Mechanisms of inherited neurodegenerative diseases

遗传性神经退行性疾病的机制

• 批准号: 10265225
• 负责人: Michael Ward
• 金额: $ 368.37万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10265225
• 关键词: Address; Amyotrophic Lateral Sclerosis; Annexins; Axon; Axonal Transport; Binding; Bioinformatics; Biology; C-terminal; Carrier Proteins; Cell Line; Cell Survival; Cell physiology; Cells; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communities; Complex; Cytoplasmic Granules; Development; Disease; Disease Pathway; Distal; Docking; Double-Stranded RNA; Drug Targeting; Embryo; Endosomes; Extramural Activities; Gel; Genes; Genetic; Genetic Transcription; Growth Cones; Human; Impairment; In Vitro; Individual; Infrastructure; Inherited; International; Knowledge; Label; Libraries; Link; Lysosomes; Mediating; Membrane; Metabolic; Methods; Microscopy; Microtubules; Mitochondria; Molds; Molecular; Monitor; Morphology; Motor; Mutate; Mutation; N-terminal; National Institute of Child Health and Human Development; National Institute of Neurological Disorders and Stroke; Nature; Neurites; Neurodegenerative Disorders; Neurons; Organelles; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Phosphotransferases; Population; Property; Proteins; Proteomics; Protocols documentation; Publishing; RNA; RNA Transport; RNA analysis; RNA delivery; Research; Research Personnel; Research Project Grants; Science; Site; Stimulus; Structure; Synapses; System; Technology; Transgenes; Translating; Translations; Zebrafish; aged; base; biophysical properties; cell type; druggable target; effective therapy; experimental study; forward genetics; frontotemporal lobar dementia-amyotrophic lateral sclerosis; gene discovery; gene function; imaging study; improved; in vivo; induced pluripotent stem cell; knock-down; live cell microscopy; neuronal cell body; neuronal survival; protein complex; protein function; response; screening; single cell sequencing; stress granule; transcriptomics; ward

Identification of a new mechanism of axonal RNA granule transport: Neurons are highly polarized and compartmentalized cells, with complex dendritic networks and far-reaching axons. To effectively and efficiently respond to stimuli and metabolic demands far from the soma, neurons rely heavily on local translation of proteins. Within axons, RNAs are locally translated at a number of sites, including growth cones, pre-synapses, and at intra-axonal organelles such as mitochondria. Though we know that axonal RNA transport requires microtubules and motor proteins, the precise mechanisms by which RNAs are transported from the soma to these distal regions of the neuron had been unclear. Unlike membrane-bound cargos, such as mitochondria and endosomes, RNAs usually exist within membraneless phase-separated ribonuclear protein complexes known as RNA granules. Therefore, the adapter machinery utilized by membraneless and membrane-bound cargos are likely different. In collaboration with Jennifer Lippincott Schwartz (HHMI) and Peter Hyslop (U. Cambridge), we made a surprising discovery that RNA granules are indirectly transported long distances in axons by hitchhiking on moving lysosomes (Liao Y, Lippincott-Schwartz J* & Ward ME*, 2019, Cell (*co-corresponding authors)). To identify the potential adapters linking lysosomes to RNA granules, we used a Lamp1-APEX proximity labeling proteomic strategy in human iPSC-derived neurons. Comparing hits from our lysosome-APEX experiment against a recently-published APEX analysis of RNA granule composition revealed Annexin A11 (ANXA11), a recently discovered ALS-associated protein, as a potential adapter. In collaboration with Lucy Forrest (NINDS), structural prediction of ANXA11 revealed C-terminal membrane binding annexin domains as well as an N-terminal low-complexity (LC) domain. In vitro experiments showed that purified ANXA11 could interact with artificial lysosomes. In addition, purified ANXA11 could phase-separate in vitro, a property conferred by its N-terminal LC domain. Live cell microscopy experiments showed that ANXA11 interacted with lysosomes and RNA granules in cells, and co-trafficked in axons in cultured neurons. Purified ANXA11 was sufficient to mediate docking of purified stress granules with artificial lysosomes in vitro. In collaboration with Katie Drerup (NICHD), we showed that RNA granule/lysosome co-transport occurred in vivo in zebrafish embryos. Knockdown of ANXA11 in neurons impaired transport of RNA granules and delivery of RNA to growth cones. ALS-associated mutations in ANXA11 changed its biophysical properties (resulting in more gel-like states) and reduced its interactions with lysosomes and RNA granules. These mutations also interfered with axonal RNA granule transport and delivery of RNA to growth cones. Our observations of endolysosome-dependent axonal RNA transport is consistent with recent observations from other groups. Interestingly RNA granules hitchhike on endosomes during long-distance transport in filamentous fungi, suggesting that this mode of RNA transport is evolutionarily conserved. Together, these experiments identified a molecular relationship between lysosomes, RNA granules, and axonal transport. Our findings further suggest the possibility that dysfunctional RNA transport may be a converging mechanism of FTD/ALS, potentially disrupted by mutations in different classes of disease-associated proteins that alter lysosomal, RNA granule, or transport protein functions. Development of a CRISPRi screening platform in iPSC-derived neurons: In collaboration with Martin Kampmann at UCSF, we developed a new method to perform large-scale, forward-genetic CRISPR-inhibition (CRISPRi) screens in human iPSC-derived neurons (Ruin T, Gachechiladze M, & Ludwig C, Ward ME* & Kampmann M*, 2019, Neuron (*co-corresponding authors)). Here, we merged our highly-scalable i3Neuron differentiation technology with a potent CRISPRi screening approach developed by the Kampmann lab. Integration of a CAG-dCas9-BFP-KRAB transgene at the CLYBL safe harbor locus allowed for durable knockdown of targeted genes in both iPSCs and NGN2-differentiated neurons through lentiviral sgRNA delivery. We then performed three CRISPRi-based screens in differentiated neurons, using readouts of survival, single-cell transcriptomics, and morphology: a) A pooled survival screen in iPSCs versus neurons using a lentiviral sub-library of sgRNAs targeting 2300 kinases and druggable targets. By quantifying enrichment or depletion of sgRNAs in aged iPSCs/neurons versus the starting cell population, we identified genes for which knockdown either improved survival (protective) or reduced survival (essential). We found a shared set of genes that was essential for survival of iPSCs and neurons. Interestingly, we also discovered genes whose knockdown-related survival phenotype was cell-type specific. b) A single cell sequencing combined with CRISPRi knockdown (CROPseq) screen to interrogate how knockdowns of genes identified in our survival screen altered cellular transcriptional states. CROPseq analysis revealed cell-type specific transcriptomic responses to gene knockdowns. c) A high-content microscopy approach to characterize how gene knockdown of individual genes identified in our survival screen altered neuronal morphology and survival. These imaging studies