Elucidating a microgliaassociated role for SORLA in modulating AD pathogenesis

阐明 SORLA 在调节 AD 发病机制中的小胶质细胞相关作用

• 批准号: 10455261
• 负责人: Timothy Yikai Huang
• 金额: $ 281.85万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10455261
• 关键词: Abeta clearance; Affect; Alleles; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Alzheimer' s disease risk; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Behavior; Biological Assay; Brain; CRISPR/Cas technology; Cell Line; Cells; Coculture Techniques; Defect; Disease; ES Cell Line; Early Onset Alzheimer Disease; Epigenetic Process; Functional disorder; Gene Expression; Gene Expression Profile; Generations; Genes; Glial Differentiation; Gliosis; Hematopoietic stem cells; Histology; Homeostasis; Human; Impairment; In Vitro; Label; Late Onset Alzheimer Disease; Link; Location; Mediating; Methods; Microdialysis; Microglia; Modeling; Mus; Mutation; Nature; Neurons; Newborn Infant; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Peptides; Play; Proteomics; Recombinants; Role; Senile Plaques; Synapses; System; TREM2 gene; Testing; Up-Regulation; Variant; Xenograft Model; Xenograft procedure; age related; behavior in vitro; cytokine; embryonic stem cell; exome sequencing; extracellular; gain of function; genetic signature; genome wide association study; human embryonic stem cell; human embryonic stem cell line; in vivo; insight; mouse model; mutant; nano-string; neglect; neuroinflammation; proteotoxicity; pup; receptor; risk variant; sortilin; trafficking; transcriptome sequencing; transcriptomics; uptake

PROJECT SUMMARY Mutations in SORLA (encoded by SORL1) identified through GWAS and whole exome sequencing analysis have been linked to increased Alzheimer’s disease (AD) risk. Although a neuronal role for SORLA in suppressing amyloidogenic APP processing and consequent Aβ generation has been established, SORLA expression is ~8-fold higher in human microglia compared to neurons, thus implicating a microglial role for SORLA in AD pathogenesis. So far, functional roles for SORLA in microglia have not yet been described. Here, we used CRISPR/Cas9 editing methods to integrate AD-associated A528T and R744X mutations into the SORL1 (encoding SORLA) locus in human H9 embryonic stem cells, which were subsequently differentiated into human microglia-like (hMGL) cells. Comparing transcriptomic profiles between wildtype and AD-associated SORL1 (A528T, R744X) and TREM2 (R47H) mutant hMGLs reveals pathogenic microglia gene signatures such as induced APOE expression previously described in AD mouse models and human AD brain. Our results also show that cultured SORL1R744X and TREM2R47H hMGLs feature defects in Aβ uptake in an APOE-dependent manner, along with impaired Aβ clearance and plaque association in mouse brain xenotransplants by microdialysis/histology. These results provide pioneering evidence that SORLA dysfunction can confer pathogenic expression signatures and impair microglial function. We hypothesize that microglial dysfunction will vary according to domain-specific mutations in the SORLA extracellular region, and that early and late onset SORLA mutations may show differential effects on microglia dysregulation and microglia/neuron interaction. Using our gene editing/human microglial modeling and analysis platform, we will expand our SORLA mutant embryonic stem cell (ESC) panel to include representative early and late AD onset mutations within each of the functional domains in the SORLA extracellular region. We will characterize gene expression profiles of wildtype (WT) and SORLA mutant ESC-derived microglia (“xhMGs”) in vivo by RNAseq/proteomic analysis, as well as functional aspects of microglial function (Aβ uptake, cytokine profiles) in xhMGs xenotransplanted in AD mouse brain. As our results indicate that SORLA mutations such as R744X and A528T upregulate APOE which may trigger certain aspects of microglia function, we will also test whether APOE and other potential drivers of microglia dysfunction epistatically mediate downstream pathogenic behavior in SORLA mutant microglia xenotransplants in AD mouse brain. We will also explore cellular mechanisms associated with various SORLA mutations that drive cellular dysfunction in ESC-derived microglia and neurons; to this end, we will investigate how SORLA mutations in human microglia (xhMGs) interact with neurons in mouse AD brain xenotransplants, as well as WT and SORLA mutant hMGLs/neurons in co-culture. These results will provide us with mechanistic insight into how various mutations can trigger microglia dysfunction, and potentially describe how aspects of microglial and neuronal-related SORLA pathways are affected to alter age-related onset in AD pathogenesis.

