Neuroinflammation, Protein Aggregates, ApoE4 Drug Targeting, and Autophagy Rescue

神经炎症、蛋白质聚集体、ApoE4 药物靶向和自噬拯救

• 批准号: 10768318
• 负责人: Sue Tilton Griffin
• 金额: $ 47.03万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10768318
• 关键词: Acute-Phase Proteins; Alzheimer' s Disease; Alzheimer' s neuropathogenesis; Amyloid beta-Protein; Apolipoprotein E; Astrocytes; Autophagocytosis; Binding; Brain; Brain Diseases; Brain region; Chemical Agents; Chronic; DNA; DNA Sequence; Development; Drug Targeting; Event; Exhibits; Feedback; Functional disorder; Genes; Genetic Transcription; Genotype; Glial Fibrillary Acidic Protein; Histologic; Individual; Inherited; Interleukin-1 beta; Lead; Microglia; Modification; Molecular; Motion; Neurofibrillary Tangles; Neurons; Outcome; Parents; Pathogenesis; Patients; Pharmaceutical Preparations; Phosphotransferases; Play; Prevention strategy; Production; Protein Inhibition; Proteins; Proteomics; Reporting; Repression; Role; Stress; Testing; Work; apolipoprotein E-4; brain tissue; chemical binding; cytokine; drug candidate; gain of function; hyperphosphorylated tau; neuroinflammation; novel; protein aggregation; protein protein interaction; small molecule; transcription factor

We established the pluripotent interleukin-1β (IL-1 β) cytokine as a significant player in the pathogenesis of Alzheimer’s disease as it sets in motion a self-amplifying positive-feedback cycle in which neuronal stress induces synthesis of the neuron's acute phase protein β APP and release of its fragments sAPPa and Aβ. Both these proteins activate microglia and a progressive elevation of IL-1 β, which drives chronically enhanced formation of the hallmark aggregates of AD: A β plaques and, via IL-1 β -induced synthesis and activation of specific kinases, hyperphosphorylation of tau in neurofibrillary tangles in neurons and glial fibrillary acidic protein (GFAP) in astrocytes. Interestingly, both events are dramatically enhanced in AD patients who inherit the Alzheimer gene from both parents (genotype ApoE4,4). Although these IL-1 β and ApoE genotype driven events favor negative outcomes, our progress in drug-development initiatives shows that they are amenable to treatment. Specifically, GFAP-binding chemical agents were shown to inhibit protein aggregation. Further, we discovered a novel function of the APOE£4 gene, which is a toxic gain-of- function exhibited by its protein product, ApoE4. We have demonstrated that ApoE4 competes with Transcriptional Factor EB (TFEB) for binding to the CLEAR DNA motifs, thus, hindering the transcription of three proteins crucial for lysosomal autophagy. We show this to be the case in brain tissues from AD 4,4, but not AD 3,3 patients. Now, importantly, we have identified a lead compound that binds to ApoE4 protein, obviating its interactions with CLEAR DNA and restoring the expression of three autophagy genes, that encode for production of p62, LC3B, and LAMP2 proteins. Now, we are prepared to further elucidate the role of IL-1 β in cellular pathophysiology, establishing its effects on kinases and kinase targets that manifest the modifications which drive predominant aggregate nucleation or propagation events. Our advanced molecular and histological approaches will be applied to confirm predictions of protein-protein interactions derived from proteomics approaches, particularly those involving GFAP. Moreover, these interactions can now be evaluated across brain regions and disease states to test their concordance with known AD parameters. Finally, we will elucidate the mechanisms of action of our identified novel small-molecule drugs targeted to ApoE4 in inhibiting all its pathognomonic interactions with other ApoE4 targeted DNA sequences. Successful completion of our proposed work promises a preventive strategy for foiling the known dramatic role that ApoE4 plays in Alzheimer neuropathogenesis. RELEVANCE: Successful completion of this proposed work will obviate the many pathognomonic aspects of inheritance of the Alzheimer gene (APOEE4) in the 1 in 4 individuals in the US, i.e., 80 million, who inherit one or both copies of this gene. Through the action of our specific drug candidates to inhibit the ApoE4 protein we will, therefore, restore the lysosomal autophagy necessary for efficient clearance of large aggregates, which, as we reported, is repressed in the brains of those who inherit one or both APOE4 genes.

我们发现，多能性白细胞介素-1β (IL-1 β)细胞因子在阿尔茨海默病的发病机制中发挥着重要作用，因为它启动了一个自我放大的正反馈循环，在这个循环中，神经元应激诱导神经元急性期蛋白β APP的合成，并释放其片段sAPPa和a β。这两种蛋白激活小胶质细胞和IL-1 β的进行性升高，从而导致AD: a β斑块标志性聚集体的长期增强形成，并通过IL-1 β诱导的特定激酶的合成和激活，导致神经元中神经原纤维缠结和胶质纤维酸性蛋白（GFAP）中的tau过度磷酸化。有趣的是，在从父母双方遗传了阿尔茨海默病基因（基因型apoe4,4）的AD患者中，这两个事件都显著增强。尽管这些IL-1 β和ApoE基因型驱动的事件倾向于负面结果，但我们在药物开发方面的进展表明，它们是可以治疗的。具体来说，gfap结合的化学试剂被证明可以抑制蛋白质聚集。此外，我们还发现了ApoE4基因的一种新功能，即其蛋白产物ApoE4所表现出的毒性功能增益。我们已经证明，ApoE4与转录因子EB （TFEB）竞争，以结合CLEAR DNA基序，从而阻碍三种对溶酶体自噬至关重要的蛋白质的转录。我们在AD 4,4患者的脑组织中发现了这种情况，但AD 3,3患者则没有。现在，重要的是，我们已经确定了一种与ApoE4蛋白结合的先导化合物，消除了它与CLEAR DNA的相互作用，并恢复了三种自噬基因的表达，这些基因编码p62、LC3B和LAMP2蛋白的产生。现在，我们准备进一步阐明IL-1 β在细胞病理生理中的作用，确定其对激酶和激酶靶点的影响，这些激酶和靶点表现出驱动主要聚集成核或繁殖事件的修饰。我们先进的分子和组织学方法将应用于证实蛋白质组学方法衍生的蛋白质-蛋白质相互作用的预测，特别是那些涉及GFAP的预测。此外，这些相互作用现在可以跨大脑区域和疾病状态进行评估，以测试它们与已知AD参数的一致性。最后，我们将阐明我们鉴定的靶向ApoE4的新型小分子药物的作用机制，以抑制其与其他ApoE4靶向DNA序列的所有病理相互作用。我们所提出的工作的成功完成有望阻止ApoE4在阿尔茨海默病神经发病机制中发挥的已知戏剧性作用的预防策略。