Anne Gjøen Simonsen

• Dahl, Anette Kathinka; Mann, Daniel; Lystad, Alf Håkon Lohsen; Sachse, Carsten; Simonsen, Anne Gjøen & Pankiv, Serhiy (2026). Characterizing the membrane recruitment domain of BDCPs and its role in gray platelet syndrome. Journal of Cell Biology. ISSN 0021-9525. 225(10). doi: 10.1083/jcb.202510098. Full text in Research Archive
• Løchen, Arja Katrina Lea Arnesen; Søreng, Kristiane; Veroni, Chiara; Moncho, Laura Cristina Trachsel; Asp, Nagham Theres & Gaupset, Robin [Show all 10 contributors for this article] (2026). Interaction of HS1BP3 with cortactin modulates TKS5 localisation, cell secretion and cancer malignancy. Molecular Oncology. ISSN 1574-7891. doi: 10.1002/1878-0261.70248. Full text in Research Archive Show summary

  The HCLS1-Binding Protein 3 (HS1BP3) interacts with the SH3 domain of cortactin (CTTN), a protein that contributes to a malignant phenotype in cancers. Here, we demonstrate that high expression of HS1BP3 is associated with reduced survival for gastric adenocarcinoma and triple negative breast carcinoma patients and that HS1BP3 is specifically upregulated in these cancers. We mapped the HS1BP3-cortactin interaction site to the third proline-rich region (PRR3.1) of HS1BP3 and show that this interaction is important for cancer cell proliferation, extracellular matrix degradation and secretion. HS1BP3 expression was found to correlate with expression of the invadopodia scaffold protein TKS5 and we show that the localisation of TKS5 inside multivesicular endosomes is increased in cells expressing an HS1BP3 PRR3.1 mutant. Overall, our results highlight the importance of the direct interaction between HS1BP3 and cortactin in cancer development by regulating cell proliferation, secretion and invasion, which may provide an explanation for the negative correlation between HS1BP3 levels and the survival of gastric adenocarcinoma and triple negative breast cancer patients.

• Lapao, Ana Sofia Da Silva; Johnson, Lauren Sophie; Moncho, Laura Cristina Trachsel; Rodgers, Samuel J; Singh, Sakshi & Ng, Matthew [Show all 9 contributors for this article] (2025). The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism. eLIFE. 14. doi: 10.7554/elife.105541. Full text in Research Archive Show summary

  SYTL5 is a member of the Synaptotagmin-Like (SYTL) protein family that differs from the Synaptotagmin family by having a unique N-terminal Synaptotagmin homology domain that directly interacts with the small GTPase RAB27A. Several SYTL protein family members have been implicated in plasma membrane transport and exocytosis, but the specific function of SYTL5 remains unknown. We here show that SYTL5 is a RAB27A effector and that both proteins localise to mitochondria and vesicles containing mitochondrial material. Mitochondrial recruitment of SYTL5 depends on its interaction with functional RAB27A. We demonstrate that SYTL5-RAB27A positive vesicles containing mitochondrial material, autophagy proteins and LAMP1 form during hypoxia and that depletion of SYTL5 and RAB27A reduces mitophagy under hypoxia mimicking conditions, indicating a role for these proteins in mitophagy. Indeed, we find that SYTL5 interacts with proteins involved in vesicle-mediated transport and cellular response to stress and that its depletion compromises mitochondrial respiration and increases glucose uptake. Intriguingly, SYTL5 expression is significantly reduced in tumours of the adrenal gland and correlates positively with survival for patients with adrenocortical carcinoma.

• Croce, Katherine R; Ng, Christopher; Pankiv, Serhiy; Albarran, Eddy; Langfelder, Peter & Jesus, Ana Ramos de [Show all 22 contributors for this article] (2025). A rare genetic variant confers resistance to neurodegeneration across multiple neurological disorders by augmenting selective autophagy - PMC. Neuron. ISSN 0896-6273. 113(22), p. 3780–3797. doi: 10.1016/j.neuron.2025.08.018. Full text in Research Archive Show summary

  The study of disease modifiers is a powerful way to identify patho-mechanisms associated with disease. Using the strong genetic traits of Huntington’s disease (HD), we identified a rare, single-nucleotide polymorphism (SNP) in WDFY3 associated with a delayed age of onset of up to 23 years. Remarkably, the introduction of the orthologous SNP into mice recapitulates this neuroprotection, significantly delaying neuropathological and behavioral dysfunction in two models of HD. The SNP increases expression of the protein autophagy-linked Fab1, YOTB, Vac1, and EEA1 (FYVE) protein (Alfy), an autophagy adaptor protein for the clearance of aggregated proteins, whose ectopic overexpression is sufficient to capture the neuroprotective effects of the variant. Increasing Alfy expression protects not only against HD but also against the toxicity due to phospho-α-synuclein and AT8-positive accumulation. By combining human and mouse genetics, we have uncovered a pathway that protects against multiple proteinopathies, revealing a much-sought-after, shared therapeutic target across a broad range of neurodegenerative diseases.

