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.

项目摘要 通过GWAS和整个外显子组测序确定的Sorla中的突变（由SORL1编码） 分析与阿尔茨海默氏病（AD）风险增加有关。虽然Sorla在 已经建立了抑制淀粉样蛋白生成的应用程序处理和随之而来的Aβ产生，索拉 与神经元相比，人类小胶质细胞的表达高约8倍，因此隐含了小胶质细胞的作用 AD发病机理中的索拉。到目前为止，尚未描述索拉在小胶质细胞中的功能作用。这里， 我们使用CRISPR/CAS9编辑方法将与AD相关的A528T和R744X突变集成到SORL1中 （编码索拉）人类H9胚胎干细胞中的基因座，随后被分化为人类 小胶质细胞（HMGL）细胞。比较野生型和与AD相关的SORL1之间的转录组曲线 （A528T，R744X）和TREM2（R47H）突变体HMGLS揭示了致病的小胶质细胞基因特征，例如 先前在AD小鼠模型和人类AD大脑中描述的诱导APOE表达。我们的结果也是如此 证明培养的Sorl1R744X和Trem2R47H HMGLS在APOE依赖性的Aβ摄取中特征缺陷 方式，以及小鼠脑部异种移植中的Aβ清除率和斑块关联受损 微透析/组织学。这些结果提供了先驱证据，表明索拉功能障碍可以进行会议 致病性表达特征和损害小胶质细胞功能。我们假设小胶质功能障碍将 根据索拉外细胞外区域的域特异性突变而变化，早期和晚期发作 Sorla突变可能显示出对小胶质细胞失调和小胶质细胞/神经元相互作用的不同影响。 使用我们的基因编辑/人类小胶质模型和分析平台，我们将扩展我们的索拉 突变的胚胎干细胞（ESC）面板，包括每个内部的早期和晚期发作突变 索拉细胞外区域的功能域。我们将表征基因表达曲线 wildtype（WT）和Sorla突变体Esc衍生的小胶质细胞（“ XHMGS”）在RNASEQ/蛋白质组学分析中为 以及在AD中XHMGS的小胶质细胞功能（Aβ摄取，细胞因子曲线）的功能方面 小鼠大脑。由于我们的结果表明，诸如R744X和A528T之类的索拉突变上调了ApoE 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在索拉突变体小胶质细胞中院长介导下游的致病行为 AD小鼠大脑中的异种移植植物。我们还将探索与各种索拉相关的细胞机制 ESC衍生的小胶质细胞和神经元中驱动细胞功能障碍的突变；为此，我们将调查 人类小胶质细胞（XHMG）中的索拉突变如何与小鼠AD大脑异种移植植物中的神经元相互作用， 以及共同培养中的WT和Sorla突变体HMGLS/神经元。这些结果将为我们提供机械 深入了解各种突变如何触发小胶质细胞功能障碍，并有可能描述 小胶质细胞和神经元相关的索拉途径受到影响，以改变AD发病机理中与年龄相关的发作。