No abstract

Lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the fly nervous system.

Interaction of the autophagosomal SNARE Syntaxin 17 (Syx17) with the homotypic fusion and vacuole protein–sorting (HOPS) tethering complex is necessary for the fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic degradation and biosynthetic transport to lysosomes and eye pigment granules.

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 conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient‐ and disease‐specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient‐derived material for large‐scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST‐controlled neuron‐specific microRNAs miR‐9 and miR‐124, we show that the effect of REST inhibition is only partially mediated via microRNA up‐regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one‐step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single‐vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease.

DNA methylation contributes to the maintenance of genomic integrity in somatic cells, in part through the silencing of transposable elements. In this study, we use CRISPR-Cas9 technology to delete DNMT1 , the DNA methyltransferase key for DNA methylation maintenance, in human neural progenitor cells (hNPCs). We observe that inactivation of DNMT1 in hNPCs results in viable, proliferating cells despite a global loss of DNA CpG-methylation. DNA demethylation leads to specific transcriptional activation and chromatin remodeling of evolutionarily young, hominoid-specific LINE-1 elements (L1s), while older L1s and other classes of transposable elements remain silent. The activated L1s act as alternative promoters for many protein-coding genes involved in neuronal functions, revealing a hominoid-specific L1-based transcriptional network controlled by DNA methylation that influences neuronal protein-coding genes. Our results provide mechanistic insight into the role of DNA methylation in silencing transposable elements in somatic human cells, as well as further implicating L1s in human brain development and disease.

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.

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.