Establishment of a causal link between AD and L1 retrotransposons

AD 和 L1 反转录转座子之间因果关系的建立

• 批准号: 10519029
• 负责人: Alysson R. Muotri
• 金额: $ 78.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10519029
• 关键词: Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Alzheimer' s disease patient; Astrocytes; Autopsy; Biological Models; Brain; CRISPR/Cas technology; Cell Aging; Cell Death; Cells; Chronic; Collection; Cytoplasm; DNA; Data; Disease; Disease Progression; Elements; Evolution; Gene Expression; Genome; Genomics; Goals; Human; Immune system; Immunization; Individual; Inflammation; Inflammatory; Interferon Activation; Interferons; L1 Elements; Lead; Link; Measures; Microglia; Modeling; Molecular; Neuroglia; Neurons; Nucleosides; Organoids; Outcome Study; Pathogenesis; Pathway interactions; Phenotype; Protective Agents; Protocols documentation; RNA-Directed DNA Polymerase; Retrotransposon; Reverse Transcriptase Inhibitors; Series; Single-Stranded DNA; Site; Sterility; Synapses; Syndrome; Testing; Therapeutic; age effect; base; brain cell; cell type; cytokine; design; disorder control; experimental study; human disease; human model; human tissue; improved; induced pluripotent stem cell; insight; mutant; neural network; neurotoxic; neurotoxicity; novel; overexpression; premature; response; synaptogenesis; tau aggregation; tau phosphorylation; transcriptomics; whole genome

Abstract Our overall goal is to determine a potential causal link between L1, an active retrotransposon element, in human brain cells and Alzheimer's Disease (AD). L1s are involved in numerous human diseases with different molecular and cellular mechanisms. L1 expression was also found to be upregulated in AD postmortem human tissues, leading to the speculation that L1s could contribute, at least partially, to the mechanisms leading to AD in humans. However, despite all these correlations, there is no experimental-based causal link between L1s and AD so far. Here, we hypothesized that L1 expression in AD-derived brain cells can contribute to AD pathology, using both cell-autonomous and non-cell-autonomous mechanisms. Accumulation of L1-derived ssDNA molecules in the cytoplasm of glial cells might be responsible for a chronic low-level stimulation of the immune system, aka “sterile” inflammation, and can aggravate molecular and cellular phenotypes in neurons derived from AD patients. Moreover, a cell autonomous mechanism, triggered by L1 ssDNA accumulation in neurons could accelerate and exacerbate molecular and cellular phenotypes related to AD pathogenesis. Thus, the combinatory impact of L1 expression in the different cell types could causally contribute to AD. To experimentally test this hypothesis, we will use induced pluripotent stem cells (iPSC) from controls and AD to overexpress L1 ssDNA molecules and analyze their impact on different neuronal types individually or in combination using a brain organoid model system. The use of iPSC-derived cells and organoid model is perfectly suited as we can isolate the aging effect and capture the entire genome of AD individuals in relevant cell types. We have designed the following specific aims to test our hypothesis: Aim 1: Determine the impact of L1 retrotransposons in AD-derived astrocytes. We will measure alterations in transcriptomics, cytokines/interferon release levels and interferon-stimulated gene (ISGs) expression in AD/isogenic-derived astrocytes upon L1 ssDNA overexpression. We will also evaluate neurotoxicity using astrocyte conditioned media from AD-derived astrocytes overexpressing L1 ssDNA. Aim 2: Determine the impact of L1 retrotransposons in AD-derived cortical neurons. We will measure Tau aggregation and phosphorylation, and synaptic loss, all early hallmarks of the AD progression. Aim 3: To model AD progression with a brain organoid model. We will use our optimized protocol to generate brain cortical organoids (BCO) infused with human microglia from AD- and control individuals. BCO with L1 ssDNA but treated with nucleoside reverse transcriptase inhibitors (NRTI) as a protective agent, will be compared. Our proposal aims to demonstrate an eventual causal contribution of L1-derived ssDNA to molecular, cellular and network phenotypes in brain cells and organoids derived from AD individuals. Our data will reveal unexplored pathways with immediate therapeutic relevance that could lead to transformative treatments for AD and other aging-related syndromes.

抽象的 我们的总体目标是确定 L1（一种活跃的逆转录转座子元件）与 人类脑细胞和阿尔茨海默病 (AD) 与许多不同的人类疾病有关。 分子和细胞机制也被发现在 AD 死后人类中表达上调。 组织，导致人们推测 L1 可能至少部分地促成 AD 的机制 然而，尽管存在所有这些相关性，L1 之间不存在基于实验的因果关系。 到目前为止，我们发现 AD 来源的脑细胞中的 L1 表达可能有助于 AD。 病理学，使用细胞自主和非细胞自主机制的 L1 衍生的积累。 神经胶质细胞细胞质中的单链DNA分子可能对神经胶质细胞的慢性低水平刺激负责。 免疫系统，又名“无菌”炎症，可以加剧神经元的分子和细胞表型 此外，由 L1 ssDNA 积累触发的细胞自主机制。 神经元可以加速和加剧与 AD 发病机制相关的分子和细胞表型。 因此，不同细胞类型中 L1 表达的综合影响可能会导致 AD。 通过实验检验这一假设，我们将使用来自对照和 AD 的诱导多能干细胞 (iPSC) 过表达 L1 ssDNA 分子并单独或分批分析它们对不同神经类型的影响 使用脑类器官模型系统的组合 iPSC 衍生细胞和类器官模型的使用是 非常适合，因为我们可以分离衰老效应并捕获相关 AD 个体的整个基因组 我们设计了以下具体目标来检验我们的假设： 目标 1：确定影响。 我们将测量 AD 衍生星形胶质细胞中 L1 逆转录转座子的转录组学变化， AD/同基因衍生细胞因子/干扰素释放水平和干扰素刺激基因 (ISG) 表达 我们还将使用星形胶质细胞条件化来评估 L1 ssDNA 过表达的神经毒性。 来自过度表达 L1 ssDNA 的 AD 衍生星形胶质细胞的培养基 目标 2：确定 L1 的影响。 AD 来源的皮质神经元中的逆转录转座子我们将测量 Tau 聚集和磷酸化， 目标 3：用大脑模拟 AD 进展。 我们将使用我们的优化方案来生成注入了脑皮质类器官（BCO）。 来自 AD 和对照个体的人类小胶质细胞，具有 L1 ssDNA，但经过核苷逆转处理。 我们的建议旨在证明转录酶抑制剂（NRTI）作为保护剂的效果。 L1 衍生的 ssDNA 对脑细胞分子、细胞和网络表型的最终因果贡献 我们的数据将直接揭示未探索的途径。 治疗相关性可能会导致 AD 和其他衰老相关综合征的变革性治疗。