Mechanisms of cellular clearance in the retinal pigment epithelium

 描述（由申请人提供）：视网膜色素上皮（RPE）是一种高度代谢活性的有丝分裂后组织，具有许多视觉不可或缺的功能。RPE的关键功能是脱落的感光细胞外节的日常吞噬和降解。这两个特征-被吞噬的外节的高代谢和昼夜节律消化-对RPE中的细胞清除机制造成沉重负担。RPE对碎片的无效处理促进了称为脂褐质和玻璃疣的不溶性聚集体的积累，其与黄斑变性的发病机制有关。自噬是一种清除机制，可以清除受损的细胞成分、病原体和其他碎片。虽然自噬是RPE健康和功能障碍的一个新兴核心参与者，但我们对RPE中如何实现这种基本的分解代谢功能以及如何在年龄相关性黄斑变性等难治性疾病中进行治疗靶向的了解有限。本研究的目的是详细剖析RPE中自噬的分子调控和执行机制。我们将使用极化的初级成人RPE单层的高速实时成像，基因破坏和Stargardt病的Abca 4-/-小鼠模型来测试关于自噬，吞噬和溶酶体途径如何在RPE中交叉以及如何受到先天应激源如脂褐素双维甲酸积累的影响的特定假设。自噬的执行可以分为三个阶段，我们的目标是针对：自噬的起始（目标1），自噬体的生物发生（目标2）和自噬的完成（目标3）。目的1将检验外节吞噬作用抑制RPE中雷帕霉素复合物1（mTORC 1）的机制靶点以激活转录因子TFEB和TFE 3的假设，所述转录因子TFEB和TFE 3诱导溶酶体和自噬基因的综合网络的表达。目的2将研究逐步的生物发生和成熟的自噬体在实时和测试的假设，外段含有吞噬体招募到新生的自噬体作为自噬货物。自噬体和溶酶体的基于微管的运输对于自噬货物的降解是必需的。目的3将检验微管蛋白的翻译后修饰调节自噬体/溶酶体转运的效率从而控制自噬通量的假设。这些实验将通过解决自噬-溶酶体功能如何缩放以满足外节降解的负担，自噬和吞噬-溶酶体途径如何相互作用以维持“清洁”RPE，以及自噬是否促进或预防RPE功能障碍来回答关于RPE中细胞清除的基本问题。缺乏机制的见解，显着阻碍了视网膜变性中的自噬治疗靶向。我们预计，这项研究将产生crucia信息的自噬清除的RPE和援助的发展战略，以保持RPE的健康和功能，在人类的寿命。

 DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is a highly metabolically active, post-mitotic tissue, which performs numerous functions that are indispensable for vision. A key function of the RPE is the daily phagocytosis and degradation of shed photoreceptor outer segments. These two features - high metabolism and circadian digestion of phagocytosed outer segments - place a heavy burden on cellular clearance mechanisms in the RPE. Inefficient disposal of debris by the RPE promotes the accumulation of insoluble aggregates called lipofuscin and drusen, which are implicated in the pathogenesis of macular degenerations. Autophagy is a clearance mechanism that removes damaged cellular components, pathogens and other debris. Although autophagy is an emerging central player in the health and dysfunction of the RPE, we have limited insight into how this essential catabolic function is accomplished in the RPE and how it can be therapeutically targeted in intractable diseases like age-related macular degeneration. The goal of this research is to dissect the molecular regulation and execution of autophagy in the RPE in mechanistic detail. We will use high-speed live imaging of polarized primary adult RPE monolayers, gene disruption and the Abca4-/- mouse model of Stargardt disease to test specific hypotheses regarding how autophagic, phagocytic and lysosomal pathways intersect in the RPE and how this is affected by innate stressors like lipofuscin bisretinoid accumulation. The execution of autophagy can be divided into three phases that our Aims are directed to: initiation of autophagy (Aim 1), biogenesis of autophagosomes (Aim 2) and completion of autophagy (Aim 3). Aim 1 will test the hypothesis that outer segment phagocytosis inhibits the mechanistic target of Rapamycin complex 1 (mTORC1) in the RPE to activate the transcription factors TFEB and TFE3, which induce the expression of a comprehensive network of lysosomal and autophagy genes. Aim 2 will investigate the step-wise biogenesis and maturation of autophagosomes in real-time and test the hypothesis that outer segment-containing phagosomes are recruited into nascent autophagosomes as autophagic cargo. Microtubule-based transport of autophagosomes and lysosomes is required for degradation of autophagic cargo. Aim 3 will test the hypothesis that post-translational modifications of tubulin regulate the efficiency of autophagosome/lysosome transport and thereby control autophagic flux. These experiments will answer fundamental questions about cellular clearance in the RPE by addressing how autophagic-lysosome functions are scaled to meet the burden of outer segment degradation, how autophagic and phago-lysosomal pathways interact to maintain "clean" RPE, and whether autophagy promotes or prevents RPE dysfunction. Lack of mechanistic insight has significantly hampered therapeutic targeting of autophagy in retinal degenerations. We anticipate that this research will yield crucia information regarding autophagic clearance in the RPE and aid the development of strategies to preserve RPE health and function over the human lifespan.