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.

摘要 阿尔茨海默病（AD）的一个突出病理特征是β淀粉样蛋白的细胞外积累 神经炎斑块中激活小胶质细胞/巨噬细胞的Aβ肽。使用定量蛋白质组学方法 我们鉴定了许多蛋白质，这些蛋白质显示Aβ刺激的人单核细胞来源的 巨噬细胞/小胶质细胞（hMDM），包括我们新鉴定的神经毒性蛋白MMP 9， 概括了AD病理学的神经炎性tau串珠。然而，人们对导致AD的原因知之甚少。 这些神经毒性蛋白在Aβ激活的hMDM中的翻译和分泌机制。 临床病理学数据显示，AD发病与组蛋白H3赖氨酸9水平升高直接相关。 二甲基化（H3 K9 me 2）。与这一事实相一致，酶 抑制组蛋白甲基转移酶G9 a催化增加的H3 K9 me 2拯救突触， AD小鼠的认知功能，暗示AD中的组成性活性G9 a和G9 a相关通路 病因学我们的化学蛋白质组学方法使用生物素化的相同G9 a抑制剂捕获， 鉴定了G9 a与N6-甲基腺苷（m6 A）RNA甲基化酶L3的相互作用和其他翻译 Aβ刺激的hMDM和AD小鼠海马中的调节蛋白。考虑到L3 促进癌基因翻译促进癌细胞生长，我们从Aβ刺激的 hMDMs表明，除了其在“抗AD”转录沉默中的典型功能外， 基因，G9 a激活某些神经毒性基因的翻译。因此，本项目的目标是 以表征G9 a在AD病因学中的这种新的翻译调节功能。我们发现，在 在长期Aβ刺激的hMDM中，（1）G9 a显示出更高的酶活性，并与 （2）AD相关神经毒性蛋白的m6 A被胃L3修饰;（3）G9 a或 同样，胃L3导致这些蛋白质的过度表达减少，表明G9 a和胃L3均 在相同的翻译调节途径中工作;（4）胃L3是G9 a的非组蛋白底物，并且 甲基化赖氨酸的消除降低了胃L3与翻译起始因子eIF 3的结合， 否则对胃L3介导的帽非依赖性翻译至关重要。我们提出了两个目标来测试中央 假设通过Aβ诱导的与胃L3的相互作用，组成型活性G9 a激活翻译 某些神经毒性炎症蛋白的分泌促进AD。我们将（1）确定 组成型活性G9 a和胃L3协同激活翻译的分子机制 Aβ诱导的神经毒性蛋白，和（2）确定组成型活性G9 a如何促进胃L3介导的 Aβ诱导的神经毒性蛋白的翻译，这反过来又有助于AD发病机制。我们将揭开 G9 a在Aβ诱导的、胃L3介导的神经毒性炎症中的这种新的非经典功能，可能是 AD病因学基础的早期病理机制，用于AD的早期治疗干预。