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.

项目摘要 目前，美国有超过570万阿尔茨海默氏病（AD）患者，这一数字是 预计到2050年将超过1400万。尽管进行了数十年的研究，但治疗的有效疗法 AD仍然缺乏，最近针对β-淀粉样斑块（Aβ）的临床试验在很大程度上没有成功。 近三分之二的AD患者是失去神经疾病的绝经后妇女 雌激素。关于马酮替代疗法提出的研究希望17β-雌二醇（E2）可能会提供 有效预防神经元丧失的治疗方法，但一项大型临床试验发现，共轭进化引起 乳腺癌和血小板事件的风险增加。作为替代方案，我们已经合成了 表征了非甾体类雌激素配体的STX，该配体有选择地靶向未识别的受体 中枢神经系统（CNS），但重要的是STX不吸引“经典”的核雌激素受体， 这使STX避免了雌激素的不利作用。口服的STX交叉血液 脑屏障并激活CNS神经元中的神经保护信号通路。 STX改善了线粒体 功能并增强神经元突触传播。此外，STX可预防淀粉样蛋白的毒性 培养的海马神经元，而持续的口服STX可以预防小鼠模型中的淀粉样蛋白毒性 广告。因此，STX具有很高的治疗潜力，但需要对接收器进行识别以进一步 开发作为AD的治疗方法。根据物理 /药物数据，我们假设STX 靶向中枢神经系统神经元中的GQ耦合膜雌激素受体，以提供神经保护作用。我们 建议使用照片跨链接隔离该接收器，然后单击化学物质，并通过体内确认其身份 CRISPR/CAS9诱变研究。我们设计并合成了一种新型的双功能STX衍生物 （BF-STX）包含一个photo-crosslinkablediazirine组和一个炔烃组，该组允许特定标记 STX蛋白与荧光团结合的偶联，并从细胞裂解物中分离出STX蛋白结合物 蛋白质组学分析。在体外，照片连接的BF-STX标签POMC表达下丘脑（MHYPO43）细胞。 因此，我们将：（1）在MHYPO43细胞裂解物和分离BF-中的Photo-Crosslink BF-STX到候选受体 STX蛋白通过单击化学从化学到叠叠珠的膜分数结合，并通过 蛋白质组学。最有希望的命中将通过MHYPO43细胞中的siRNA敲低来验证 测量STX信号的丢失； （2）使用CRISPR/CAS9在体内验证承诺受体候选者 POMCCRE小鼠的诱变。单腺相关病毒（AAV）载体含有重组酶依赖性 金黄色葡萄球菌CAS9和针对每个候选受体的单个指南RNA将针对POMC 神经元。分子生物学（单细胞RT-PCR）和电生理实验将进行至 验证mRNA表达的降低和对STX的物理反应的丧失。这些结果 研究将有助于开发STX作为治疗绝经后妇女和AD的新型疗法。