项目概要 通过 GWAS 和全外显子组测序鉴定出 SORLA 突变（由 SORL1 编码） 分析表明，与阿尔茨海默病（AD）风险增加有关。尽管 SORLA 的神经元作用 抑制淀粉样蛋白 APP 加工和随后的 Aβ 生成已被证实，SORLA 与神经元相比，人类小胶质细胞中的表达量高约 8 倍，因此暗示小胶质细胞在 AD 发病机制中的 SORLA。迄今为止，SORLA 在小胶质细胞中的功能作用尚未被描述。这里， 我们使用 CRISPR/Cas9 编辑方法将 AD 相关的 A528T 和 R744X 突变整合到 SORL1 中 （编码 SORLA）人类 H9 胚胎干细胞中的位点，随后分化为人类 小胶质细胞样 (hMGL) 细胞。比较野生型和 AD 相关 SORL1 之间的转录组谱 (A528T、R744X) 和 TREM2 (R47H) 突变 hMGL 揭示了致病性小胶质细胞基因特征，例如 先前在 AD 小鼠模型和人类 AD 大脑中描述过诱导 APOE 表达。我们的结果也 表明培养的 SORL1R744X 和 TREM2R47H hMGL 在 APOE 依赖性中具有 Aβ 摄取缺陷 方式，以及小鼠脑异种移植物中 Aβ 清除和斑块关联受损 微透析/组织学。这些结果提供了开创性的证据，证明 SORLA 功能障碍可以导致 致病性表达特征并损害小胶质细胞功能。我们假设小胶质细胞功能障碍会 根据 SORLA 胞外区的域特异性突变而变化，并且早发和晚发 SORLA 突变可能对小胶质细胞失调和小胶质细胞/神经元相互作用表现出不同的影响。 利用我们的基因编辑/人类小胶质细胞建模和分析平台，我们将扩展我们的 SORLA 突变胚胎干细胞 (ESC) 组包括每个组中具有代表性的早期和晚期 AD 发病突变 SORLA 胞外区的功能域。我们将表征基因表达谱 通过 RNAseq/蛋白质组分析对野生型 (WT) 和 SORLA 突变型 ESC 衍生的小胶质细胞 (“xhMGs”) 进行体内分析，如 AD 异种移植的 xhMG 中小胶质细胞功能（Aβ 摄取、细胞因子谱）的功能方面 老鼠的大脑。我们的结果表明，R744X 和 A528T 等 SORLA 突变会上调 APOE，从而 可能会触发小胶质细胞功能的某些方面，我们还将测试 APOE 和其他潜在驱动因素是否 小胶质细胞功能障碍上位介导 SORLA 突变小胶质细胞的下游致病行为 AD小鼠大脑的异种移植。我们还将探索与各种 SORLA 相关的细胞机制 导致 ESC 衍生的小胶质细胞和神经元细胞功能障碍的突变；为此，我们将调查 人类小胶质细胞 (xhMG) 中的 SORLA 突变如何与小鼠 AD 大脑异种移植物中的神经元相互作用， 以及共培养中的 WT 和 SORLA 突变型 hMGL/神经元。这些结果将为我们提供机制 深入了解各种突变如何引发小胶质细胞功能障碍，并可能描述这些突变的各个方面 小胶质细胞和神经元相关的 SORLA 通路受到影响，从而改变 AD 发病机制中与年龄相关的发病。