多细胞生物发育过程中组织特异的自噬分子机制及活性调控机理

Autophagy is an evolutionarily conserved lysosome-mediated degradation process. It acts as a cell survival mechanism in response to various metabolic stresses and functions as a quality control system by selectively removing unwanted proteins and organelles. Dysfunctional autophagy has been linked to the development of diseases including tumorigenesis and neurodegeneration. Our knowledge of the molecular machinery and regulation of the autophagy pathway has mainly come from analyses of budding yeast and transformed cell lines. However, these studies fail to consider unique multi-cellular animal-specific mechanisms for the regulation and function of autophagy. Multicellular organisms have different cell types which are physiologically distinct. The autophagic machinery may have evolved to accommodate the specific characteristics of each cell type, but little emphasis has been placed on such potential cell type-specific differences. Moreover, the autophagic machinery must also integrate signaling from other cells and environmental conditions to maintain cell, tissue and organism homeostasis. Thus, different cell types may respond differentially to a variety of stresses. Cellular metabolism following nutritional resource restriction may be distinct from that during development.In multicellular organism development, cell context, intercellular communication, organismal stress and homeostasis may all impact the regulation and function of this catabolic process. How autophagy activity is regulated in physiological contexts during multicellular organism development remains largely unknown...Here we established C. elegans as a genetic model system to study tissue-specific variants of the autophagic machinery, including the differential use of core autophagy genes in different tissues and the identification of specific factors involved in tissue-specific variants of autophagy. We are also investigating regulation of autophagy in developmental contexts, and answer questions such as how the autophagic machinery integrates signaling and nutrient availability in multicellular organisms and how autophagy regulation is coordinated between different tissues. Reporters for the autophagy substrate SQST-1/p62 will be expressed specifically in different tissues and then forward genetic screens will be carried out to isolate mutants that cause defective degradation of substrates. Neuronal, intestinal, epidermal and muscle-specific SQST-1 reporters have been generated and more than 50 mutants exhibiting tissue-specific phenotypes in autophagy have been identified from pioneer genetic screens. These genes may define tissue-specific components of the autophagic machinery or factors that positively control autophagy activity. We showed that impaired function of ribosomal protein RPL-43 causes accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we have isolated more than 100 genes that promote autophagy activity upon inactivation. We will also carry out suppressor screens for partial loss of function of autophagy mutants. During the course of this study, we will focus on cell-non-autonomous mechanisms regulating autophagy activity. We will use CRISPR/Cas9 to generate tissue-specific knockouts of autophagy genes, and autophagy activity will be analyzed in the target tissues and in other tissues. Unexpectedly, we have found that loss of function of the worm cuticle structure, which is mainly composed of extracellular matrix, activates autophagy activity. We also found that impairment of neuronal function affects autophagy flux in muscle cells. We will further expand our study to mammalian cells to investigate the identified genes in autophagy regulation. Our study will provide a framework for understanding the regulation of autophagy under physiological conditions and greatly advance our understanding of autophagy in mammals.

自噬可降解胞内物质为细胞提供物质和能量，维持细胞稳态平衡。自噬异常可导致多种疾病的发生。人们对自噬的认识主要源于酵母和细胞系，但这些并不能揭示多细胞生物发育过程中特有的自噬作用和调控机制。我们将利用线虫模型，在不同组织中特异表达自噬底物的报告基因，筛选参与线虫不同发育阶段的特异组织的自噬基因及正调控因子；并筛选功能部分缺失的自噬突变体的抑制子以及抑制rpl-43突变体幼虫肠道细胞中自噬底物聚集的抑制子，鉴定线虫发育过程中自噬的负调控因子。我们还将利用CRISPR/Cas9技术，在线虫不同发育阶段的特异组织中敲除必需自噬基因，研究其对自身及其它组织的影响。我们还将在细胞中探讨这些新基因的功能，并利用小鼠疾病模型研究其与神经退行性疾病的关系。本项目结合线虫筛选体系、细胞系及小鼠疾病模型研究组织特异的自噬分子机制及活性调控机理，这将极大地丰富人们对多细胞生物自噬的理解，并揭示自噬与疾病的关系。

细胞自噬是一种高度保守的由溶酶体介导的降解途径，对维持细胞的稳态平衡至关重要。自噬异常导致包括神经退行性疾病等多种人类疾病的发生发展。以往人们对自噬分子机制的了解主要源自对单细胞酵母遗传筛选得到的自噬基因的研究，但对更为复杂的多细胞生物自噬的特有步骤的机制及调控知之甚少。张宏课题组建立了为研究多细胞生物自噬的线虫模型，发现了系列多细胞生物特有的新自噬基因，在多细胞生物自噬的分子机制、调控机理及生理功能方面取得了重要进展。在项目执行期间，课题组探究了线虫不同发育阶段的特异组织的自噬基因及自噬活性的调控机制，如发现线虫TGF-Beta蛋白DAF-7 协同调控不同组织的自噬，还鉴定了新的自噬基因钙调蛋白酶clp-2参与胚胎后发育时期的神经细胞和表皮细胞的自噬过程。项目组还揭示了内质网膜蛋白Atlastin及VAPs介导自噬起始的ULK1复合体的内质网上定位，以及内质网和自噬小体互作的建立。通过遗传筛选，项目组阐明了多个调控自噬小体成熟的机制，比如发现内质网定位的膜蛋白SUSR2通过调节PI(4)P水平而调控自噬小体与溶酶体融合；还发现蛋白质相变调控自噬的降解机制，并阐述了肌醇多磷酸激酶（IPMK）通过调节转录因子TFEB的“相分离”调控自噬小体成熟的机制。我们还利用小鼠疾病模型研究自噬异常与神经退行性疾病的关系。发现人类神经系统疾病BPAN和ID致病基因WDR45和WDR45B在自噬小体成熟晚期的关键作用及其分子机制，为阐明相关人类疾病的致病机理提供了理论基础。课题组还发现COVID-19病毒SARS-CoV-2通过阻断自噬小体与溶酶体的融合来抑制自噬活性，从而逃脱自噬监控的分子机理。课题组所取得的系列成果突破了前人的认知，在科学研究上开拓了新的方向，具有很高的国际影响力。项目执行五年来共发表高水平论文26篇，全面阐述了多细胞生物自噬的特有步骤的分子机制。

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