Cui Jun, Carey Joseph, Reijo Pera Renee A
Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT, United States.
McLaughlin Research Institute, Great Falls, MT, United States.
Front Aging Neurosci. 2022 Dec 12;14:1026468. doi: 10.3389/fnagi.2022.1026468. eCollection 2022.
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra region of the midbrain. Diagnostic criteria for PD require that at least two of three motor signs are observed: tremor, rigidity, and/or bradykinesia. The most common and effective treatment for PD is Levodopa (L-DOPA) which is readily converted to DA and has been the primary treatment since the 1960's. Dopamine agonists have also been developed but are less effective than L-DOPA. Although the lack of a model system to study PD has hampered efforts to identify treatments, diverse screening strategies have been proposed for identification of new pharmaceutical candidates. Here, we describe a pilot screen to identify candidate molecules from a bioactive compound library, that might increase formation, maintenance and/or survival of DA neurons . The screen used a previously characterized reporter construct consisting of the luciferase gene inserted downstream of the endogenous tyrosine hydroxylase (TH) gene and neurons differentiated from human pluripotent stem cells for 18 days. The reporter mimics expression of TH and includes a secreted luciferase whose activity can be measured non-invasively over multiple timepoints. Screening of the bioactive compound library resulted in the identification of a single molecule, SGC0946, that is an inhibitor of DOT1L (Disruptor Of Telomeric silencing 1-Like) which encodes a widely-conserved histone H3K79 methyltransferase that is able to both activate and repress gene transcription. Our results indicate that SGC0946 increased reporter luciferase activity with a single treatment for 48-h post-plating being equivalent to continuous treatment. Moreover, data suggested that the total number of neurons differentiated in the assays was comparable from experiment to experiment under different SGC0946 treatments over time. In contrast, data suggested that the survival and/or maintenance of DA neurons might be specifically enhanced by SGC0946 treatment. These results document the feasibility of a set of tools for further exploration of small molecules that may impact DA neuron differentiation, maintenance and/or survival. Results provide evidence in support of other reports that indicate inhibition of DOT1L may play an important role in maintenance and survival of neural progenitor cells (NPCs) and their lineage-specific differentiation.
帕金森病（PD）是一种常见的神经退行性疾病，其特征是中脑黑质区域的多巴胺能（DA）神经元进行性丧失。帕金森病的诊断标准要求观察到三种运动体征中的至少两种：震颤、僵硬和/或运动迟缓。帕金森病最常见且有效的治疗方法是左旋多巴（L-DOPA），它很容易转化为多巴胺，自20世纪60年代以来一直是主要治疗方法。多巴胺激动剂也已研发出来，但效果不如L-DOPA。尽管缺乏研究帕金森病的模型系统阻碍了寻找治疗方法的努力，但已经提出了多种筛选策略来鉴定新的药物候选物。在此，我们描述了一项初步筛选，以从生物活性化合物库中鉴定可能增加DA神经元形成、维持和/或存活的候选分子。该筛选使用了一种先前表征的报告构建体，其由插入内源性酪氨酸羟化酶（TH）基因下游的荧光素酶基因和从人多能干细胞分化18天的神经元组成。该报告基因模拟TH的表达，并包括一种分泌型荧光素酶，其活性可以在多个时间点进行非侵入性测量。对生物活性化合物库的筛选导致鉴定出一种单一分子SGC0946，它是DOT1L（端粒沉默破坏因子1样）的抑制剂，DOT1L编码一种广泛保守的组蛋白H3K79甲基转移酶，能够激活和抑制基因转录。我们的结果表明，SGC0946单次处理48小时后可增加报告荧光素酶活性，这与连续处理相当。此外，数据表明，在不同的SGC0946处理下，随着时间的推移，实验中分化的神经元总数在不同实验之间具有可比性。相比之下，数据表明SGC0946处理可能特异性增强DA神经元的存活和/或维持。这些结果证明了一套工具对于进一步探索可能影响DA神经元分化、维持和/或存活的小分子的可行性。结果为其他报告提供了证据支持，这些报告表明抑制DOT1L可能在神经祖细胞（NPC）的维持和存活及其谱系特异性分化中起重要作用。
