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

• 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
• 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' 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

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 包括帕金森氏病和阿尔茨海默氏症在内的神经退化的一般机制是 错误折叠的神经元蛋白的分解和随后的聚集。例如，在“联体核病”中 神经毒性与错误折叠的α-突触核蛋白(ASYN)的切割有关，可能主要是通过 天冬酰胺内肽酶(AEP)[Zhang等2017]。这会产生神经毒肽，然后聚集在一起 溶酶体中含有ASYN，形成与神经退行性变相关的路易氏包涵体。一个非常相似的 植物细胞中存在一种机制，即植物AEP分解错误折叠的蛋白质以产生液泡 与程序性细胞死亡(PCD)相关的聚集体[Hatsugai等人，2015]。然而，植物不会 含有ASYN，因此，为了模拟ASYN的毒性，我们将植物细胞转化为表达错误折叠- 人类ASYN的易感A53T变体(I期)。然后用百里香酚引发植物PCD[Hassanien 等人2013]，表达ASYN-A53T的植物细胞被证明对此更敏感 毒性高于对照组。在第二阶段，这些转基因(ASYN)植物细胞的突变群体将被挑选出来用于 在这个程序下存活。这是“靶向进化”的一个例子，在这种进化中，存活下来的突变体 选择应该朝着增加代谢产物的生物合成的方向进化，以抑制AEP和/或ASYN毒性。 具有ASYN保护活性的单个突变植物细胞克隆将通过筛选 然后将使用耐药培养和微量分析方法[Kelley等人，2019年]来鉴定活性 代谢物作为先导。红半边莲细胞培养在第一阶段使用，因为我们之前 用人多巴胺转运蛋白基因转化这些细胞以模拟MPP+诱导的多巴胺能 神经毒性[Brown等人2016年]。在第二阶段，我们还将使用药用植物何首乌，它 包含一种抑制ASYN毒性的二苯乙烯类化合物[Zhang等人2018年]。第二阶段旨在确定新的线索， 启动AEP和ASYN目标，并在细胞和动物模型中测试其中最有希望的目标 联核症。将与肯塔基大学帕金森氏病研究中心共同开发Leads 中心和一个制药伙伴。识别与这些目标接洽的线索也将支持目标- 指导突变植物细胞的进化，作为药物发现的商业平台。