Identification of the Neuroprotective STX Receptor in the Brain

大脑中神经保护性 STX 受体的鉴定

• 批准号: 10571667
• 负责人: Martin Jeffrey Kelly
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10571667
• 关键词: Academy; Acceleration; Alkynes; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; American; Amyloid; Amyloid beta-Protein; Azides; Biological; Brain; Breast Cancer Risk Factor; CRISPR/Cas technology; Calcium Signaling; Cells; Central Nervous System; Chemistry; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Conjugated Estrogens; Core Facility; Coupled; Data; Dementia; Development; Diazomethane; Electrophysiology (science); Endocrinologist; Estradiol; Estrogen Nuclear Receptor; Estrogen Receptors; Estrogens; Event; Exhibits; Gene Deletion; Goals; Guide RNA; Hippocampus; Hormone replacement therapy; Hypothalamic structure; In Vitro; Isomerism; Label; Ligands; Measures; Membrane; Mitochondria; Molecular; Mus; Mutagenesis; Neurology; Neurons; Oral; Oral Administration; Physiological; Postmenopause; Pro-Opiomelanocortin; Process; Proteins; Proteomics; Receptor Activation; Recommendation; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Selective Estrogen Receptor Modulators; Senile Plaques; Shiga Toxin; Signal Pathway; Signal Transduction; Slice; Small Interfering RNA; Staphylococcus aureus; Structure of nucleus infundibularis hypothalami; Synaptic Transmission; Therapeutic; Toxic effect; United States; Virus; Woman; adeno-associated viral vector; blood-brain barrier crossing; candidate identification; crosslink; design; effective therapy; estrogenic; experimental study; fluorophore; improved; in vivo; knock-down; mRNA Expression; mouse model; neuron loss; neuroprotection; nonhuman primate; novel; novel therapeutics; pharmacologic; prevent; receptor; recombinase; response; side effect; thrombotic

PROJECT SUMMARY Currently, there are over 5.7 million Alzheimer’s disease (AD) patients in the United States, and this number is predicted to reach over 14 million by the year 2050. Despite decades of research, effective therapies for treating AD remain lacking, and recent clinical trials targeting β-amyloid plaques (Aβ) have been largely unsuccessful. Nearly two-thirds of AD patients are postmenopausal women who have lost the neuropr otective effects of estrogens. Studies on hormone replacement therapy raised hopes that 17β-estradiol (E2) might provide an effective treatment for preventing neuronal loss, but a large clinical trial found that conjugated estrogens caused an increased risk of breast cancer and thrombotic events. As an alternative, we have synthesized and characterized a non-steroidal estrogenic ligand, STX that selectively targets an unidentified receptor in the central nervous system (CNS), but importantly STX does not engage the “classical” nuclear estrogen receptors, which enables STX to avoid the unfavorable effects of estrogen. Orally administered STX crosses the blood brain barrier and activates neuroprotective signaling pathways in CNS neurons. STX improves mitochondrial function and enhances neuronal synaptic transmission. Moreover, STX protects against amyloid toxicity in cultured hippocampal neurons, while sustained oral STX protects against amyloid toxicity in a mouse model of AD. Therefore, STX has a high therapeutic potential, but identification of the receptor is needed for further development as a treatment for AD. Based on physiological / pharmacological data, we hypothesize that STX targets a Gq-coupled membrane estrogen receptor in CNS neurons to provide neuroprotective actions. We propose to isolate this receptor using photo-crosslinking and click chemistries and confirm its identity via in vivo CRISPR/Cas9 mutagenesis studies. We have designed and synthesized a novel bifunctional STX derivative (BF-STX) that contains a photo-crosslinkablediazirine group and an alkyne group, which permits specific tagging of STX-protein conjugates with a fluorophore and the isolation of STX-protein conjugates from cell lysates for proteomic analysis. Photo-crosslinked BF-STX labels POMC-expressing hypothalamic (mHypo43) cells in vitro. Therefore, we will: (1) photo-crosslink BF-STX to candidate receptors in mHypo43 cell lysates and isolate BF- STX-protein conjugates from the membrane fraction via click chemistry to azide-bearing beads and analyze by proteomics. The most promising hits will be validated by siRNA knock-down in mHypo43 cells followed by measuring the loss of STX signaling; and (2) validate promising receptor candidates in vivo using CRISPR/Cas9 mutagenesis in POMCCre mice. Single adeno-associated viral (AAV) vectors containing recombinase-dependent Staphylococcus aureus Cas9 and a single guide RNA against each candidate receptor will be targeted to POMC neurons. Molecular biological (single cell RT-PCR) and electrophysiological experiments will be conducted to validate the reduction in mRNA expression and loss of physiological responses to STX. The results from these studies will help to develop STX as a novel therapeutic for treating postmenopausal women and AD.

项目总结 目前，美国有超过570万阿尔茨海默病(AD)患者，这个数字是 预计到2050年将达到1400多万。尽管进行了几十年的研究，但有效的治疗方法 AD仍然缺乏，最近针对β-淀粉样斑块(Aβ)的临床试验基本上没有成功。 近三分之二的AD患者是绝经后的女性，她们已经失去了药物的神经保护作用 雌激素。激素替代疗法的研究增加了人们对17β-雌二醇(E_2)可能提供 预防神经元丢失的有效治疗方法，但一项大型临床试验发现，结合雌激素导致 乳腺癌和血栓形成事件的风险增加。作为另一种选择，我们已经合成了 鉴定了一种非甾体雌激素配体STX，它选择性地靶向 中枢神经系统(CNS)，但重要的是，STX不接触“经典的”核雌激素受体， 这使STX能够避免雌激素的不利影响。口服STX跨越血液 脑屏障并激活中枢神经系统神经元的神经保护性信号通路。STX改善线粒体 功能和增强神经元突触传递。此外，STX对淀粉样蛋白毒性具有保护作用。 持续口服STX对小鼠海马神经元淀粉样蛋白毒性的保护作用 广告。因此，STX具有很高的治疗潜力，但其受体的鉴定有待于进一步研究。 开发作为治疗阿尔茨海默病的方法。基于生理/药理学数据，我们假设STX 靶向中枢神经系统神经元中GQ偶联的膜雌激素受体，以提供神经保护作用。我们 建议采用光交联法和点击化学法分离该受体，并通过体内实验确认其身份 CRISPR/Cas9突变研究。我们设计并合成了一种新型的双功能STX衍生物 (BF-STX)，其包含可光交联的二氮杂基和炔基，其允许特定的标记 将STX-蛋白结合物与荧光团结合，并从细胞裂解物中分离STX-蛋白结合物 蛋白质组学分析。光交联型BF-STX体外标记表达POMC的下丘脑(MHypo43)细胞。 因此，我们将：(1)将BF-STX光交联到mHypo43细胞裂解产物中的候选受体上，并分离BF-STX。 STX-蛋白质从膜部分通过点击化学连接到叠氮化珠上，并通过 蛋白质组学。最有希望的命中将通过在mHypo43细胞中敲除siRNA来验证，然后 测量STX信号的丢失；以及(2)使用CRISPR/Cas9在体内验证有前景的候选受体 POMCCRE小鼠的诱变作用。含重组酶依赖的单型腺相关病毒载体 金黄色葡萄球菌Cas9和针对每个候选受体的单一引导RNA将靶向POMC 神经元。将进行分子生物学(单细胞RT-PCR)和电生理实验 验证STX对mRNA表达的降低和生理反应的丧失。这些研究的结果 研究将有助于开发STX作为治疗绝经后妇女和阿尔茨海默病的新疗法。