Mechanisms of inherited neurodegenerative diseases

遗传性神经退行性疾病的机制

• 批准号: 10708629
• 负责人: Michael Ward
• 金额: $ 273.52万
• 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/10708629
• 关键词: Active Sites; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Autopsy; Axon; Axonal Transport; Base Excision Repairs; Binding; Biological Markers; Biology; Biophysics; Biotin; Brain; CRISPR interference; Cell Differentiation process; Cell Line; Cell Nucleus; Cell model; Cell physiology; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communities; Conceptions; Cytoplasmic Granules; DNA; DNA Single Strand Break; Data Set; Development; Disease; Distal; Doxycycline; Enhancers; Ensure; Event; Exons; Frontotemporal Dementia; Functional disorder; Funding; Future; Gene Expression; Genes; Genetic Screening; Genome; Genome engineering; Genomics; Genotype; Glutamates; Human; Image; Induced pluripotent stem cell derived neurons; Inherited; Institutes; International; Lead; Link; Location; Lysosomes; Manuscripts; Maps; Mediating; Methods; Mitotic; Modeling; Molecular; Molecular Biology; Monitor; Motor; Mutation; National Institute of Neurological Disorders and Stroke; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurosciences; Paper; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Plasma; Play; Process; Production; Proteins; Proteomics; Protocols documentation; Publishing; RNA; RNA Splicing; RNA Transport; RNA-Binding Proteins; Recurrence; Reporting; Repression; Reproducibility; Research; Research Personnel; Risk; Robotics; Rodent; Role; Seminal; Specimen; System; Techniques; Technology; Thymidine; Transcript; Transcriptional Activation; Translations; analog; base; cell type; chromatin remodeling; demethylation; disease phenotype; effective therapy; familial Alzheimer disease; forward genetics; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genome editing; genome wide association study; genome-wide; improved; induced pluripotent stem cell; multiple omics; mutant; next generation sequencing; novel; protein TDP-43; repaired; stem cells; synaptic function; ward

1) Disrupted RNA transport in ALS/FTD. In close collaboration with Jennifer Lippincott Schwartz (HHMI), our group made the surprising discovery that RNA granules are indirectly transported long distances in axons by hitchhiking on moving lysosomes (Liao et al, Cell, 2019), and that this process is disrupted by an ALS-associated mutant protein. Using proteomics, imaging, and biophysics, we showed that this hitchhiking is mediated by a newly-discovered ALS protein, ANXA11, which serves as a regulatable molecular tether between RNA granules and lysosomes during transport. ALS-associated mutations in ANXA11 block RNA transport and local translation of RNA in distal neuronal processes. These findings suggest that dysfunctional local translation may contribute to ALS, and that molecular convergences exist between lysosomal and RNA biology in ALS pathogenesis. We are currently studying why RNA granules hitchhike on lysosomes, rather than direct transport on motor proteins, in neurons. 2) Dysregulated RNA splicing in ALS/FTD. As part of a multi-lab collaboration between my group, Pietro Frattas team (UCL), and Len Petrucellis team (Mayo), we have used a combination of iPSC cellular models and human datasets to identify and characterize pathological splicing events in ALS/FTD that occur in the setting of TDP-43 mislocalization. TDP-43 normally functions as a splicing repressor. However, TDP-43 becomes depleted from the nucleus in most cases of ALS and FTD, and the resulting loss of splicing repression leads to the inclusion