• Zapata-Muñoz, Juan; Jiménez-Loygorri, Juan Ignacio; Stumpe, Michael; Villarejo-Zori, Beatriz; Alonso-Gil, Sandra & Terešak, Petra [Show all 11 contributors for this article] (2025). The Developing Retina Undergoes Mitochondrial Remodeling via PINK1/PRKN-dependent Mitophagy. Journal of Molecular Biology (JMB). ISSN 0022-2836. 437(18). doi: 10.1016/j.jmb.2025.169263. Full text in Research Archive Show summary

  Mitophagy, the selective degradation of mitochondria, is essential for retinal ganglion cell (RGC) differentiation and retinal homeostasis. However, the specific mitophagy pathways involved and their temporal dynamics during retinal development and maturation remain poorly understood. Using proteomics analysis of isolated mouse retinas across developmental stages and the mitophagy reporter mouse line, mito-QC, we characterized mitophagy throughout retinogenesis. While mitolysosomes were more prevalent in the mature retina, we observed two distinct mitophagy peaks during embryonic development. The first, independent of PTEN-induced kinase 1 (PINK1) activation, was associated with RGCs. The second, PINK1-dependent peak was triggered after an increase in retinal oxidative stress. This PINK1-dependent, oxidative stress-induced mitophagy pathway is conserved in mice and zebrafish, providing the first evidence of programmed, PINK1-dependent mitophagy during development.

• Mauthe, Mario; Beek, Nicole van de; Mari, Muriel; Korsten, Giel; Nobari, Parisa & Castelino, Kennith B. [Show all 17 contributors for this article] (2025). A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy. Nature Cell Biology. ISSN 1465-7392. 27(9), p. 1448–1464. doi: 10.1038/s41556-025-01747-1. Full text in Research Archive Show summary

  Perturbations in protein quality control lead to the accumulation of misfolded proteins and protein aggregates, which can compromise health and lifespan. One key mechanism eliminating protein aggregates is aggrephagy, a selective type of autophagy. Here we reveal that fragmentation is required before autophagic clearance of various types of amorphous aggregates. This fragmentation requires both the 19S proteasomal regulatory particle and the DNAJB6-HSP70-HSP110 chaperone module. These two players are also essential for aggregate compaction that leads to the clustering of the selective autophagy receptors, which initiates the autophagic removal of the aggregates. We also found that the same players delay the formation of disease-associated huntingtin inclusions. This study assigns a novel function to the 19S regulatory particle and the DNAJB6-HSP70-HSP110 module, and uncovers that aggrephagy entails a piecemeal process, with relevance for proteinopathies.

• Croce, Katherine R.; Ng, Christopher; Pankiv, Serihy; Albarran, Eddy; Langfelder, Peter & Jesus, Ana Ramos de [Show all 22 contributors for this article] (2025). A rare genetic variant confers resistance to neurodegeneration across multiple neurological disorders by augmenting selective autophagy. Neuron. ISSN 0896-6273. 113(22), p. 3780–3797. doi: 10.1016/j.neuron.2025.08.018. Full text in Research Archive Show summary

  The study of disease modifiers is a powerful way to identify patho-mechanisms associated with disease. Using the strong genetic traits of Huntington’s disease (HD), we identified a rare, single-nucleotide polymorphism (SNP) in WDFY3 associated with a delayed age of onset of up to 23 years. Remarkably, the introduction of the orthologous SNP into mice recapitulates this neuroprotection, significantly delaying neuropathological and behavioral dysfunction in two models of HD. The SNP increases expression of the protein autophagy-linked Fab1, YOTB, Vac1, and EEA1 (FYVE) protein (Alfy), an autophagy adaptor protein for the clearance of aggregated proteins, whose ectopic overexpression is sufficient to capture the neuroprotective effects of the variant. Increasing Alfy expression protects not only against HD but also against the toxicity due to phospho-α-synuclein and AT8-positive accumulation. By combining human and mouse genetics, we have uncovered a pathway that protects against multiple proteinopathies, revealing a much-sought-after, shared therapeutic target across a broad range of neurodegenerative diseases.

