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

项目总结 Duchenne肌营养不良症(DMD)是由产生蛋白质的DMD基因突变引起的 营养不良蛋白。尽管该基因产生不同大小的异构体，但只有最大的异构体Dp427执行 通过将细胞外基质(ECM)连接到细胞骨架来发挥骨骼肌的关键功能。DMD突变 也会导致神经功能障碍，但这些变化的细胞和分子基础很差- 站着。有趣的是，DMD患者的认知缺陷的严重性随着较小的认知缺陷的连续丢失而恶化 不能连接细胞外基质和细胞骨架的异构体，意味着这些较短的营养不良蛋白具有额外的功能。 DMD患者的总大脑和灰质体积减少，突变会影响基因转录 中等大小的异构体Dp140与这种还原联系最强16。我建议在这里探讨DYS的作用- 发育中的脑室/室下区(V-SVZ)中的营养素，是脑内主要的神经干细胞巢(NSC) 成年哺乳动物的大脑。V-SVZ在出生后脑发育过程中的一个关键输出是少突胶质细胞前体. 生殖器细胞(OPC)，它继续使前脑髓鞘形成。髓鞘形成的时机和适当的实施 在许多与DMD相同的神经过程中起着关键作用。室管膜细胞(ECs) 是V-SVZ中特殊的多纤毛细胞，排列在脑室周围的神经干细胞和 调节NSC的静止和激活。我的赞助人的实验室最近报告说，营养不良的葡聚糖，结合部分- NER调节V-SVZ神经干细胞中的缺口信号，调节NSC的命运决定和发育。 《欧洲共同市场评论》24。营养不良多糖和营养不良蛋白也都被发现影响出生后OPC的发育， 包括延缓白质束髓鞘形成。据报道，肌肉中存在异常的缺口信号。 然而，在DMD41动物模型中，Dstrophin是否调节NSCs中的缺口仍不清楚。 在我的第一个目标中，我将研究dystrophin亚型如何通过以下方式调节出生后早期V-SVZ生态位的形成 检查电子商务的发展和组织，使之成为风车。在第二个目标中，我将探索dystrophin是如何 异构体调节V-SVZ神经干细胞的功能以及神经元和神经胶质前体细胞的产生。从头到尾我都会 检测dystrophin在V-SVZ神经干细胞中调节缺口信号的能力，并检测dystrophin是否缺乏 通过调节缺口通路可以挽救细胞表型。我将使用小的抗肌营养不良蛋白结构和 DMD小鼠模型(mdx、mdx4cv、mdx3cv)与缺口活动报告鼠相结合。耐人寻味的是，小 据报道，肌营养不良蛋白在肌肉细胞中转移到细胞核，这表明了新的 小的营养不良蛋白在神经干细胞核中的功能作用，这将通过Se-2的修饰来评估。 核进出口所需的顺序。最后，作为补充，我将使用新生儿脑室 电穿孔策略预防或挽救发育中的V-SVZ和使用V-SVZ中的dystrophin表达 模拟NSC和EC发育的细胞培养。总之，我的研究将调查抗肌营养不良蛋白在 为出生后的大脑产生神经前体细胞的关键干细胞利基的形成和功能。

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.