Investigation of fmnl2 in cerebellar development

项目摘要 先天性共济失调通常是由小脑的功能障碍和畸形引起的，特别是 内侧蚓部这些疾病可能是由于缺乏适当的发育信号级联， 决定了神经元的增殖和形成。初级纤毛为各种发育信号提供枢纽 蛋白质如SHH或WNT。纤毛蛋白功能障碍导致影响人类的罕见遗传疾病 神经系统、视觉系统、肝脏、肾脏和骨骼系统的发育。患者 睫状体病如Joubert综合征和相关疾病显示小脑蚓部发育不全，上级增厚， 小脑脚和加深的脚间窝。细胞骨架的成分，例如肌动蛋白 和微管，在纤毛发生和维持现有纤毛成分中起着至关重要的作用， 支撑脚手架虽然许多纤毛相关基因已被发现是这些疾病的原因， 细胞骨架调节因子的家族及其与纤毛的关系尚未完全确定， 这些分子以前是否与异常的大脑发育有关。 我们的实验室使用正向遗传方法来确定小脑发育和退化的关键途径 神经元的数量通过化学诱变筛选，我们发现了一种共济失调小鼠 表型与某些纤毛病变相似的突变体：小脑海马发育不全， 小叶异常、小脑沿前后轴沿着伸长以及上级的失败 小脑脚到交叉。通过定位克隆，我们鉴定了Fmnl2中剪接受体的突变， 导致Fmnl2转录物中的外显子跳跃。有趣的是，突变小鼠脑中Fmnl2转录物的水平 与WT相比没有变化，但蛋白质水平降低，这表明框内缺失编码了 是该蛋白质稳定性所必需的。 FMNL 2是一种自抑制的细胞骨架效应物，先前已显示在200 ℃时驱动肌动蛋白聚合。 培养细胞的丝状伪足和片状伪足顶端。尽管这个家族中的其他蛋白质已经显示出结合和 调节微管，肌动蛋白，并影响纤毛的形成，无论这种蛋白质在微管和 肌动蛋白在大脑发育中的作用尚不清楚。使用这种新的小鼠模型，我将研究FMNL 2的作用， 在肌动蛋白和微管的稳定，并确定如何亚型损失这种蛋白质可能会影响 纤毛发生和纤毛维持。这些研究将启发我们对小脑畸形的认识 并影响我们对微管和肌动蛋白在人体内作用机制的理解， 纤毛病变

PROJECT SUMMARY Congenital ataxias generally result from dysfunctions and malformations of the cerebellum, particularly the medial vermis. These disorders may result from the lack of proper developmental signaling cascades which dictate the proliferation and formation of neurons. Primary cilia provide a hub for various developmental signaling proteins such as SHH or WNT. Dysfunction in ciliary proteins leads to rare genetic disorders affecting human development in the nervous system, optical system, and liver, kidney, and skeletal systems. Patients with ciliopathies like Joubert Syndrome and related disorders display cerebellar vermis hypoplasia, thickened superior cerebellar peduncles, and a deepened interpeduncular fossa. Components of the cytoskeleton, such as actin and microtubules, play a vital role in ciliogenesis and the maintenance of existing ciliary components and supporting scaffold. Although many cilia-related genes have been found to be causal for these disorders, cytoskeletal regulators of the formin family and their relationship with cilia has not yet been fully defined, nor have these molecules been previously associated with abnormal brain development. Our lab uses a forward genetic approach to identify pathways critical to cerebellar development and degeneration of neurons in this brain region. Through a chemical mutagenesis screening, we discovered an ataxic mouse mutant with phenotypes similar to those observed in some ciliopathies: cerebellar hippocampal hypoplasia, abnormal foliation, cerebellar elongation along the anterior-posterior axis, as well as the failure of the superior cerebellar peduncle to decussate. By positional cloning, we identified a mutation at a splice acceptor in Fmnl2, leading to exon skipping in Fmnl2 transcripts. Interestingly, levels of Fmnl2 transcripts in the brain of mutant mice are unchanged compared to WT, but protein levels are reduced, suggesting that the in-frame deletion encoded by this exon are necessary for stability of this protein. FMNL2 is an autoinhibited cytoskeletal effector that has been previously shown to drive actin polymerization at filopodia and lamellipodia tips of cultured cells. Although other proteins in this family have shown to bind and regulate microtubules, actin, and influence cilia formation, whether this protein functions in microtubules and actin during brain development is unknown. Using this novel mouse model, I will investigate the role of FMNL2 in actin and microtubule stabilization and determine how the hypomorphic loss of this protein may impact ciliogenesis and cilia maintenance. These studies will enlighten our understanding of cerebellar malformations and impact our understanding of the mechanisms underlying the role of microtubules and actin in human ciliopathies.