• Moncho, Laura Cristina Trachsel; Veroni, Chiara; Mathai, Benan John; Lapao, Ana Sofia Da Silva; Singh, Sakshi & Asp, Nagham Theres [Show all 9 contributors for this article] (2025). SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics. Journal of Cell Biology. ISSN 0021-9525. 224(5). doi: 10.1083/jcb.202404009. Full text in Research Archive Show summary

  We here identify the endosomal protein SNX10 as a negative regulator of piecemeal mitophagy of OXPHOS machinery components. In control conditions, SNX10 localizes to early endocytic compartments in a PtdIns3P-dependent manner and modulates endosomal trafficking but also shows dynamic connections with mitochondria. Upon hypoxia-mimicking conditions, SNX10 localizes to late endosomal structures containing selected mitochondrial proteins, including COX-IV and SAMM50, and the autophagy proteins SQSTM1/p62 and LC3B. The turnover of COX-IV was enhanced in SNX10-depleted cells, with a corresponding reduced mitochondrial respiration and citrate synthase activity. Importantly, zebrafish larvae lacking Snx10 show reduced levels of Cox-IV, as well as elevated ROS levels and ROS-mediated cell death in the brain, demonstrating the in vivo relevance of SNX10-mediated modulation of mitochondrial bioenergetics.

• Moncho, Laura Cristina Trachsel; Mathai, Benan John; Veroni, Chiara & Simonsen, Anne Gjøen (2025). SNX10 at the crossroad of endocytosis and piecemeal mitophagy. Autophagy. ISSN 1554-8627. 21(9), p. 2077–2079. doi: 10.1080/15548627.2025.2499641. Full text in Research Archive Show summary

  Mitophagy targets damaged or dysfunctional mitochondria for lysosomal degradation. While canonical mitophagy pathways target the whole mitochondria for lysosomal degradation, it has become clear that selected mitochondrial components can be targeted for lysosomal degradation via other pathways, such as piecemeal mitophagy or mitochondria-derived vesicles. In a recent study, we identified the PX domain-containing endosomal protein SNX10 as a negative modulator of piecemeal mitophagy. Endosomal SNX10-positive vesicles dynamically interact with mitochondria and acquire selected mitochondrial proteins upon hypoxia. Zebrafish larvae lacking Snx10 show elevated Cox-IV degradation, increased levels of reactive oxygen species (ROS), and ROS-dependent neuronal death.

• Pankiv, Serhiy; Dahl, Anette Kathinka; Aas, Aleksander; Andersen, Rosa Linn; Brech, Andreas & Holland, Petter [Show all 9 contributors for this article] (2024). BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways. Journal of Cell Biology. ISSN 0021-9525. 223(12). doi: 10.1083/jcb.202408173. Full text in Research Archive Show summary

  We identify BEACH domain–containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.

• Sporbeck, Katharina; Haas, Maximilian L.; Pastor-Maldonado, Carmen J.; Schüssele, David S.; Hunter, Catherine & Takacs, Zsuzsanna [Show all 18 contributors for this article] (2023). The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension. Communications Biology. 6(1). doi: 10.1038/s42003-023-05236-9. Full text in Research Archive Show summary

  Abstract Human WIPI β-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.

• Kaur, Namrita; Ballina, Laura R de la; Haukaas, Håvard Styrkestad; Torgersen, Maria Lyngaas; Radulovic, Maja & Munson, Michael J [Show all 11 contributors for this article] (2023). TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy. EMBO Journal. ISSN 0261-4189. 42(17), p. 1–19. doi: 10.15252/embj.2022113105. Full text in Research Archive Show summary

  Abstract Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin‐like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1‐containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1‐containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage‐induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1‐ATG5‐ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1‐ATG5‐ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

• Charsou, Chara; Ng, Matthew & Simonsen, Anne Gjøen (2022). Regulation of autophagosome biogenesis and mitochondrial bioenergetics by the cholesterol transport protein GRAMD1C. Autophagy. ISSN 1554-8627. doi: 10.1080/15548627.2022.2155020. Full text in Research Archive
• Sikkeland, Jørgen; Ng, Matthew; Nenseth, Hatice Zeynep; Ünal, Bilal; Qu, Su & Jin, Yang [Show all 8 contributors for this article] (2022). STAMP2 suppresses autophagy in prostate cancer cells by modulating the integrated stress response pathway. American Journal of Cancer Research. 12(1), p. 327–336. Full text in Research Archive Show summary

  Six Transmembrane Protein of Prostate 2 (STAMP2) is critical for prostate cancer (PCa) growth. We previously showed that STAMP2 regulates the expression of stress induced transcription factor ATF4, which is implicated in starvation-induced autophagy. We therefore investigated whether STAMP2 is involved in the regulation of autophagy in PCa cells. Here we show that STAMP2 suppresses autophagy in PCa cells through modulation of the integrated stress response axis. We also find that STAMP2 regulates mitochondrial respiration. These findings suggest that STAMP2 has significant metabolic effects through mitochondrial function and autophagy, both of which support PCa growth.

