Deciphering the non-canonical function of the histone methyltransferase G9a in the etiology of AD

破译组蛋白甲基转移酶 G9a 在 AD 病因学中的非典型功能

• 批准号: 10491670
• 负责人: XIAN CHEN
• 金额: $ 19.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10491670
• 关键词: Alzheimer' s Disease; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease related dementia; Amyloid beta-Protein; Automobile Driving; Binding; Cancer Cell Growth; Chromatin; Chronic; Clinical Trials; Cognitive; Cytoplasm; Cytosol; Data; Early Intervention; Epigenetic Process; Etiology; Event; G9a histone methyltransferase; Gene Silencing; Genes; Genetic Translation; Hippocampus (Brain); Histone H3; Human; Inflammation; Inflammatory; Label; Lysine; MMP9 gene; Mediating; Messenger RNA; Methylation; Microglia; Modification; Molecular; Mus; Oncogenes; Onset of illness; Pathogenesis; Pathologic; Pathway interactions; Patients; Peptide Initiation Factors; Peptides; Pharmaceutical Preparations; Prefrontal Cortex; Prognostic Marker; Protein Overexpression; Protein Secretion; Proteins; Proteomics; Regulation; Regulatory Pathway; Role; Sampling; Senile Plaques; Specificity; Synapses; Testing; Therapeutic Intervention; Tissues; Translational Regulation; Translations; Virulence Factors; Work; abeta accumulation; base; chemoproteomics; cognitive function; diagnostic biomarker; extracellular; genetic regulatory protein; inhibitor; innovation; macrophage; monocyte; neuroinflammation; neurotoxic; neurotoxicity; new therapeutic target; novel; novel diagnostics; precision medicine; promoter; synaptic function; tRNA Methyltransferases; targeted treatment; tau Proteins

Abstract A prominent pathological feature of Alzheimer’s disease (AD) is extracellular accumulation of amyloid-beta (Aβ) peptides in neuritic plaques that activate microglia/macrophages. Using a quantitative proteomic approach we identified numerous proteins that showed increased secretion from Aβ-stimulated human monocyte-derived macrophages/microglia (hMDMs), including our newly characterized neurotoxic protein MMP9 that recapitulates neuritic tau beading for AD pathology. However, little is known about the AD-causing mechanisms underlying the translation and secretion of these neurotoxic proteins in Aβ-activated hMDMs. Clinicopathologic data show that AD onset is directly associated with an increased level of histone H3 lysine 9 dimethylation (H3K9me2) in prefrontal cortex tissue of AD patients. Consistent with this fact, enzymatic inhibition of the histone methyltransferases G9a that catalyze the increased H3K9me2 rescued synaptic and cognitive functions in AD mice, implicating constitutively active G9a and G9a-associated pathways in AD etiology. Our chemoproteomic approach with a biotinylated version of the same G9a inhibitor captured and identified G9a interactions with the N6-methyladenosine (m6A) RNA methylase METTL3 and other translation regulatory proteins in both Aβ-stimulated hMDMs and the hippocampus of AD mice. Considering that METTL3 promoted oncogene translation for cancer cell growth, our chemoproteomic discovery from Aβ-stimulated hMDMs indicated that, in addition to its canonical function in transcriptional silencing of ‘anti-AD’ genes, G9a activates translation of certain neurotoxic genes. Consequently, the objective of this project is to characterize this new translation regulatory function of G9a in AD etiology. We have found that, in the hMDMs with prolonged Aβ stimulation, (1) G9a showed a higher enzymatic activity and interacted with METTL3; (2) mRNAs of AD-related neurotoxic proteins were m6A-modified by METTL3; (3) depletion of G9a or METTL3 led to similarly reduced overexpression of these proteins, suggesting that G9a and METTL3 both work in the same translation regulatory pathways; (4) METTL3 is a non-histone substrate of G9a, and elimination of methylated lysines decreased METTL3 binding to translation initiation factor eIF3 that is otherwise critical for METTL3-mediated, cap-independent translation. We propose two Aims to test the central hypothesis that, via Aβ-induced interaction with METTL3, constitutively active G9a activates translation of certain neurotoxic inflammatory proteins whose secretion promotes AD. We will (1) Determine the molecular mechanism by which constitutively active G9a and METTL3 cooperate to activate the translation of Aβ-induced neurotoxic proteins, and (2) Determine how constitutively active G9a promotes METTL3-mediated translation of Aβ-induced neurotoxic proteins, which, in turn, contributes to AD pathogenesis. We will uncover this new non-canonical function of G9a in Aβ-induced, METTL3-mediated neurotoxic inflammation, a likely early-stage pathological mechanism that underlies AD etiology, for early therapeutic intervention of AD.

抽象的 阿尔茨海默氏病（AD）的重要病理特征是淀粉样蛋白β的细胞外积累 （Aβ）在激活小胶质细胞/巨噬细胞的神经质斑块中的肽。使用定量蛋白质组学方法 我们确定了许多蛋白质，这些蛋白质显示出对Aβ刺激的人单核细胞衍生的分泌增加 巨噬细胞/小胶质细胞（HMDMS），包括我们新表征的神经毒性蛋白MMP9 概括为AD病理学的神经质Tau串珠。但是，对引起广告的了解知之甚少 这些神经毒性蛋白在Aβ激活的HMDM中的翻译和分泌的机制。 临床病理数据表明，AD发作直接与组蛋白H3赖氨酸的水平升高直接相关 AD患者的前额叶皮层组织中的二甲基化（H3K9ME2）。与这个事实一致，酶促 抑制组蛋白甲基转移酶G9A，催化增加的H3K9me2拯救了突触和 AD小鼠的认知功能，暗示着始终活跃的G9a和与G9a相关的途径 病因。我们的化学蛋白质组学方法采用捕获的同一G9A抑制剂的生物素化版本，并 鉴定出与N6-甲基拉丹代氨酸（M6A）RNA甲基酶METTL3和其他翻译的G9A相互作用 Aβ刺激的HMDM和AD小鼠海马的调节蛋白。考虑到该mettl3 促进了癌细胞生长的癌基因翻译，我们从Aβ刺激的化学蛋白质组学发现 HMDMS表明，除了其在“抗AD”的转录沉默中的规范功能外 基因，G9a激活了某些神经毒性基因的翻译。因此，该项目的目的是 为了表征G9A在AD病因中的新翻译调节功能。我们发现，在 延长Aβ刺激的HMDMS（1）G9a显示出较高的酶促活性，并与 mettl3; （2）通过METTL3对AD相关神经毒性蛋白的mRNA进行M6A修饰； （3）G9A或 METTL3导致这些蛋白质的过度表达类似，这表明G9A和METTL3都 在相同的翻译调节途径中工作； （4）METTL3是G9A的非histone基板，并且 消除甲基化歌词降低了Mettl3与翻译起始因子EIF3的结合，即 否则对于Mettl3介导的，独立的翻译至关重要。我们提出了两个目标，以测试中央 假设通过Aβ诱导的Mettl3相互作用，组成性活性G9A激活了翻译 分泌促进AD的某些神经毒性炎症蛋白。我们将（1）确定 组成性活跃的G9A和METTL3合作以激活的翻译的分子机制 Aβ诱导的神经毒性蛋白，（2）确定组成性活性G9A如何促进Mettl3介导 Aβ诱导的神经毒性蛋白的翻译反过来又导致了AD发病机理。我们将发现 G9a在Aβ诱导的，Mettl3介导的神经毒性感染中的新型非典型功能 AD病因的基础的早期病理机制，用于AD的早期治疗干预。