Regulators of Autophagosome Formation in Drosophila Muscles

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.     

Autophagy and human diseases

Autophagy is a major intracellular degradative process that delivers cytoplasmic materials to the lysosome for degradation. Since the discovery of autophagy-related (Atg) genes in the 1990s, there has been a proliferation of studies on the physiological and pathological roles of autophagy in a variety of autophagy knockout models. However, direct evidence of the connections between ATG gene dysfunction and human diseases has emerged only recently. There are an increasing number of reports showing that mutations in the ATG genes were identified in various human diseases such as neurodegenerative diseases, infectious diseases, and cancers. Here, we review the major advances in identification of mutations or polymorphisms of the ATG genes in human diseases. Current autophagy-modulating compounds in clinical trials are also summarized.     

Differences in the starvation-induced autophagy response in MDA-MB-231 and MCF-7 breast cancer cells

Breast cancer is a heterogeneous disease with distinct subtypes that have made targeted therapy of breast cancer challenging. Previous studies have demonstrated that an altered autophagy capacity can influence the development of breast cancer. However, the molecular differences in starvation-induced autophagic responses in MDA-MB-231 and MCF-7 cells have not been fully elucidated. In this study, we found that an increase of LC3B-II protein expression level and a decrease of the p62 protein expression level in both cells treated by Earle’s balanced salt solution. Meanwhile, we observed an increase of autophagosome using transmission electron microscopy and an enhancement in the green fluorescence intensity of LC3B protein by confocal microscopy. Furthermore, we detected the expression of 13 autophagy-related (ATG) genes and 11 autophagy signaling pathway-related genes using qPCR. Among 13 ATG genes, we found that 6 genes were up-regulated in treated MDA-MB-231 cells, while 4 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. In addition, among 11 autophagy signaling pathway-related genes, 7 genes were up-regulated in treated MDA-MB-231 cells, while 5 genes were up-regulated and 1 gene was down-regulated in treated MCF-7 cells. These findings suggest that the autophagic response to starvation was different in the two treated cell lines, which will contribute to further study on the molecular mechanism of starvation-induced autophagy and improve the targeted therapy of breast cancer.