Signaling pathways of necroptosis

 描述(由申请人提供)：我们已出版(Re等人)。神经元，2014)，野生型脊髓运动神经元(MN)被表达的小鼠家族性突变体SOD1(MSOD1)和人类散发性ALS星形胶质细胞-或他们的条件培养液(CM)选择性地杀死-通过caspase不依赖的机制。在这项研究中，我们还表明，在我们的体外模型中，受体相互作用蛋白-1(RIP1)的激酶功能的变构抑制剂NEC-1(NEC-1)对MN死亡具有完全的保护作用。与RIP1的发现一致，我们的试点数据表明，沉默RIP3和抑制混合谱系激酶结构域样(MLKL)，这两个已知的坏死下垂的决定因素，也提供了MN保护。这些发现提供了第一个实验证据，证明在慢性人类神经疾病模型中，坏死性下垂调节神经元死亡。然而，值得注意的是，除了RIP1、RIP3和MLKL之外，坏死性下垂的分子网络，特别是在神经元中，仍然难以捉摸。因此，作为揭开坏死性下垂神经元分子网络的初始阶段，我们提出了一个为期两年的工作范围，包括两个连续的步骤：首先，为了将坏死性下垂过程中发生的复杂基因表达变化归因于有限数量的调控基因的作用，我们将使用：(I)我们的MN互动组(一个细胞类型特异的调控网络)；以及(Ii)从纯化的小鼠胚胎干细胞来源的MN获得的RNAseq数据，这些MN暴露在CM中，用mSOD1星形胶质细胞(中毒条件)或野生型SOD1星形胶质细胞(无毒对照条件)制成。为了区分由星形胶质细胞引起的MN的一般变化和与坏死性下垂有关的变化，每个实验都将在有或没有拮抗剂NEC-1的情况下进行。将使用Marina算法识别坏死性下垂的候选主调控子(MRS；即与表型相关的转录因子和信号通路基因集)。其次，为了验证在步骤1中确定的候选MRS在MN死亡表型中的作用，将使用我们的试点数据所示的shRNA策略将每个MRS作为靶点。这一验证将在我们的体外小鼠和人类模型中通过监测MN存活率和轴突长度来完成。在步骤2中还将对MN样品进行多重qRT-PCR实验，在每个样品中将分别击倒一个MR。这里的目标是检查是否有任何已识别的MRS调节其他MRS的表达。作为未来的研究，我们建议：(1)利用这里产生的信息来开发基因组特征和新的试剂/工具来研究死后小鼠模型和患有神经退行性疾病(如但不限于ALS)的患者的坏死性下垂；以及(2)评估最有希望的MRS在ALS转基因小鼠模型中疾病表型表达中的作用。

 DESCRIPTION (provided by applicant): We have published (Re et al. Neuron, 2014) that wild-type spinal motor neurons (MNs) are selectively killed by both mouse familial mutant SOD1 (mSOD1)-expressing astrocytes and human sporadic ALS astrocytes-or their conditioned medium (CM)-through a caspase-independent mechanism. In this study, we also show that necrostatin-1 (Nec-1), an allosteric inhibitor of the kinase function of the receptor interacting protein-1 (RIP1), which is an obligatory mediator of necroptosis, affords full protection against MN death in our in vitro models. Consistent with the RIP1 finding, our pilot data demonstrate that silencing RIP3 and inhibiting mixed lineage kinase domain-like (MLKL), two other known determinants of necroptosis, also provide MN protection. These findings offer the first experimental evidence that necroptosis regulates neuronal death in a model of chronic human neurological disorder. Yet, remarkably, aside from RIP1, RIP3, and MLKL, the molecular network of necroptosis, especially in neurons, remains elusive. Thus, as an initial phase toward unraveling the neuronal molecular network of necroptosis, we propose a 2 year scope of work consisting of two sequential steps: First, to assign the complex gene expression changes that occur during necroptosis to the actions of a limited number of regulatory genes, we will use: (i) our MN interactome (a cell type-specific regulatory network); and (ii) RNAseq data obtained from purified mouse embryonic stem cell-derived MNs exposed to CM made with either mSOD1-astrocytes (toxic condition) or wild-type SOD1 astrocytes (non-toxic control condition). So as to differentiate between general changes in MNs induced by astrocytes and those involved in necroptosis, each experiment will be performed in the presence or the absence of the antagonist Nec-1. Candidate master regulators (MRs; i.e. sets of transcription factor and signaling pathway genes causally associated with the phenotype) of necroptosis will be identified using the MARINa algorithm. Second, to validate the role of the candidate MRs-identified in Step 1-in the MN death phenotype, each will be targeted using shRNA strategies as shown in our pilot data. This validation will be done in both our in vitro mouse and human models by monitoring both MN survival and neurite length. Also to be performed in Step 2 are multiplex qRT-PCR experiments on MN samples, in each a MR will be individually knocked-down. The goal here is to examine whether any of the identified MRs regulate the expression of the others. As future studies, we propose: (1) to use the information generated herein to develop a genomic signature and new reagents/tools to study necroptosis in both post-mortem mouse models of and patients with neurodegenerative disorders such as, but not limited to ALS; and, (2) to assess the role of the most promising MRs in the expression of the disease phenotype in transgenic mouse model of ALS.