of cryptic exons in numerous transcripts. We used our CRISPRi i3Neuron platform to deplete TDP-43 from cortical neurons, discovering hundreds of new cryptic-exon containing transcripts in addition to well-described cryptic exons in genes such as STMN2. One novel cryptic exon was found in UNC13A, a gene previously implicated in ALS through GWAS studies. We found that the risk-associated SNPs were near the cryptic exon, and interfered with TDP-43 binding, thereby increasing the pathologic inclusion of cryptic exons in the setting of TDP-43 mislocalization. In addition, with the Fratta and Petrucelli labs, we showed that a classic cryptic exon in STMN2 can be detected in post-mortem brains from FTLD-TDP and FTD-MND patients, and is specific for patients with TDP-43 pathology (i.e. not present in those with FUS/MAPT pathology). This study suggests that CSF/plasma based identification of cryptic exon transcripts or protein products facilitate biomarkers of TDP-43 mislocalization in ALS/FTD. Our current studies focus on cell-specific cryptic exon formation, and whether cryptic exons can be used as biomarkers in patient specimens to detect and monitor TDP-43 dysfunction. 3) iPSC neuron models to study neurodegenerative diseases. I co-developed an improved method to differentiate large numbers of human iPSCs into neurons (Wang & Ward, Stem Cell Reports, 2017). Through single-copy integration of a doxycycline-inducible neurogenin 2 (iNGN2), we created a clonal iPSC line that enables simple, rapid, reproducible, and scalable production of mature glutamatergic neurons. We termed this cellular platform i3Neurons (inducible x integrated x isogenic). i3Neurons can be produced less expensively and faster than primary rodent cultures, are genomically stable, and can be readily manipulated with genome-editing technologies. We have shared this technology broadly with the neuroscience community and have sent our cell lines to over 100 national and international labs. I was a co-first author on the original paper describing the technique, conceived of and developed the inducible NGN2 iPSC line, and optimized the methods for scalable i3Neuron production (Fernandopulle et al, Curr Protoc Cell Biol, 2018; also available at protocols.io). In collaboration with Martin Kampmann's team at UCSF, we expanded upon our iPSC neuron technology to develop a new CRISPR-inhibition forward genetics screening platform. Scalable production of iPSC-derived neurons that co-express dCas9 allowed us to discover a host of new neuron survival related pathways (Tian et al, Neuron, 2019), and will enable future synthetic lethality screens aimed at uncovering neurodegeneration-related pathways. Current studies focus on leveraging new perturb-seq platforms to perform large scale screens of genotype:phenotype associations in disease-relevant iPSC derived cell types. Finally, I co-direct the largest-ever genome engineering project to date, the iPSC Neurodegenerative Disease Initiative (iNDI). iNDI is a multi-institute project funded by the NIA/NINDS that will genome-engineer over 100 isogenic iPSC lines harboring familial mutations implicated in neurodegenerative diseases, and then phenotype disease-relevant differentiated cells such as neurons using multi-omic robotic platforms (Ramos et al, Neuron, In Press). iPSC lines will be widely available to the research community and distributed by JAX, which started in August 2022 and will continue to expand as new lines are available. 