• Cable, Jennifer; Weber-Ban, Eilika; Clausen, Tim; Walters, Kylie J.; Sharon, Michal & Finley, Daniel J. [Show all 32 contributors for this article] (2022). Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report. Annals of the New York Academy of Sciences. ISSN 0077-8923. 1510(1), p. 79–99. doi: 10.1111/nyas.14745. Full text in Research Archive
• Simonsen, Anne Gjøen & Wollert, Thomas (2022). Don't forget to be picky – selective autophagy of protein aggregates in neurodegenerative diseases. Current Opinion in Cell Biology. ISSN 0955-0674. 75, p. 1–7. doi: 10.1016/j.ceb.2022.01.009. Full text in Research Archive Show summary

  The homeostasis of cells depends on the selective degradation of damaged or superfluous cellular components. Autophagy is the major pathway that recognizes such components, sequesters them in de novo formed autophagosomes and delivers them to lysosomes for degradation. The recognition of specific cargo and the biogenesis of autophagosomes involve a dedicated machinery of autophagy related (ATG) proteins. Intense research over the past decades has revealed insights into the function of autophagy proteins and mechanisms that govern cargo recognition. Other aspects including the molecular mechanisms involved in the onset of human diseases are less well understood. However, autophagic dysfunctions, caused by age related decline in autophagy or mutations in ATG proteins, are directly related to a large number of human pathologies including neurodegenerative disorders. Here, we review most recent discoveries and breakthroughs in selective autophagy and its relationship to neurodegeneration.

• Ganley, Ian G. & Simonsen, Anne Gjøen (2022). Diversity of mitophagy pathways at a glance. Journal of Cell Science. ISSN 0021-9533. 135(23), p. 1–8. doi: 10.1242/jcs.259748. Full text in Research Archive Show summary

  ABSTRACT Mitochondria are crucial organelles that play a central role in various cell signaling and metabolic pathways. A healthy mitochondrial population is maintained through a series of quality control pathways and requires a fine-tuned balance between mitochondrial biogenesis and degradation. Defective targeting of dysfunctional mitochondria to lysosomes through mitophagy has been linked to several diseases, but the underlying mechanisms and the relative importance of distinct mitophagy pathways in vivo are largely unknown. In this Cell Science at a Glance and the accompanying poster, we describe our current understanding of how parts of, or whole, mitochondria are recognized by the autophagic machinery and targeted to lysosomes for degradation. We also discuss how this might be regulated under different physiological conditions to maintain mitochondrial and cellular health.

• Søreng, Kristiane; Pankiv, Serhiy; Bergsmark, Camilla; Haugsten, Ellen Margrethe; Dahl, Anette Kathinka & Ballina, Laura R de la [Show all 9 contributors for this article] (2022). ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration. Journal of Cell Science. ISSN 0021-9533. 135(4). doi: 10.1242/jcs.259138. Full text in Research Archive Show summary

  ABSTRACT Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.

• Ng, Matthew; Charsou, Chara; Lapao, Ana Sofia Da Silva; Singh, Sakshi; Moncho, Laura Cristina Trachsel & Schultz, Sebastian Wolfgang [Show all 9 contributors for this article] (2022). The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics. Nature Communications. 13(1). doi: 10.1038/s41467-022-33933-2. Full text in Research Archive Show summary

  Abstract During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the membranes that form the autophagosome, remain to be fully characterized. Here, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and a corresponding increase in autophagy initiation events. We further show that the ER-localized cholesterol transport protein GRAMD1C functions as a negative regulator of starvation-induced autophagy and that both its cholesterol transport VASt domain and membrane binding GRAM domain are required for GRAMD1C-mediated suppression of autophagy initiation. Similar to its yeast orthologue, GRAMD1C associates with mitochondria through its GRAM domain. Cells lacking GRAMD1C or its VASt domain show increased mitochondrial cholesterol levels and mitochondrial oxidative phosphorylation, suggesting that GRAMD1C may facilitate cholesterol transfer at ER-mitochondria contact sites. Finally, we demonstrate that expression of GRAMD family proteins is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.

