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Mimicking synuclein toxicity in plant cells to identify novel neuroprotective leads

模拟植物细胞中的突触核蛋白毒性以鉴定新型神经保护先导化合物

基本信息

批准号：
10267035
负责人：
JOHN M. LITTLETON
金额：
$ 61.75万
依托单位：
NAPROGENIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10267035
关键词：
1-Methyl-4-phenylpyridinium Agrobacterium Alzheimer&apos s Disease Anabolism Animal Model Apoptosis Asparagine Biotechnology Cell Culture Techniques Cell model Cells Chemicals Cleaved cell Clone Cells Complementary DNA Development Directed Molecular Evolution Endopeptidases Enzymes Exposure to Foundations Genes Human In Vitro Inclusion Bodies Individual Kentucky Lead Lobelia Lysosomes Materials Testing Mediating Medicinal Plants Methods Modification Molecular Mutagenesis Nerve Degeneration Neurodegenerative Disorders Neurons Neurotoxins Parkinson Disease Pathologic Peptide Hydrolases Peptides Pharmacologic Substance Phase Plant Roots Plants Polygonum Population Procedures Process Proteins Research Resistance Risk Stilbenes Substance Use Disorder Technology Transfer Testing Therapeutic Toxic effect Transgenic Organisms United States National Institutes of Health Universities Variant aged alpha synuclein analytical method asparaginylendopeptidase base commercialization cytotoxic dopamine transporter drug discovery expectation gain of function mutation inhibitor/antagonist misfolded protein mutant neurotoxic neurotoxicity novel novel therapeutics programs screening substance use treatment synuclein synucleinopathy

项目摘要

Abstract A general mechanism for neurodegeneration, including Parkinson's disease and Alzheimer's dementia, is the breakdown and subsequent aggregation of misfolded neuronal proteins. In the “synucleinopathies” for example neurotoxicity is associated with the cleavage of mis-folded alpha-synuclein (ASYN), probably mainly by asparagine endopeptidase (AEP) [Zhang et al 2017]. This generates neurotoxic peptides that then aggregate with ASYN in lysosomes, forming the Lewy inclusion bodies associated with neurodegeneration. A very similar mechanism exists in plant cells in which plant AEP breaks down misfolded proteins to produce vacuolar aggregates associated with programmed cell death (PCD) [Hatsugai et al, 2015]. However, plants do not contain ASYN, so, in order to mimic ASYN toxicity, plant cells were transformed to express the misfolding- prone A53T variant of human ASYN (phase I). Thymoquinone was then used to trigger plant PCD [Hassanien et al 2013] and the plant cells expressing ASYN-A53T were shown to be significantly more susceptible to this toxicity than controls. In phase II a mutant population of these transgenic (ASYN) plant cells will be selected for survival under this procedure. This is an example of “target-directed evolution” in which mutants that survive selection should “evolve” toward increased biosynthesis of metabolites that inhibit AEP and/or ASYN toxicity. Individual mutant plant cell clones with ASYN-protective activity will be identified by screening extracts of resistant cultures, and micro-analytical methods [Kelley et al, 2019] will then be used to identify active metabolites as leads. Lobelia cardinalis cell cultures were used in phase I because we had previously transformed these with the human dopamine transporter gene to mimic MPP+-induced dopaminergic neurotoxicity [Brown et al 2016]. In phase II we will also use the medicinal plant, Polygonum multiflorum, which contains a stilbene that inhibits ASYN toxicity [Zhang et al 2018]. Phase II aims to identify novel leads that engage the AEP and ASYN targets, and to test the most promising of these in cellular and animal models of synucleinopathy. Leads will be developed with the University of Kentucky Parkinson's Disease Research Center and a pharmaceutical partner. Identification of leads that engage these targets will also support target- directed evolution in mutant plant cells as a commercial platform for drug discovery.

抽象的神经变性（包括帕金森病和阿尔茨海默氏痴呆）的一般机制是错误折叠的神经元蛋白质的分解和随后的聚集。例如，在“突触核蛋白病”中神经毒性与错误折叠的 α-突触核蛋白 (ASYN) 的裂解有关，可能主要是通过天冬酰胺内肽酶 (AEP) [Zhang et al 2017]。这会产生神经毒性肽，然后聚集与溶酶体中的 ASYN 结合，形成与神经变性相关的路易包涵体。一个非常相似的植物细胞中存在植物 AEP 分解错误折叠蛋白质以产生液泡的机制与程序性细胞死亡 (PCD) 相关的聚集体 [Hatsugai et al, 2015]。然而，植物并不含有 ASYN，因此，为了模拟 ASYN 毒性，植物细胞被转化以表达错误折叠- 人类 ASYN 的易发 A53T 变体（I 期）。然后使用百里醌引发植物 PCD [Hassanien et al 2013]并且表达ASYN-A53T的植物细胞被证明更容易受到这种影响毒性高于对照。在第二阶段，将选择这些转基因（ASYN）植物细胞的突变群体在此程序下的生存。这是“定向进化”的一个例子，其中存活下来的突变体选择应该“进化”以增加抑制 AEP 和/或 ASYN 毒性的代谢物的生物合成。具有 ASYN 保护活性的单个突变植物细胞克隆将通过筛选提取物来鉴定然后将使用抗性培养物和微分析方法 [Kelley et al, 2019] 来识别活性代谢物作为先导物。半边莲细胞培养物用于第一阶段，因为我们之前用人类多巴胺转运蛋白基因改造它们以模拟 MPP+ 诱导的多巴胺能神经毒性 [Brown et al 2016]。在第二阶段，我们还将使用药用植物何首乌，它含有抑制 ASYN 毒性的二苯乙烯 [Zhang et al 2018]。第二阶段旨在确定新的线索参与 AEP 和 ASYN 目标，并在细胞和动物模型中测试其中最有前途的目标突触核蛋白病。将与肯塔基大学帕金森病研究中心共同开发线索中心和制药合作伙伴。识别与这些目标相关的线索也将支持目标突变植物细胞的定向进化作为药物发现的商业平台。