showed that the vast majority of genes identified in our primary pooled survival screen indeed influenced cell survival, and in a number of cases also influenced neurite morphology. Finally, in a separate collaboration with Len Petrucellis lab (Mayo Clinic) we showed that CRISPRi i3Neurons could be used to reliably knockdown individual genes in neurons and assess for specific cellular phenotypes. Therefore, this system is also useful for hypothesis-directed research projects, such as monitoring accumulation of dsRNA and reduced neuronal survival in the setting of HP1alpha knockdown (Zhang YJ, Ward ME, Petrucelli L (2019) Science). We have shared these CRISPRi i3Neuron cell lines with multiple labs from the intramural, extramural, and international community. Though we continue to collaborate with the Kampmann lab, we have now built infrastructure within the IRP to independently conduct CRISPRi screens. A consortium of investigators at NCATS (Ken Cheng), NICHD (Porter, Le Pichon, Bonifacino), and NINDS (Ward) currently meet monthly as an i3Neuron workgroup to coordinate projects and refine cellular and bioinformatic protocols related to CRISPRi screens.

鉴定轴突 RNA 颗粒运输的新机制：神经元是高度极化和区室化的细胞，具有复杂的树突网络和影响深远的轴突。为了有效且高效地响应远离体细胞的刺激和代谢需求，神经元严重依赖蛋白质的局部翻译。在轴突内，RNA 在多个位点进行局部翻译，包括生长锥、突触前和轴突内细胞器（如线粒体）。尽管我们知道轴突 RNA 运输需要微管和运动蛋白，但 RNA 从体细胞运输到神经元远端区域的精确机制尚不清楚。与线粒体和内体等膜结合货物不同，RNA 通常存在于称为 RNA 颗粒的无膜相分离核糖核蛋白复合物中。因此，无膜和膜结合货物使用的适配器机制可能不同。 我们与 Jennifer Lippincott Schwartz (HHMI) 和 Peter Hyslop (U. Cambridge) 合作，得出了一个令人惊讶的发现，即 RNA 颗粒通过搭移动溶酶体的便车在轴突中间接长距离运输（Liao Y, Lippincott-Schwartz J* & Ward ME*, 2019, Cell（*共同通讯作者））。为了识别连接溶酶体与 RNA 颗粒的潜在接头，我们在人类 iPSC 衍生的神经元中使用了 Lamp1-APEX 邻近标记蛋白质组策略。将我们的溶酶体 APEX 实验的命中结果与最近发表的 RNA 颗粒组成的 APEX 分析进行比较，发现膜联蛋白 A11 (ANXA11)（一种最近发现的 ALS 相关蛋白）是一种潜在的接头。与 Lucy Forrest (NINDS) 合作，ANXA11 的结构预测揭示了 C 端膜结合膜联蛋白结构域以及 N 端低复杂性 (LC) 结构域。体外实验表明，纯化的 ANXA11 可以与人工溶酶体相互作用。此外，纯化的 ANXA11 可以在体外发生相分离，这是由其 N 端 LC 结构域赋予的特性。活细胞显微镜实验表明，ANXA11 与细胞中的溶酶体和 RNA 颗粒相互作用，并在培养的神经元的轴突中共同运输。纯化的 ANXA11 足以介导纯化的应激颗粒与体外人工溶酶体的对接。我们与 Katie Drerup (NICHD) 合作，证明了斑马鱼胚胎体内发生 RNA 颗粒/溶酶体共转运。神经元中 ANXA11 的敲低会损害 RNA 颗粒的运输以及 RNA 向生长锥的递送。 ANXA11 中与 ALS 相关的突变改变了其生物物理特性（导致更多的凝胶状状态）并减少了其与溶酶体和 RNA 颗粒的相互作用。这些突变还干扰轴突 RNA 颗粒的运输以及 RNA 向生长锥的递送。 我们对内溶酶体依赖性轴突 RNA 转运的观察与其他组最近的观察结果一致。有趣的是，在丝状真菌的长距离运输过程中，RNA 颗粒会搭上内体的便车，这表明这种 RNA 运输模式在进化上是保守的。这些实验共同确定了溶酶体、RNA 颗粒和轴突运输之间的分子关系。我们的研究结果进一步表明，功能失调的 RNA 转运可能是 FTD/ALS 的一种聚合机制，可能会被不同类别的疾病相关蛋白的突变所破坏，这些蛋白会改变溶酶体、RNA 颗粒或转运蛋白的功能。 开发 iPSC 衍生神经元中的 CRISPRi 筛选平台：我们与 UCSF 的 Martin Kampmann 合作，开发了一种新方法，可在人类 iPSC 衍生神经元中进行大规模、前向遗传 CRISPR 抑制 (CRISPRi) 筛选 (Ruin T, Gachechiladze M, & Ludwig C, Ward ME* & Kampmann M*, 2019, Neuron （*共同通讯作者））。在这里，我们将高度可扩展的 i3Neuron 分化技术与 Kampmann 实验室开发的有效 CRISPRi 筛选方法相结合。在 CLYBL 安全港位点整合 CAG-dCas9-BFP-KRAB 转基因，可以通过慢病毒 sgRNA 递送持久敲低 iPSC 和 NGN2 分化神经元中的靶基因。然后，我们使用存活率、单细胞转录组学和形态学读数，对分化神经元进行了三项基于 CRISPRi 的筛选： a) 使用针对 2300 个激酶和可药物靶标的 sgRNA 慢病毒子文库对 iPSC 与神经元进行汇总生存筛选。通过量化老化 iPSC/神经元与起始细胞群中 sgRNA 的富集或消耗，我们确定了敲除可提高存活率（保护性）或降低存活率（必需）的基因。我们发现了一组对于 iPSC 和神经元生存至关重要的共享基因。有趣的是，我们还发现了与敲低相关的存活表型具有细胞类型特异性的基因。 b) 单细胞测序与 CRISPRi 敲低 (CROPseq) 筛选相结合，以探究我们的生存筛选中发现的基因敲低如何改变细胞转录状态。 CROPseq 分析揭示了对基因敲低的细胞类型特异性转录组反应。 c) 采用高内涵显微镜方法来表征我们的生存筛选中确定的单个基因的基因敲除如何改变神经元形态和生存。这些成像研究表明，在我们的初级合并生存筛选中鉴定出的绝大多数基因确实影响细胞生存，并且在许多情况下还影响神经突形态。 最后，在与 Len Petrucellis 实验室（梅奥诊所）的单独合作中，我们表明 CRISPRi i3Neurons 可用于可靠地敲低神经元中的单个基因并评估特定的细胞表型。因此，该系统对于假设导向的研究项目也很有用，例如在 HP1alpha 敲低的情况下监测 dsRNA 的积累和神经元存活率的降低（Zhang YJ, Ward ME, Petrucelli L (2019) Science）。 我们已与来自校内、校外和国际社会的多个实验室共享这些 CRISPRi i3Neuron 细胞系。尽管我们继续与坎普曼实验室合作，但我们现在已经在 IRP 内建立了基础设施来独立进行 CRISPRi 筛选。 NCATS（Ken Cheng）、NICHD（Porter、Le Pichon、Bonifacino）和 NINDS（Ward）的研究人员联盟目前作为 i3Neuron 工作组每月举行一次会议，以协调项目并完善与 CRISPRi 筛选相关的细胞和生物信息协议。