shown that these proteins play important roles in nuclear positioning and migration during retinal development. Studies performed in this thesis indicate that Syne-2, SUN1 and SUN2 proteins mediate the rod cell nuclear positioning and cone cell nuclear migration. In addition, SUN1 and SUN2 proteins might play a redundant function in cone cell nuclear migration during retinal development. Staining of the retinal sections with Syne-2, SUN1 and SUN2 antibodies, we found that Syne-2, SUN1 and SUN2 proteins are localized to the NE of most cell types in the mouse retina. Both HE and immunofluorescent staining revealed a severe loss of photoreceptors in the outer nuclear layer (ONL) and a group of mislocalized nuclei lying between the inner plexiform layer (IPL) and inner nuclear layer (INL). The mislocalized nuclei were identified to belong to rods both in Syne-2〓 and Sun1〓 mice with immunofluorescent staining. Physiological ERG test also implied potential problems in photoreceptors and some other cell types in adult Syne-2〓 and Sun1〓 mice. Immunofluorescent staining of specific cell markers in the retina displayed a severe loss of outer segment (OS) in both rods and cones in the retina of Syne-2〓 mice while only a loss of rod OS only in the retina of Sun1〓 mice. Furthermore, the normal cone cell nuclear migration is found to be blocked in the retinas from Syne-2〓 and Sun1〓;Sun2〓;NSE-SUN1 mice. This thesis also describes the success generation of both Syne-1 and Sun1 conditional knockout mice. Both Syne-1/2 and Sun 1/2 double knockout mice died soon after birth. These conditional knockout mice would be helpful for the further investigation of the KASH and SUN protein functions in various tissues and different organs. The Syne-1〓 mice from Syne-1 targeted alleles exhibited myonuclear mislocalization defects in skeletal muscle. The Syne-1〓;Syne-2〓 mice died immediately after birth as expected, but no obvious defects were observed on the Syne-1〓;Syne-2〓 mice. Meanwhile, Sun1〓 mice from Sun1 targeted alleles exhibited sterility phenotype similar to that of Sun1〓 mice. Sun1〓;Sun2〓 mice died shortly after birth, while no gross abnormalities were observed on Sun1〓;Sun2〓 mice. Sun1 and Sun2 conditional double knockout mice were generated for study of the interatction between the two genes. Strikingly, the Sun1〓;Sun2〓;Nestin-Cre mice died in one or two days after birth due to a lack of the instinct of milk sucking. Histological analysis revealed that the cerebral cortex of Sun1〓;Sun2〓;Nestin-Cre mice displayed severe laminary defects, which was similar with those of Sun1〓;Sun2〓 mice. Our studies on Drosophila MSP-300 and mouse Syne-1/2 and SUN1/2 proteins discovered their crucial functions in nurse cell and oocyte nuclei anchorage during cytoplasmic transport process in fly oogenesis and in photoreceptor cell nuclear migration during retinal development and neuronal migration of brain cortex in mice. These results would advance our knowledge on the molecular mechanism of nuclear positioning and migration during multiple processes, and provide new insights into the development of retina, brain and related diseases. Key words: KASH domain, SUN domain, Nuclear envelope protein, MSP-300, Syne-1, Syne-2, SUN1, SUN2, cytoplasmic transport, rod, cone, OS, ERG, retinal development, conditional gene knockout Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant hereditary neurodegenerative disorder caused by the expansion of a poly-glutamine (poly-Q) repeat in Atrophin-1 protein. Ectopic expression of a poly-Q expanded human Atrophin-1 protein is sufficient to induce DRPLA phenotypes in transgenic mice. However, it is still unclear whether the dominant effect of poly-Q expansion is due to its functional interference with wild-type Atrophin-1 proteins, which are present in both human patients and transgenic mouse models. Here, we report the generation and analysis of an Atrophin-1 gene targeting allele. Homozygous mutant mice exhibit growth retardation and progressive male infertility, but no obvious signs of neurodegeneration. Disruption of wild-type Atrophin-1 gene neither blocked nor enhanced the neurodegenerative phenotypes caused by a poly-Q expanded transgene. Our result supports the model that the DRPLA disease is induced by poly-Q expanded proteins in a manner independent of any functional interaction with the non-poly-Q expanded proteins present in patients. Key words: DRPLA, poly-Q, transgene, knockout, neurodegeneration