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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.

摘要神经变性，包括帕金森氏病和阿尔茨海默氏痴呆症的一般机制是，错误折叠的神经元蛋白的分解和随后的聚集。例如在“共核蛋白病”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，在溶酶体中与ASYN结合，形成与神经变性相关的路易包涵体。一个非常相似在植物细胞中存在这样一种机制，即植物AEP分解错误折叠的蛋白质，产生液泡，与程序性细胞死亡（PCD）相关的聚集体[Hatsugai et al，2015]。然而，植物不含有ASYN，因此，为了模拟ASYN毒性，转化植物细胞以表达错误折叠- 人ASYN的倾向性A53 T变体（I期）。然后使用百里醌触发植物PCD [Hassanien 等人2013]，并且表达ASYN-A53 T的植物细胞显示出对此显著更敏感。毒性高于对照组。在第II阶段，将选择这些转基因（ASYN）植物细胞的突变群体，在这个过程中生存。这是一个“目标导向进化”的例子，选择应该朝着增加抑制AEP和/或ASYN毒性的代谢物的生物合成“进化”。具有ASYN保护活性的单个突变体植物细胞克隆将通过筛选然后将使用耐药培养物和微量分析方法[Kelley et al，2019]来鉴定活性代谢物作为线索。半边莲细胞培养物用于第一阶段，因为我们以前将这些与人类多巴胺转运蛋白基因转化，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，含有抑制ASYN毒性的芪[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。共核蛋白病电极导线将与肯塔基州大学帕金森病研究中心共同开发中心和一个医药合作伙伴。确定与这些目标接触的线索也将支持目标- 在突变植物细胞中定向进化作为药物发现的商业平台。