• Rahman, Mohummad Aminur; Engelsen, Agnete; Sarowar, Shahin; Bindesbøll, Christian; Birkeland, Even & Goplen, Dorota [Show all 10 contributors for this article] (2022). Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway. Frontiers in Cell and Developmental Biology. 10, p. 1–23. doi: 10.3389/fcell.2022.1022191. Full text in Research Archive Show summary

  Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5−/− (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

• Xu, Jing; Yang, Kevin C; Go, Nancy Erro; Colborne, Shane; Ho, Cally J & Hosseini-Beheshti, Elham [Show all 11 contributors for this article] (2022). Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations. Autophagy. ISSN 1554-8627. 18(11), p. 2547–2560. doi: 10.1080/15548627.2022.2039535. Full text in Research Archive Show summary

  Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.

• Munson, Michael Joseph; Mathai, Benan John; Ng, Matthew; Moncho, Laura Cristina Trachsel; Ballina, Laura R de la & Simonsen, Anne Gjøen (2022). GAK and PRKCD kinases regulate basal mitophagy. Autophagy. ISSN 1554-8627. 18(2), p. 467–469. doi: 10.1080/15548627.2021.2015154. Full text in Research Archive Show summary

  The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal “housekeeping” mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings.

• Maestro, Inés; Ballina, Laura R de la; Porras, Gracia; Corrochano, Silvia; Lago, Eva de & Simonsen, Anne Gjøen [Show all 8 contributors for this article] (2022). Discovery of Mitophagy Inhibitors with Therapeutic Potential in Different Familial Amyotrophic Lateral Sclerosis Mutations. International Journal of Molecular Sciences. ISSN 1661-6596. 23(20). doi: 10.3390/ijms232012676. Full text in Research Archive Show summary

  Mitophagy is the selective degradation of mitochondria by autophagy. It promotes the turnover of mitochondria and prevents the accumulation of dysfunctional mitochondria, which can lead to cellular degeneration. Mitophagy is known to be altered in several pathological conditions, especially in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We recently demonstrated an increase in autophagy flux in lymphoblasts from ALS patients bearing a mutation in SOD1. Thus, the identification of mitophagy inhibitors may be a therapeutic option to recover mitochondrial homeostasis. Here, using a phenotypic mitophagy assay, we identified a new mitophagy inhibitor, the small molecule named IGS2.7 from the MBC library. Interestingly, the treatment of different cellular and in vivo models of ALS with mutations on SOD1 and TARDBP with this inhibitor restores autophagy to control levels. These results point mitophagy inhibitors, especially IGS2.7, to a new therapeutic approach for familial ALS patients.

• Durgan, Joanne; Lystad, Alf Håkon Lohsen; Sloan, Katherine; Carlsson, Sven R.; Wilson, Michael I. & Marcassa, Elena [Show all 14 contributors for this article] (2021). Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine. Molecular Cell. ISSN 1097-2765. 81(9), p. 2031–2040.e8. doi: 10.1016/j.molcel.2021.03.020. Full text in Research Archive Show summary

  Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

• Munson, Michael Joseph; Mathai, Benan J.; Ng, Matthew; Moncho, Laura Cristina Trachsel; Ballina, Laura R de la & Schultz, Sebastian W. [Show all 13 contributors for this article] (2021). GAK and PRKCD are positive regulators of PRKN-independent mitophagy. Nature Communications. 12(1). doi: 10.1038/s41467-021-26331-7. Full text in Research Archive Show summary

  The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN- dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

• Ng, Matthew; Wai, Timothy & Simonsen, Anne Gjøen (2021). Quality control of the mitochondrion. Developmental Cell. ISSN 1534-5807. 56(7), p. 881–905. doi: 10.1016/j.devcel.2021.02.009. Full text in Research Archive
• Giovannucci, Tatiana A.; Salomons, Florian A.; Stoy, Henriette; Herzog, Laura K.; Xu, Shanshan & Qian, Weixing [Show all 15 contributors for this article] (2021). Identification of a novel compound that simultaneously impairs the ubiquitin-proteasome system and autophagy. Autophagy. ISSN 1554-8627. doi: 10.1080/15548627.2021.1988359. Full text in Research Archive Show summary

  The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.

