Establishment of a causal link between AD and L1 retrotransposons

摘要 我们的总体目标是确定L1（一种活跃的反转录转座子元件）与 人类脑细胞和阿尔茨海默病（AD）。L1与许多人类疾病有关， 分子和细胞机制。L1表达在AD尸检中也被发现上调， 组织，导致推测L1可能有助于，至少部分地，导致AD的机制 在人类身上。然而，尽管有这些相关性，L1之间并没有基于实验的因果联系 AD至今在这里，我们假设AD衍生脑细胞中的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。L1衍生的蓄积 胶质细胞胞质中的ssDNA分子可能是导致慢性低水平刺激胶质细胞的原因。 免疫系统，又名“无菌”炎症，并可加重神经元中的分子和细胞表型 来自AD患者。此外，L1 ssDNA积累触发的细胞自主机制， 神经元可加速和加重与AD发病机制相关的分子和细胞表型。 因此，L1在不同细胞类型中表达的组合影响可能导致AD。到 为了通过实验验证这一假设，我们将使用来自对照组和AD的诱导多能干细胞（iPSC）， 过表达L1 ssDNA分子，并分析它们对不同神经元类型的影响， 使用脑类器官模型系统的组合。iPSC衍生的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， 细胞类型。我们设计了以下具体目标来验证我们的假设：目标1：确定影响 L1反转录转座子在AD衍生的星形胶质细胞中的表达。我们将测量转录组学的变化， 细胞因子/干扰素释放水平和干扰素刺激的基因（ISG）表达在AD/等基因源性 星形胶质细胞L1 ssDNA过表达。我们还将评估神经毒性使用星形胶质细胞条件 来自过表达L1 ssDNA的AD衍生的星形胶质细胞的培养基。目标2：确定L1的影响 逆转录转座子在AD衍生的皮质神经元中的表达。我们将测量Tau的聚集和磷酸化， 和突触丢失，这些都是AD进展的早期标志目的3：用大脑模拟AD进展 类器官模型我们将使用我们的优化方案来生成注入有 来自AD和对照个体的人小胶质细胞。使用L1 ssDNA但经核苷逆转治疗的BCO 转录酶抑制剂（NRTI）作为保护剂，将进行比较。我们的提案旨在展示 L1衍生的ssDNA对脑细胞中分子、细胞和网络表型的最终因果作用 和源自AD个体的类器官。我们的数据将揭示未探索的途径， 这可能导致AD和其他衰老相关综合征的变革性治疗。

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.