Identification of the Neuroprotective STX Receptor in the Brain
大脑中神经保护性 STX 受体的鉴定
基本信息
- 批准号:10571667
- 负责人:
- 金额:$ 23.1万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AcademyAccelerationAlkynesAlzheimer&aposs DiseaseAlzheimer&aposs disease modelAlzheimer&aposs disease patientAmericanAmyloidAmyloid beta-ProteinAzidesBiologicalBrainBreast Cancer Risk FactorCRISPR/Cas technologyCalcium SignalingCellsCentral Nervous SystemChemistryClinicalClinical TrialsClustered Regularly Interspaced Short Palindromic RepeatsConjugated EstrogensCore FacilityCoupledDataDementiaDevelopmentDiazomethaneElectrophysiology (science)EndocrinologistEstradiolEstrogen Nuclear ReceptorEstrogen ReceptorsEstrogensEventExhibitsGene DeletionGoalsGuide RNAHippocampusHormone replacement therapyHypothalamic structureIn VitroIsomerismLabelLigandsMeasuresMembraneMitochondriaMolecularMusMutagenesisNeurologyNeuronsOralOral AdministrationPhysiologicalPostmenopausePro-OpiomelanocortinProcessProteinsProteomicsReceptor ActivationRecommendationResearchReverse Transcriptase Polymerase Chain ReactionRodentSelective Estrogen Receptor ModulatorsSenile PlaquesShiga ToxinSignal PathwaySignal TransductionSliceSmall Interfering RNAStaphylococcus aureusStructure of nucleus infundibularis hypothalamiSynaptic TransmissionTherapeuticToxic effectUnited StatesVirusWomanadeno-associated viral vectorblood-brain barrier crossingcandidate identificationcrosslinkdesigneffective therapyestrogenicexperimental studyfluorophoreimprovedin vivoknock-downmRNA Expressionmouse modelneuron lossneuroprotectionnonhuman primatenovelnovel therapeuticspharmacologicpreventreceptorrecombinaseresponseside effectthrombotic
项目摘要
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.
项目概要
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,这一数字将超过 1400 万。尽管进行了数十年的研究,但有效的治疗方法
AD 仍然缺乏,最近针对 β-淀粉样蛋白斑 (Aβ) 的临床试验基本上不成功。
近三分之二的 AD 患者是绝经后女性,她们已经失去了神经保护作用
雌激素。激素替代疗法的研究引发了人们的希望:17β-雌二醇 (E2) 可能会提供一种
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,结合雌激素会导致
乳腺癌和血栓事件的风险增加。作为替代方案,我们合成并
表征了一种非甾体雌激素配体,STX,它选择性地靶向未识别的受体
中枢神经系统 (CNS),但重要的是 STX 不参与“经典”核雌激素受体,
这使得STX能够避免雌激素的不利影响。口服 STX 会穿过血液
脑屏障并激活中枢神经系统神经元的神经保护信号通路。 STX 改善线粒体
功能并增强神经元突触传递。此外,STX 可防止淀粉样蛋白毒性
培养的海马神经元,而持续口服 STX 可防止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,但需要进一步鉴定其受体
开发作为 AD 的治疗方法。根据生理/药理学数据,我们假设 STX
靶向 CNS 神经元中的 Gq 偶联膜雌激素受体,提供神经保护作用。我们
建议使用光交联和点击化学分离该受体,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双功能STX衍生物
(BF-STX) 包含可光交联的二氮丙啶基团和炔烃基团,允许进行特定标记
STX-蛋白缀合物与荧光团的结合以及从细胞裂解液中分离 STX-蛋白缀合物
蛋白质组分析。光交联 BF-STX 在体外标记表达 POMC 的下丘脑 (mHypo43) 细胞。
因此,我们将:(1)将 BF-STX 光交联到 mHypo43 细胞裂解物中的候选受体并分离 BF-
通过点击化学将膜组分中的 STX-蛋白缀合物连接到带有叠氮化物的珠子上,并通过以下方法进行分析
蛋白质组学。最有希望的命中将通过 mHypo43 细胞中的 siRNA 敲低进行验证,然后
测量STX信令的丢失; (2) 使用 CRISPR/Cas9 在体内验证有希望的候选受体
POMCCre 小鼠中的诱变。含有重组酶依赖性的单个腺相关病毒 (AAV) 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)和电生理学实验
验证 mRNA 表达的减少和对 STX 生理反应的丧失。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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Martin Jeffrey Kelly其他文献
Martin Jeffrey Kelly的其他文献
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{{ truncateString('Martin Jeffrey Kelly', 18)}}的其他基金
Cross-talk between Leptin and Estrogen Signaling in Hypothalamic Arcuate Neurons
下丘脑弓状神经元中瘦素和雌激素信号传导之间的串扰
- 批准号:
7993025 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-Talk Between Estrogen and Metabolic Hormone Signaling in Arcuate Neurons
弓状神经元中雌激素和代谢激素信号传导之间的串扰
- 批准号:
9174776 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-Talk between Leptin and Estrogen Signaling in Hypothalamic Arcuate Neurons
下丘脑弓状神经元中瘦素和雌激素信号传导之间的串扰
- 批准号:
8307979 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Crosstalk between Estrogen and Metabolic Hormone Signaling in Kisspeptin Neurons
Kisspeptin 神经元中雌激素和代谢激素信号传导之间的串扰
- 批准号:
10246663 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-talk between Estrogen and Metabolic Hormone Signaling in Kisspeptin Neurons
Kisspeptin 神经元中雌激素和代谢激素信号传导之间的串扰
- 批准号:
10295726 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-talk between Estrogen and Metabolic Hormone Signaling in Kisspeptin Neurons
Kisspeptin 神经元中雌激素和代谢激素信号传导之间的串扰
- 批准号:
10473890 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-talk between Leptin and Estrogen Signaling in Hypothalamic Arcuate Neurons
下丘脑弓状神经元中瘦素和雌激素信号传导之间的串扰
- 批准号:
8113859 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
Cross-Talk between Leptin and Estrogen Signaling in Hypothalamic Arcuate Neurons
下丘脑弓状神经元中瘦素和雌激素信号传导之间的串扰
- 批准号:
8488293 - 财政年份:2005
- 资助金额:
$ 23.1万 - 项目类别:
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