• Terešak, Petra; Lapao, Ana Sofia Da Silva; Subic, Nemanja; Boya, Patricia; Elazar, Zvulun & Simonsen, Anne Gjøen (2021). Regulation of PRKN-independent mitophagy. Autophagy. ISSN 1554-8627. 18(1), p. 24–39. doi: 10.1080/15548627.2021.1888244. Full text in Research Archive
• Kumar, Suresh; Javed, Ruheena; Mudd, Michal H.; Pallikkuth, Sandeep; Lidke, Keith A. & Jain, Ashish [Show all 22 contributors for this article] (2021). Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes. Cell. ISSN 0092-8674. 184(24), p. 5950–5969. doi: 10.1016/j.cell.2021.10.017. Full text in Research Archive Show summary

  The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins

• Klionsky, Daniel J.; Petroni, Giulia; Amaravadi, Ravi K; Baehrecke, Eric H; Ballabio, Andrea & Boya, Patricia [Show all 59 contributors for this article] (2021). Autophagy in major human diseases. EMBO Journal. ISSN 0261-4189. 40, p. e108863–e108863. doi: 10.15252/embj.2021108863. Full text in Research Archive Show summary

  Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

• Sharma, Kulbhushan; Asp, Nagham Theres; Harrison, Sean; Siller, Richard; Baumgarten, Saphira Felicitas & Gupta, Swapnil [Show all 10 contributors for this article] (2021). Autophagy modulates cell fate decisions during lineage commitment. Autophagy. ISSN 1554-8627. p. 1–17. doi: 10.1080/15548627.2021.2008691. Full text in Research Archive Show summary

  Early events during development leading to exit from a pluripotent state and commitment toward a specific germ layer still need in depth understanding. Autophagy has been shown to play a crucial role in both development and differentiation. This study employs human embryonic and induced pluripotent stem cells to understand the early events of lineage commitment with respect to the role of autophagy in this process. Our data indicate that a dip in autophagy facilitates exit from pluripotency. Upon exit, we demonstrate that the modulation of autophagy affects SOX2 levels and lineage commitment, with induction of autophagy promoting SOX2 degradation and mesendoderm formation, whereas inhibition of autophagy causes SOX2 accumulation and neuroectoderm formation. Thus, our results indicate that autophagy-mediated SOX2 turnover is a determining factor for lineage commitment. These findings will deepen our understanding of development and lead to improved methods to derive different lineages and cell types.

• Maestro, Inés; Ballina, Laura R de la; Simonsen, Anne Gjøen; Boya, Patricia & Martinez, Ana (2021). Phenotypic Assay Leads to Discovery of Mitophagy Inducers with Therapeutic Potential for Parkinson's Disease. ACS Chemical Neuroscience. 12(24), p. 4512–4523. doi: 10.1021/acschemneuro.1c00529. Full text in Research Archive Show summary

  Mitophagy, the selective degradation of mitochondria by autophagy, involved in important physiological processes and defects in pathways has been reported in pathological conditions, such as neurodegeneration. Thus, mitophagy is an interesting target for drug discovery programs. In this investigation, we used robust phenotypic assay to screen a set of 50 small heterocyclic compounds to identify inducers of mitophagy. We identified two compounds, VP07 and JAR1.39, that induce Parkin-dependent mitophagy. Based on structure–activity relationship studies, we proposed the ability of the compounds to act as light chain 3 (LC3) interactors, similar to cardiolipin or ceramide, triggering mitophagy via Pink1/Parkin. Finally, we show promising therapeutic applicability in a cellular model of Parkinson’s disease.

• Corkery, Dale P.; Nadeem, Aftab; Aung, Kyaw Min; Hassan, Ahmed; Liu, Tao & Cervantes-Rivera, Ramón [Show all 14 contributors for this article] (2021). Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy. Journal of Cell Science. ISSN 0021-9533. 134(5). doi: 10.1242/jcs.252015. Full text in Research Archive
• Bindesbøll, Christian; Aas, Aleksander; Ogmundsdottir, Margret Helga; Pankiv, Serhiy; Reine, Trine M. & Zoncu, Roberto [Show all 7 contributors for this article] (2020). NBEAL1 controls SREBP2 processing and cholesterol metabolism and is a susceptibility locus for coronary artery disease. Scientific Reports. 10(1). doi: 10.1038/s41598-020-61352-0. Full text in Research Archive Show summary

  Dysregulated cholesterol homeostasis promotes the pathology of atherosclerosis, myocardial infarction and strokes. Cellular cholesterol is mainly regulated at the transcriptional level by SREBP2, but also through uptake of extracellular cholesterol from low density lipoproteins (LDL) via expression of LDL receptors (LDLR) at the cell surface. Identification of the mechanisms involved in regulation of these processes are thus key to understand the pathology of coronary artery disease. Here, we identify the large and poorly characterized BEACH domain protein Neurobeachin-like (NBEAL) 1 as a Golgi- associated protein required for regulation of cholesterol metabolism. NBEAL1 is most abundantly expressed in arteries. Genetic variants in NBEAL1 are associated with decreased expression of NBEAL1 in arteries and increased risk of coronary artery disease in humans. We show that NBEAL1 regulates cholesterol metabolism by modulating LDLR expression in a mechanism involving interaction with SCAP and PAQR3 and subsequent SREBP2-processing. Thus, low expression of NBEAL1 may lead to increased risk of coronary artery disease by downregulation of LDLR levels.

