Exploration of developmental brain abnormalities in mouse models of Duchenne muscular dystrophy

项目摘要 杜氏肌营养不良症（DMD）是由DMD基因突变引起的，该基因产生蛋白质 抗肌萎缩蛋白。虽然该基因产生各种大小的同种型，但只有最大的同种型Dp 427， 通过将细胞外基质（ECM）连接到细胞骨架而在骨骼肌中发挥关键作用。DMD突变 也可能导致神经功能障碍，但这些变化的细胞和分子基础并不充分- 站着。有趣的是，在DMD患者中观察到的认知缺陷的严重程度与较小的 不能连接ECM和细胞骨架的同种型，这意味着这些较短的肌营养不良蛋白的额外功能。 DMD患者的总脑和灰质体积减少，突变影响了DMD基因的转录。 中等大小的同种型Dp 140与这种减少最密切相关16。我在这里建议探讨dys的作用- 在发育中的脑室/脑室下区（V-SVZ）中的营养因子，主要的神经干细胞龛（NSC）， 成年哺乳动物的大脑在出生后的大脑发育过程中，V-SVZ的一个关键输出是少突胶质细胞前体， 生殖细胞（OPCs），继续为前脑形成髓鞘。髓鞘形成的时机和正确执行 在DMD中受影响的许多相同的神经过程中起着关键作用。室管膜细胞 是V-SVZ中的特化多纤毛细胞，其排列在围绕NSC的脑室中， 调节NSC的静止和激活。我担保人的实验室最近报告说肌营养不良蛋白聚糖，结合部分- 抗肌萎缩蛋白的一种，调节V-SVZ神经干细胞中的notch信号，以调节NSC的命运决定和发育。 ECS 24的操作肌营养不良聚糖和肌营养不良蛋白也被发现影响出生后OPC的发育， 包括延迟白色物质束髓鞘形成。据报道，在肌肉中， 然而，DMD动物模型中的干细胞41，肌营养不良蛋白是否调节NSC中的Notch仍然未知。 在我的第一个目标中，我将研究肌营养不良蛋白亚型如何调节出生后早期V-SVZ龛的形成， 把电子商务的发展和组织检查成风车。在第二个目标中，我将探讨肌营养不良蛋白 同种型调节V-SVZ NSC功能以及神经元和神经胶质祖细胞的产生。我将自始至终 检查抗肌萎缩蛋白调节V-SVZ神经干细胞中notch信号传导的能力， 细胞表型可以通过调节Notch途径来挽救。我将使用小的抗肌萎缩蛋白结构， DMD小鼠模型（mdx、mdx 4cv、mdx 3cv）与notch活性报告小鼠组合。有趣的是， 据报道，肌营养不良蛋白在肌肉细胞中易位到细胞核，表明新的肌营养不良蛋白的潜力。 神经干细胞核中小的肌营养不良蛋白的功能作用，这将通过修饰硒- 核武器进出口所需的许可证。最后，作为补充方法，我将使用新生儿心室 电穿孔策略，以防止或挽救发育中的V-SVZ中的肌营养不良蛋白表达并使用V-SVZ 模拟NSC和EC发育的细胞培养物。我的研究将共同探讨抗肌萎缩蛋白在免疫系统中的作用。 一个关键的干细胞龛的形成和功能，为出生后的大脑产生神经祖细胞。

PROJECT SUMMARY Duchenne Muscular Dystrophy (DMD) results from mutations in the DMD gene, which generates the protein dystrophin. Although the gene produces various sized isoforms, only the largest isoform, Dp427, performs a critical function in skeletal muscle by linking the extracellular matrix (ECM) to the cytoskeleton. DMD mutations can also cause neurological dysfunction, but the cell and molecular basis of these changes are poorly under- stood. Interestingly, the severity of cognitive deficits seen in DMD worsens with successive loss of the smaller isoforms that cannot connect the ECM and cytoskeleton, implying additional functions these shorter dystrophins. DMD patients have reduced total brain and gray matter volume, with mutations that affect transcription of the mid-size isoform Dp140 being most strongly linked to this reduction16. I propose here to explore the role of dys- trophin in the developing ventricular/subventricular zone (V-SVZ), the major neural stem cell niche (NSC) in the adult mammalian brain. A key output of the V-SVZ during postnatal brain development is oligodendrocyte pro- genitor cells (OPCs), which go on to myelinate the forebrain. The timing and proper execution of myelination plays a critical role in many of the same neurological processes that are affected in DMD. Ependymal cells (ECs) are specialized multi-ciliated cells in the V-SVZ that line the ventricles of the brain that surround NSCs and regulate NSC quiescence and activation. My sponsor’s lab recently reported that dystroglycan, the binding part- ner of dystrophin, modulates notch signaling in V-SVZ NSCs to regulate both NSC fate decisions and the devel- opment of ECs24. Dystroglycan and dystrophin were also both found to influence postnatal OPC development, including delaying white matter tract myelination. Dysregulated notch signaling has been reported in muscle stem cells in animal models of DMD41, however, whether dystrophin regulates notch in NSCs remains unknown. In my first aim, I will investigate how dystrophin isoforms regulate early postnatal V-SVZ niche formation by examining EC development and organization into pinwheels. In the second aim, I will explore how dystrophin isoforms regulate V-SVZ NSC function and the production of neuronal and glial progenitors. Throughout I will examine dystrophin’s ability to regulate notch signaling in V-SVZ NSCs and test whether dystrophin-deficient cell phenotypes can be rescued by modulating the notch pathway. I will use small dystrophin constructs and DMD mouse models (mdx, mdx4cv, mdx3cv) in combination with notch activity reporter mice. Intriguingly, small dystrophins have been reported to translocate to the nucleus in muscle cells, indicating the potential for novel functional roles for small dystrophins in the nucleus of NSCs, which will be assessed by modification of se- quences needed for nuclear import/export. Lastly, as a complementary approach, I will use neonatal ventricle electroporation strategies to prevent or rescue dystrophin expression in the developing V-SVZ and use V-SVZ cell cultures that model NSC and EC development. Together, my studies will investigate dystrophin’s role in the formation and function of a crucial stem cell niche that generates neural progenitors for the postnatal brain.