Mechanisms that govern dopaminergic amacrine cell diversity

项目概述：视网膜中的多巴胺信号对于调节昼夜节律和电路至关重要 重新配置白天的视野。多巴胺信号传导减少与许多视觉障碍有关， 使用多巴胺类似物的药理学疗法已经显示出治疗糖尿病等疾病的前景， 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（DAC）。这个项目是 专注于阐明控制这些特化无长突的分子机制。 这些知识将使开发新的疗法，利用再生的力量， 药物再生修复或取代多巴胺能无长突细胞和恢复多巴胺丰度， 正常水平。为了实现这一目标，我们进行了一次筛选，以确定当删除时， 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信号通路- 苏氨酸激酶LKB 1。当LKB 1在胚胎中缺失时， 持续到成年期的多巴胺能无长突。伴随着多巴胺的增加， 通过高效液相色谱法测定。我们假设LKB1信号被激活， 限制DAC人口丰富的发展计划。在这里，我们开始 确定LKB 1信号传导是否是无长突细胞前体细胞以及颞叶细胞固有的需要， 要求LKB 1信令限制DAC形成。我们还将利用超分辨率 STORM显微镜观察新生成的DAC中多巴胺的分布和释放。我们 第二个目的是确定LKB 1的下游效应物，这些效应物是限制DAC形成所必需的。我们 将利用LKB 1下游已知分子（例如AMPK）的功能丧失和拯救实验 以测试它们对DAC形成的贡献。我们还将进行单细胞RNA测序， 对照与突变视网膜中DAC之间的转录景观。这将使我们能够找到更多的 限制DAC丰度所需的LKB1下游候选物。这种分析是 从基础科学的角度来看是令人信服的，但也将定义新的分子靶点， 多巴胺神经元丧失和恢复视觉能力。这些知识可能有助于预防 眼睛以外的多巴胺能神经元缺陷，如帕金森病中发生的那些。

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.