• Gawel, Kinga Aurelia; Turski, Waldemar A.; Ent, Wietske van der; Mathai, Benan John; Kirstein-Smardzewska, Karolina Joanna & Simonsen, Anne [Show all 7 contributors for this article] (2020). Phenotypic Characterization of Larval Zebrafish (Danio rerio) with Partial Knockdown of the cacna1a Gene. Molecular Neurobiology. ISSN 0893-7648. 57(4). doi: 10.1007/s12035-019-01860-x. Full text in Research Archive Show summary

  The CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.

• Lotsberg, Maria Lie; Wnuk-Lipinska, Katarzyna; Terry, Stéphane; Tan, Tuan Zea; Lu, Ning & Moncho, Laura Trachsel [Show all 26 contributors for this article] (2020). AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells. Journal of Thoracic Oncology. ISSN 1556-0864. 15(6), p. 973–999. doi: 10.1016/j.jtho.2020.01.015. Full text in Research Archive Show summary

  Introduction Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. Methods We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. Results We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). Conclusion Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

• Melia, Thomas; Lystad, Alf Håkon & Simonsen, Anne (2020). Autophagosome biogenesis: From membrane growth to closure. Journal of Cell Biology. ISSN 0021-9525. 219(6). doi: 10.1083/jcb.202002085. Full text in Research Archive Show summary

  Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an autophagosome). This process has remained enigmatic since its initial discovery >50 yr ago, but our understanding of the mechanisms involved in autophagosome biogenesis has increased substantially during the last 20 yr. Several key questions do remain open, however, including, What determines the site of autophagosome nucleation? What is the origin and lipid composition of the autophagosome membrane? How is cargo sequestration regulated under nonselective and selective types of autophagy? This review provides key insight into the core molecular mechanisms underlying autophagosome biogenesis, with a specific emphasis on membrane modeling events, and highlights recent conceptual advances in the field.

• Navarro, Andrea Gras; Espedal, Heidi; Joseph, Justin Vareecal; Moncho, Laura Trachsel; Bahador, Marzieh & Gjertsen, Bjørn Tore [Show all 12 contributors for this article] (2019). Pretreatment of glioblastoma with bortezomib potentiates natural killer cell cytotoxicity through TRAIL/DR5 mediated apoptosis and prolongs animal survival. Cancers. 11:996(7), p. 1–25. doi: 10.3390/cancers11070996. Full text in Research Archive Show summary

  Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC–MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24–46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33–76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252–0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295–0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood–brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284–0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

• Lystad, Alf Håkon; Carlsson, Sven R. & Simonsen, Anne (2019). Toward the function of mammalian ATG12-ATG5-ATG16L1 complex in autophagy and related processes. Autophagy. ISSN 1554-8627. 15(8), p. 1485–1486. doi: 10.1080/15548627.2019.1618100. Full text in Research Archive Show summary

  The machinery that decorates autophagic membranes with lipid-conjugated LC3/GABARAP is not yet fully understood. We recently reported the purification of the full-length ATG12–ATG5-ATG16L1 complex, and in reconstitution experiments with purified ATG7, ATG3, and LC3/GABARAP in vitro, together with rescue experiments in knockout cells, important aspects of the complete lipidation reaction were revealed. Hitherto unobserved membrane-binding regions in ATG16L1 were found, contributing to properties that explain the crucial role of this protein in membrane targeting and LC3/GABARAP lipidation in macroautophagy/autophagy and other related processes.

• Lystad, Alf Håkon; Carlsson, Sven R; Ballina, Laura Rodriguez de la; Kauffman, Karlina J; Nag, Shanta & Yoshimori, Tamotsu [Show all 8 contributors for this article] (2019). Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes. Nature Cell Biology. ISSN 1465-7392. 21(3), p. 372–383. doi: 10.1038/s41556-019-0274-9. Full text in Research Archive Show summary

  Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single-membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5–ATG16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy but essential for VPS34-independent LC3B lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. This C-terminal membrane-binding region is present only in the β-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

• Abudu, Yakubu Princely; Pankiv, Serhiy; Mathai, Benan John; Lystad, Alf Håkon; Bindesbøll, Christian & Brenne, Hanne Britt [Show all 14 contributors for this article] (2019). NIPSNAP1 and NIPSNAP2 act as “eat-me signal” for mitophagy. Developmental Cell. ISSN 1534-5807. 49(4), p. 509–525.e12. doi: 10.1016/j.devcel.2019.03.013. Full text in Research Archive Show summary

