Examining the role of the Lissencephaly protein, Lis1, in dynein-based transport

描述（由申请人提供）：LIS1基因突变引起的常染色体显性脑畸形，是第一个与细胞质动力蛋白功能直接相关的神经系统疾病。这一发现为哺乳动物大脑皮层发育调控事件的分子解剖开辟了道路。Lis1和一个结合伙伴Nudel直接结合动力蛋白并调节其活性。这种相互作用几乎完全是在大脑发育的背景下研究的。最近，与精神分裂症、肌萎缩侧索硬化症（ALS）、佩里综合征和亨廷顿氏病（HD）等晚发性疾病相关的基因产物已被证明与动力蛋白通路直接相互作用。Lis1和Nudel在调节培养的成年大鼠感觉神经元中动力蛋白依赖的轴突运输中的重要作用的发现，导致了对该调节网络的干扰将导致小鼠神经元功能障碍的假设。为了验证这一点，将使用floxedlis1和Cre菌株来确定发育后破坏Lis1的病理后果。将进行组织学、生化和行为学研究以确定发育后Lis1的缺失是否会导致神经功能障碍。由于这可能加重无脑畸形和其他神经系统疾病的疾病症状，这些研究可能为临床药物干预提供可行的靶点。可能的候选药物是PPAR3激动剂文迪雅和Actos，这两种胰岛素增敏剂通常用于治疗2型糖尿病。最近的研究表明，这些药物刺激了几种细胞系的动力蛋白。APC是一种微管加端相关蛋白，通常与结肠癌有关。APC也被发现在神经元中起关键作用，影响突触功能和轴突运输。值得注意的是，动力蛋白对PPAR3激动剂的反应需要PI3K活性，并由锂模拟，锂是一种有效的Gsk32抑制剂，用于治疗情绪障碍。此外，动力蛋白在体外是Gsk32的靶标，其磷酸化导致APC片段的共免疫沉淀增加5倍，这表明激酶的抑制可能会影响动力蛋白的相互作用。感觉神经元中的动力蛋白分布在PPAR3激动剂的作用下发生改变，这导致了PPAR3通路有助于调节成年神经元中动力蛋白依赖的轴突运输的假设。这将通过确定PPAR3/Gsk32/APC通路的药理学和遗传操作对成年大鼠DRG神经元细胞器运输的影响以及确定Lis1和PPAR3通路之间是否发生串扰来验证。

DESCRIPTION (provided by applicant): Lissencephaly, an autosomal dominant brain malformation caused by mutations in the LIS1 gene, was the first neurological disorder linked directly to cytoplasmic dynein function. This discovery led the way for molecular dissection of events regulating development of the mammalian cerebral cortex. Lis1 and a binding partner, Nudel, bind directly to dynein and regulate its activity. This interaction has been studied almost exclusively in the context of brain development. More recently, gene products associated with later-onset disorders such as schizophrenia, amyotrophic lateral sclerosis (ALS), Perry Syndrome, and Huntington's disease (HD) have been shown to interact directly with dynein pathways. The discovery of an important role for Lis1 and Nudel in regulating dynein-dependent axonal transport in cultured adult rat sensory neurons has led to the hypothesis that perturbing this regulatory network will cause neuronal dysfunction in mice. To test this, the pathological consequences of disrupting Lis1 in post-developmentally will be determined using floxed-Lis1 and Cre strains. Histological, biochemical, and behavioral studies will be carried out to determine if depletion of Lis1 post- developmentally causes neurological dysfunction. Because this could exacerbate disease symptoms in lissencephaly and other neurological disorders, these studies may provide a feasible target for clinical intervention by drugs. Possible candidates are the PPAR3 agonists Avandia and Actos, insulin sensitizers normally used to treat type 2 diabetes. Recent studies indicate that these drugs stimulate dynein in several cell lines. This is blocked by mutations in APC, a microtubule plus end associated protein typically linked to colon cancer. APC has also been found to have critical roles in neurons, influencing both synaptic function and axonal transport. Notably, the dynein response to PPAR3 agonists requires PI3K activity and is mimicked by lithium, a potent Gsk32 inhibitor used to treat mood disorders. Moreover, dynein is a target of Gsk32 in vitro and its phosphorylation results in a 5-fold increase in coimmunoprecipitation of an APC fragment suggesting that inhibition of the kinase could impact dynein interactions. Dynein distribution in sensory neurons is altered in response to PPAR3 agonists, leading to the hypothesis that PPAR3 pathways contribute to regulation of dynein-dependent axonal transport in adult neurons. This will be tested by determining the effect of pharmacological and genetic manipulation of PPAR3/Gsk32/APC pathways on organelle transport in adult rat DRG neurons and determining if crosstalk occurs between Lis1 and PPAR3 pathways. PUBLIC HEALTH RELEVANCE: We have proposed a discreet set of experiments to test specific hypotheses concerning mechanisms regulating the transport of cellular components in adult nerves. The work will likely reveal interactions that, when disrupted, contribute to nervous system disorders. Understanding these mechanisms will be key to design of therapeutic regimes to treat these disorders, and may explain unexpected neurological side effects when drugs go to trial.