LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models.

Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.

Disagreement among the three musketeers of the unfolded protein response.

Jipa and Juhász preview results from the lab of Tao Wang (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208147) which show a surprising antagonism between two branches of the unfolded protein response that dictates disease progression in a model of autosomal dominant retinitis pigmentosa.

Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses.

SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Here, using a genetically non-redundant Drosophila model, we show that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. We find that accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response.Abbreviations: Atg8a: Autophagy-related 8a; cnc: cap-n-collar; IFM: indirect flight muscle; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1: Phox and Bem1; ref(2)P: refractory to sigma P; SAR: selective autophagy receptor; UBA: ubiquitin-associated.

Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.

Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.

Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila.

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.

Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.

Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.

The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy.

The small GTPase Rab5 promotes recruitment of the Ccz1-Mon1 guanosine exchange complex to endosomes to activate Rab7, which facilitates endosome maturation and fusion with lysosomes. How these factors function during autophagy is incompletely understood. Here we show that autophagosomes accumulate due to impaired fusion with lysosomes upon loss of the Ccz1-Mon1-Rab7 module in starved Drosophila fat cells. In contrast, autophagosomes generated in Rab5-null mutant cells normally fuse with lysosomes during the starvation response. Consistent with that, Rab5 is dispensable for the Ccz1-Mon1-dependent recruitment of Rab7 to PI3P-positive autophagosomes, which are generated by the action of the Atg14-containing Vps34 PI3 kinase complex. Finally, we find that Rab5 is required for proper lysosomal function. Thus the Ccz1-Mon1-Rab7 module is required for autophagosome-lysosome fusion, whereas Rab5 loss interferes with a later step of autophagy: the breakdown of autophagic cargo within lysosomes.

Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay.

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.