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.

项目总结 GWAS和外显子全序列分析鉴定SORL1编码的SorLA基因突变 分析与阿尔茨海默病(AD)风险增加有关。尽管Sorla在神经细胞中的作用 抑制淀粉样变性APP的加工和由此产生的β已经建立，索拉 与神经元相比，人小胶质细胞的表达高出约8倍，因此暗示小胶质细胞在 Sorla在AD发病机制中的作用到目前为止，SorLA在小胶质细胞中的功能还没有被描述。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 人H9胚胎干细胞中的(编码SorLA)基因座，随后分化为人类 小胶质样细胞(HMGL)。野生型和AD相关SORL1转录图谱的比较 (A528T，R744X)和TREM2(R47H)突变的hMGLS显示了致病的小胶质细胞基因特征，如 先前在AD小鼠模型和人类AD脑中所描述的诱导APOE的表达。我们的结果也是 发现培养的SORL1R744X和TREM2R47H hMGL在载脂蛋白依赖的β摄取方面存在缺陷 方式，以及受损的Aβ清除和斑块关联在小鼠异种脑移植中 微透析/组织学。这些结果为SorLA功能障碍提供了开创性的证据 致病的表达特征和损害小胶质细胞的功能。我们假设小胶质细胞功能障碍会 根据Sorla胞外区的区域特异性突变而有所不同，而且发病早和晚 SorLA突变可能在小胶质细胞失调和小胶质细胞/神经元相互作用方面表现出不同的作用。 使用我们的基因编辑/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)小组，包括每个小组中具有代表性的早期和晚期AD发病突变 在Sorla胞外区的功能结构域。我们将描述基因表达谱的特征 RNAseq/蛋白质组学分析野生型(WT)和SorLA突变体ESC来源的小胶质细胞(XhMGs)在体内，AS 以及小胶质细胞功能(Aβ摄取、细胞因子谱)在AD异种移植中的作用 老鼠的大脑。我们的结果表明，SorLA突变，如R744X和A528T，上调了APOE 可能触发某些方面的小胶质细胞功能，我们还将测试APOE和其他潜在的驱动因素 小胶质细胞功能障碍在SorLA突变型小胶质细胞下游致病行为中的作用 AD小鼠脑内异种移植。我们还将探索与各种Sorla相关的细胞机制 导致胚胎干细胞来源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 人小胶质细胞(XhMGs)SorLA突变如何与小鼠AD脑移植中的神经元相互作用 以及WT和SorLA突变型hMGLS/神经元共培养。这些结果将为我们提供机械性的 洞察各种突变如何引发小胶质细胞功能障碍，并潜在地描述 在AD发病机制中，小胶质细胞和神经元相关的SorLA通路受到影响，从而改变与年龄相关的发病。