Loss of Drosophila Vps16A enhances autophagosome formation through reduced TOR activity.

The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syntaxin 17, the autophagosomal SNARE. Here we show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down Drosophila development. Mechanistically, Tor kinase is less active in Vps16A mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17/Syntaxin 17 blocks autophagic flux without affecting the induction step in Drosophila

Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster.

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc

Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila.

The Atg2-Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. Here we show that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy

Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila.

BACKGROUND Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized. RESULTS Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α. CONCLUSIONS We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity

Advantages and limitations of different p62-based assays for estimating autophagic activity in Drosophila.

Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy

Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows for the possibility of generating patient-derived neurons. A unique feature of these so-called induced neurons (iNs) is the potential to maintain aging and epigenetic signatures of the donor, which is critical given that many diseases of the CNS are age related. Here, we review the published literature on the work that has been undertaken using iNs to model human brain disorders. Furthermore, as disease-modeling studies using this direct neuronal reprogramming approach are becoming more widely adopted, it is important to assess the criteria that are used to characterize the iNs, especially in relation to the extent to which they are mature adult neurons. In particular: i) what constitutes an iN cell, ii) which stages of conversion offer the earliest/optimal time to assess features that are specific to neurons and/or a disorder and iii) whether generating subtype-specific iNs is critical to the disease-related features that iNs express. Finally, we discuss the range of potential biomedical applications that can be explored using patient-specific models of neurological disorders with iNs, and the challenges that will need to be overcome in order to realize these applications.This research has received funding from the New York Stem Cell Foundation, the European Research Council under the European Union's Seventh Framework Programme: FP/2007-2013 Neuro Stem Cell Repair (no. 602278), ERC Grant Agreement no. 30971, the Swedish Research Council treatment of the future grant agreement K2012-99X-22324-01-5, the Swedish Research Council 70862601/Bagadilico, Swedish Parkinson Foundation (Parkinsonfonden), the Strategic Research Area at Lund University Multipark and StemTherapy. JJ is supported by the Swedish Foundation for Strategic Research (#FFL12-0074). JD is supported by a Canadian Institutes of Health Research (CIHR) fellowship (#358492), and RB is supported by an NIHR Biomedical Research Centre grant to the University of Cambridge/Addenbrooke's Hospital. MP is a New York Stem Cell Foundation—Robertson Investigator

Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila

Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans