Establishment of a causal link between AD and L1 retrotransposons

摘要 我们的总体目标是确定L1，一种活性的反转录转座子元件，在 人类脑细胞与阿尔茨海默病(AD)。L1与许多人类疾病有关，具有不同的 分子和细胞机制。在死后的AD患者中也发现L1的表达上调 组织，导致推测L1可能至少部分地参与了导致AD的机制 在人类身上。然而，尽管有所有这些相关性，L1之间并没有基于实验的因果联系 到目前为止还是AD。在这里，我们假设阿尔茨海默病患者脑细胞中L1的表达可能与AD的发病有关 病理学，使用细胞自主和非细胞自主机制。L1来源的累积 胶质细胞胞浆中的单链DNA分子可能与慢性低水平刺激神经胶质细胞有关 免疫系统，也就是“无菌”炎症，会加重神经元的分子和细胞表型。 来源于阿尔茨海默病患者。此外，一种细胞自主机制，由L1单链DNA在 神经元可以加速和加重与AD发病相关的分子和细胞表型。 因此，L1在不同细胞类型中表达的联合影响可能是导致AD的原因。至 在实验上验证这一假设，我们将使用来自对照组和AD的诱导多能干细胞(IPSC)来 L1单链DNA分子的过表达及其对不同神经元类型的影响 使用脑器官模型系统进行组合。使用IPSC衍生的细胞和器官模型是 非常适合，因为我们可以分离衰老效应并捕获相关的AD患者的整个基因组 单元类型。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 AD来源的星形胶质细胞中L1反转录转座子的表达。我们将测量转录组的变化， 阿尔茨海默病患者细胞因子/干扰素释放水平与干扰素刺激基因的表达 星形胶质细胞L1单链DNA过表达。我们还将使用星形胶质细胞条件化来评估神经毒性 AD来源的星形胶质细胞培养上清液过表达L1单链DNA。目标2：确定第一语言的影响 阿尔茨海默病患者皮层神经元中的反转录转座子。我们将测量Tau聚集和磷酸化， 以及突触丧失，这些都是AD进展的早期特征。目标3：用大脑模拟AD进展 器官模型。我们将使用我们的优化方案来产生大脑皮质器官(BCO)， 人的小胶质细胞来自AD-和对照个体。BCO与L1单链DNA结合，但核苷反转处理 转录酶抑制剂(NRTI)作为保护剂，将进行比较。我们的建议旨在展示一个 L1来源的单链DNA对脑细胞分子、细胞和网络表型的最终因果贡献 和来源于AD患者的有机化合物。我们的数据将揭示未探索的途径 治疗相关性，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.