Mechanisms that govern dopaminergic amacrine cell diversity

项目摘要：视网膜中的多巴胺信号对调节昼夜节律和回路至关重要 白天视野的重新配置。多巴胺信号减弱与许多视觉障碍和 使用多巴胺类似物的药物疗法在治疗糖尿病等疾病方面显示出了希望 视网膜病变。视网膜多巴胺的主要来源是多巴胺能无长突细胞(DACs)。这个项目是 重点阐明了管理这些专门化的长春花碱规范的分子机制。 这一知识将使利用再生能力的新疗法的开发成为可能 用于再生、修复或替换多巴胺能无长突细胞并恢复多巴胺丰度的药物 正常水平。为了实现这一目标，我们进行了一项筛选，以确定当删除时，增加的分子 多巴胺能无长突细胞形成。我们发现了一条由丝氨酸控制的候选信号通路- 苏氨酸激酶LKB1。当LKB1在胚胎中被删除时， 能持续到成年期的多巴胺能阿玛卡林。随之而来的是多巴胺的增加。 用高效液相色谱仪测定。我们假设LKB1信号正在激活 限制DAC人口丰富度的发展计划。在这里，我们开始了 确定LKB1信号是否在无长突细胞前体和临床期细胞中固有地需要 限制DAC形成的LKB1信号的要求。我们还将利用超分辨率的能力 风暴显微镜观察多巴胺在新生成的DAC中的分布和释放。我们的 第二个目的是确定LKB1中限制DAC形成所需的下游效应子。我们 将利用LKB1下游已知分子的功能丧失和挽救实验，如AMPK 以测试他们对DAC形成的贡献。我们还将进行单细胞RNA测序，以比较 对照和突变视网膜中DAC之间的转录景观。这将使我们能够找到更多 限制DAC丰度所需的LKB1下游候选物。这一分析是 从基础科学的角度来看很有说服力，但也将定义新的分子靶标来缓解 多巴胺神经元丧失和视觉能力恢复。这一知识可能有助于预防 眼睛以外的多巴胺能神经元缺陷，如帕金森氏症中出现的缺陷。

Project Summary: Dopamine signaling in the retina is crucial for regulating circadian rhythms and circuit reconfiguration for daytime vision. Reduced dopamine signaling is associated with many visual disorders and pharmacological therapies using dopamine analogues have shown promise for treating diseases like diabetic retinopathy. The primary source of retinal dopamine is the dopaminergic amacrine cells (DACs). This project is focused on elucidating molecular mechanisms that govern the specification of these specialized amacrines. This knowledge will enable the development of novel therapeutics that harness the power of regenerative medicine to regrow repair or replace the dopaminergic amacrine cells and restore dopamine abundance to normal levels. Toward this goal, we conducted a screen to identify molecules that, when deleted, increase dopaminergic amacrine cell formation. We uncovered a candidate signaling pathway controlled by the serine- threonine kinase LKB1. When LKB1 is deleted embryonically there is an approximate doubling of the dopaminergic amacrines that persists into adulthood. Concomitantly there is an increase in dopamine when measured by High Performance Liquid Chromatography. We hypothesize that LKB1 signaling is activating developmental programs that restrict the abundance of the DAC population. Here we set out to determine whether LKB1 signaling is required intrinsically in amacrine cell precursors as well as the temporal requirement for LKB1 signaling to restrict DAC formation. We will also leverage the ability of super resolution STORM microscopy to visualize the distribution and release of dopamine in the newly generated DACs. Our second aim is to identify the downstream effectors from LKB1 that are required to restrict DAC formation. We will utilize loss of function and rescue experiments of known molecules downstream from LKB1 such as AMPK to test their contribution to DAC formation. We will also conduct single cell RNA sequencing to compare the transcriptional landscapes between DACs in control vs mutant retinas. This will allow us to find additional downstream candidates from LKB1 required for restriction of the abundance of DACs. This analysis is compelling from the basic science perspective but will also define novel molecular targets for mitigating dopamine neuron loss and restoring visual capacity. This knowledge may be useful for preventing dopaminergic neuron defects beyond the eye, such as those that occur in Parkinson’s disease.