4) Endogenous DNA break/repair at neuronal enhancers. In collaboration with Andre Nussenzweig's lab at the NCI, we recently discovered that neurons undergo high levels of constitutive endogenous DNA break/repair events. The Nussenzweig lab developed a new method to map endogenous DNA break/repair events genome-wide in post-mitotic cells, called SARseq through incorporation of a thymidine analog (EdU) followed by biotin enrichment and next generation sequencing. Our lab applied SARseq to our iPSC-derived i3Neuron system, and unexpectedly identified tens of thousands of recurrent DNA breaks throughout the genome. Further studies using Chip-seq, CRISPRi, and other molecular biology approaches identified the location of these DNA breaks (enhancers), the form of DNA break (single-stranded), and the mechanism of repair (base excision repair). We further showed that these ssDNA breaks occurred at genomic sites of active demethylation events, suggesting that such ssDNA breaks are critical for chromatin remodeling and control of gene expression in neurons. Current studies focus on how these DNA breaks may play role in neurodegenerative diseases, and links to formation of these breaks with activation of transcriptional machinery.

1) ALS/FTD 中 RNA 运输中断。在与 Jennifer Lippincott Schwartz (HHMI) 的密切合作中，我们的团队做出了令人惊讶的发现，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 等人，Cell，2019），并且该过程被 ALS 相关突变蛋白破坏。利用蛋白质组学、成像和生物物理学，我们证明这种搭便车是由新发现的 ALS 蛋白 ANXA11 介导的，该蛋白在运输过程中充当 RNA 颗粒和溶酶体之间的可调节分子系链。 ANXA11 中 ALS 相关突变会阻断远端神经元过程中 RNA 转运和 RNA 局部翻译。这些发现表明，功能失调的局部翻译可能导致 ALS，并且在 ALS 发病机制中溶酶体和 RNA 生物学之间存在分子趋同。我们目前正在研究为什么 RNA 颗粒在神经元中搭车在溶酶体上，而不是直接在运动蛋白上运输。 2) ALS/FTD 中 RNA 剪接失调。作为我的团队、Pietro Frattas 团队 (UCL) 和 Len Petrucellis 团队 (Mayo) 之间多实验室合作的一部分，我们结合使用 iPSC 细胞模型和人类数据集来识别和表征 TDP-43 错误定位背景下发生的 ALS/FTD 病理剪接事件。 TDP-43 通常充当剪接阻遏物。然而，在大多数 ALS 和 FTD 病例中，TDP-43 从细胞核中耗尽，由此产生的剪接抑制丧失导致在许多转录物中包含隐秘的外显子。我们使用 CRISPRi i3Neuron 平台从皮质神经元中去除 TDP-43，除了 STMN2 等基因中已充分描述的隐秘外显子之外，还发现了数百个新的包含隐秘外显子的转录本。在 UNC13A 中发现了一个新的神秘外显子，该基因先前通过 GWAS 研究发现与 ALS 相关。我们发现风险相关的 SNP 靠近隐秘外显子，并干扰 TDP-43 结合，从而增加了 TDP-43 错误定位背景下隐秘外显子的病理包含。此外，我们与 Fratta 和 Petrucelli 实验室合作，证明 STMN2 中的一个经典隐性外显子可以在 FTLD-TDP 和 FTD-MND 患者的死后大脑中检测到，并且对于 TDP-43 病理的患者具有特异性（即在 FUS/MAPT 病理的患者中不存在）。这项研究表明，基于 CSF/血浆的隐秘外显子转录本或蛋白质产物的鉴定有助于 ALS/FTD 中 TDP-43 错误定位的生物标志物。我们目前的研究重点是细胞特异性隐性外显子的形成，以及隐性外显子是否可以用作患者标本中的生物标志物来检测和监测 TDP-43 功能障碍。 3）用于研究神经退行性疾病的 iPSC 神经元模型。我与他人共同开发了一种改进方法，可将大量人类 iPSC 分化为神经元（Wang & Ward，Stem Cell Reports，2017）。通过多西环素诱导型神经原素 2 (iNGN2) 的单拷贝整合，我们创建了克隆 iPSC 系，能够简单、快速、可重复且可扩展地生产成熟的谷氨酸能神经元。我们将这个细胞平台称为 i3Neurons（诱导型 x 整合型 x 同基因型）。 i3Neurons 的生产成本比初级啮齿动物培养物更便宜、更快，基因组稳定，并且可以通过基因组编辑技术轻松操作。我们与神经科学界广泛分享了这项技术，并将我们的细胞系发送到 100 多个国家和国际实验室。我是描述该技术的原始论文的共同第一作者，构思并开发了可诱导的 NGN2 iPSC 系，并优化了可扩展 i3Neuron 生产的方法（Fernandopulle 等人，Curr Protoc Cell Biol，2018；也可在protocols.io 上获取）。 我们与 UCSF 的 Martin Kampmann 团队合作，扩展了 iPSC 神经元技术，开发了一种新的 CRISPR 抑制正向遗传学筛选平台。共表达 dCas9 的 iPSC 衍生神经元的可扩展生产使我们能够发现许多新的神经元生存相关途径（Tian 等人，Neuron，2019），并且将使未来旨在揭示神经变性相关途径的合成致死性筛选成为可能。目前的研究重点是利用新的 perturb-seq 平台对疾病相关 iPSC 衍生细胞类型中的基因型：表型关联进行大规模筛选。 最后，我共同领导了迄今为止最大的基因组工程项目，即 iPSC 神经退行性疾病计划 (iNDI)。 iNDI 是一个由 NIA/NINDS 资助的多机构项目，将对 100 多个含有与神经退行性疾病有关的家族突变的同基因 iPSC 系进行基因组工程，然后使用多组学机器人平台对与疾病相关的分化细胞（如神经元）进行表型分析（Ramos 等人，Neuron，In Press）。 iPSC 细胞系将​​广泛供研究界使用，并由 JAX 分发，该计划于 2022 年 8 月启动，并将随着新细胞系的推出而继续扩展。 4) 神经元增强子处的内源性 DNA 断裂/修复。我们与 NCI 的 Andre Nussenzweig 实验室合作，最近发现神经元经历高水平的组成性内源 DNA 断裂/修复事件。 Nussenzweig 实验室开发了一种新方法，通过掺入胸苷类似物 (EdU)，然后进行生物素富集和下一代测序，绘制有丝分裂后细胞全基因组内源性 DNA 断裂/修复事件图谱，称为 SARseq。我们的实验室将 SARseq 应用于 iPSC 衍生的 i3Neuron 系统，并意外地在整个基因组中发现了数以万计的重复 DNA 断裂。使用 Chip-seq、CRISPRi 和其他分子生物学方法进行的进一步研究确定了这些 DNA 断裂（增强子）的位置、DNA 断裂的形式（单链）以及修复机制（碱基切除修复）。我们进一步表明，这些 ssDNA 断裂发生在活跃去甲基化事件的基因组位点，表明此类 ssDNA 断裂对于神经元中染色质重塑和基因表达的控制至关重要。目前的研究重点是这些 DNA 断裂如何在神经退行性疾病中发挥作用，以及这些断裂的形成与转录机制的激活之间的联系。