The Role of Presenilin-2 in the Control and Function of Polycystin-1

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)的特点是逐渐发展的囊性压迫和损害周围的正常肾实质，这反过来又导致最终的肾功能下降。虽然有两个基因与ADPKD表型相关，即PKD1和PKD2，但大多数病例是由于编码多囊蛋白-1(PC1)的PKD1基因突变所致。为了开发ADPKD患者的治疗方法，有必要了解与PC1功能相关的信号机制。因此，本应用的目的是探索早老素-2(PSEN2)在PC1信号调控中的新作用。PSEN2是分泌酶的催化亚基，分泌酶是一种裂解PC1的蛋白酶，导致其C末端尾巴(CTT)的释放。由此产生的PC1 CTT裂解片段似乎作为信号分子参与维持细胞内环境的稳定。虽然与PC1 CTT裂解相关的信号通路已经被阐明，但激活PSEN_2介导的PC1 CTT裂解的刺激物以及PSEN_2在囊变中的潜在参与仍然不清楚。我们先前已经确定，在HEK293细胞中，PSEN2基因敲除显著减少了PC1 CTT的切割。我们以前的研究也表明，PC1 CTT裂解调控与内质网(ER)应激相关的基因的转录活性，并由肾损伤与血流量减少相关诱导。我们假设PSEN2是正确的PC1 CTT切割和信号传递所必需的，并且这一功能参与了阻止囊变的发生。我们提出以下特定的目标来支持或驳斥我们的假设：1)目标1将建立这样的概念，即PSEN2介导的PC1 CTT切割是由内质网应激刺激的，如果受到干扰，这一过程已被证明会导致包囊形成。我们将使用基于荧光素酶的分析方法在PSEN2被敲除和ER应激的条件下测量PC1 CTT的裂解，并确定与此反应相关的特定片段的大小；2)目标2将建立这样的概念，即PSEN2介导的PC1的裂解在PC1的囊性预防活动中起关键作用，并且PC1 CTT的预防增强了肾损伤诱导囊性形成的能力。为此，我们将在基线和对缺血/再灌流诱导的急性肾损伤的反应中对PSEN2-/-小鼠进行表型分析。我们期望这些分子和生理方法可以为ADPKD治疗的发展提供新的治疗靶点。 公共卫生相关性：旨在了解与常染色体多囊肾病(ADPKD)相关的囊性增长原因的研究具有很高的公共相关性，因为它是肾脏疾病的主要遗传原因，大约每1000人中就有一人受到影响。目前已知的是，产生多囊蛋白-1蛋白的基因PKD1的突变会导致包囊生长。因此，我们的研究将探索这一关键蛋白的调控机制。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of cysts that compress and damage the surrounding normal renal parenchyma, which in turn leads to an eventual decline in renal function. Although two genes have been linked to the ADPKD phenotype, PKD1 and PKD2, majority of cases are due to mutations in the PKD1 gene, which encodes for polycystin-1 (PC1). In order to develop treatments for ADPKD patients, it is necessary to understand the signaling mechanisms associated with PC1 function. Therefore, the goal of this application is to explore the novel role of presenilin-2 (PSEN2) in the control of PC1 signaling. PSEN2 is the catalytic subunit of ¿-secretase, a protease that cleaves PC1, leading to the release of its C-terminal tail (CTT). The resulting PC1 CTT cleavage fragments appear to act as signaling molecules that participate in maintaining cell homeostasis. Although aspects of the signaling pathways associated with cleavage of PC1 CTT have been elucidated, the stimuli that activate PSEN2-mediated PC1 CTT cleavage and the potential involvement of PSEN2 in cystogenesis remain unclear. We have previously determined that PSEN2 knockdown in HEK293 cells markedly reduces PC1 CTT cleavage. Our previous studies also suggest that PC1 CTT cleavage modulates the transcriptional activity of genes related to endoplasmic reticulum (ER) stress, and is induced by renal injuries associated with decreased flow. We hypothesize that PSEN2 is required for proper PC1 CTT cleavage and signaling, and that this function participates in preventing cystogenesis. We propose the following specific aims to support or refute our hypothesis: 1) Aim 1 will establish the concept that PSEN2-mediated PC1 CTT cleavage is stimulated by ER stress, a process that if perturbed has shown to lead to cyst formation. We will measure PC1 CTT cleavage using a luciferase-based assay in conditions of PSEN2 knockdown and ER stress, and identify the size of the particular fragment associated with this response; 2) Aim 2 will establish the concept that PSEN2-mediated cleavage of PC1 plays a critical role in the cyst-preventing activities of PC1, and that prevention of PC1 CTT enhances the capacity of renal injury to induce cyst formation. To do this, we will conduct phenotypic analyses on PSEN2-/- mice at baseline and in response to ischemia/reperfusion-induced acute renal injury. We expect that these molecular and physiological approaches may suggest new therapeutic targets for ADPKD treatment development. PUBLIC HEALTH RELEVANCE: Studies aimed at understanding the causes of cystic growth associated with autosomal polycystic kidney disease (ADPKD) have high public relevance since it is the leading genetic cause of renal disease, afflicting approximately 1 in every 1,000 individuals. It is currently known that mutations in PKD1, a gene that produces the polycystin-1 protein, causes cyst growth. Therefore, our studies will explore the regulatory mechanisms of this critical protein.