  The clearance of damaged or dysfunctional mitochondria by selective autophagy (mitophagy) is important for cellular homeostasis and prevention of disease. Our understanding of the mitochondrial signals that trigger their recognition and targeting by mitophagy is limited. Here, we show that the mitochondrial matrix proteins 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and NIPSNAP2 accumulate on the mitochondria surface upon mitochondrial depolarization. There, they recruit proteins involved in selective autophagy, including autophagy receptors and ATG8 proteins, thereby functioning as an “eat me” signal for mitophagy. NIPSNAP1 and NIPSNAP2 have a redundant function in mitophagy and are predominantly expressed in different tissues. Zebrafish lacking a functional Nipsnap1 display reduced mitophagy in the brain and parkinsonian phenotypes, including loss of tyrosine hydroxylase (Th1)-positive dopaminergic (DA) neurons, reduced motor activity, and increased oxidative stress.

• Moller, Katrin; Sigurbjornsdottir, Sara; Arnthorsson, Asgeir; Podenberg, Vivian; Dilshat, Ramile & Fock, Valerie [Show all 16 contributors for this article] (2019). MITF has a central role in regulating starvation-induced autophagy in melanoma. Scientific Reports. 9. doi: 10.1038/s41598-018-37522-6. Full text in Research Archive Show summary

  The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.

• Chaurasia, Madhuri; Gupta, Swapnil; Das, Asmita; Dwarakanath, B. S.; Simonsen, Anne & Sharma, Kulbhushan (2019). Radiation induces EIF2AK3/PERK and ERN1/IRE1 mediated pro-survival autophagy. Autophagy. ISSN 1554-8627. 15(8), p. 1391–1406. doi: 10.1080/15548627.2019.1582973. Full text in Research Archive Show summary

  Cellular effects of ionizing radiation include oxidative damage to macromolecules, unfolded protein response (UPR) and metabolic imbalances. Oxidative stress and UPR have been shown to induce macroautophagy/autophagy in a context-dependent manner and are crucial factors in determining the fate of irradiated cells. However, an in-depth analysis of the relationship between radiation-induced damage and autophagy has not been explored. In the present study, we investigated the relationship between radiation-induced oxidative stress, UPR and autophagy in murine macrophage cells. A close association was observed between radiation-induced oxidative burst, UPR and induction of autophagy, with the possible involvement of EIF2AK3/PERK (eukaryotic translation initiation factor 2 alpha kinase 3) and ERN1/IRE1 (endoplasmic reticulum [ER] to nucleus signaling 1). Inhibitors of either UPR or autophagy reduced the cell survival indicating the importance of these processes after radiation exposure. Moreover, modulation of autophagy affected lethality in the whole body irradiated C57BL/6 mouse. These findings indicate that radiation-induced autophagy is a pro-survival response initiated by oxidative stress and mediated by EIF2AK3 and ERN1.

• Gawel, Kinga Aurelia; Turski, Waldemar A.; Ent, Wietske van der; Mathai, Benan John; Kirstein-Smardzewska, Karolina Joanna & Simonsen, Anne [Show all 7 contributors for this article] (2019). Phenotypic Characterization of Larval Zebrafish (Danio rerio) with Partial Knockdown of the cacna1a Gene. Molecular Neurobiology. ISSN 0893-7648. doi: 10.1007/s12035-019-01860-x. Full text in Research Archive Show summary

  The CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.

• Herhaus, Lina; Bhaskara, Ramachandra M; Lystad, Alf Håkon; Gestal-Mato, Uxia; Covarrubias-Pinto, Adriana & Bonn, Florian [Show all 9 contributors for this article] (2019). TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease. EMBO Reports. ISSN 1469-221X. doi: 10.15252/embr.201948317. Full text in Research Archive Show summary

  Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post‐translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP‐L2 on surface‐exposed serine residues (LC3C S93 and S96; GABARAP‐L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP‐L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4‐mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP‐L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de‐conjugation of LC3C and GABARAP‐L2 to autophagosomes by TBK1‐mediated phosphorylation.

• Zhen, Yan; Spangenberg, Helene; Munson, Michael Joseph; Brech, Andreas; Schink, Kay Oliver & Tan, Kia Wee [Show all 13 contributors for this article] (2019). ESCRT-mediated phagophore sealing during mitophagy. Autophagy. ISSN 1554-8627. p. 1–16. doi: 10.1080/15548627.2019.1639301. Full text in Research Archive Show summary

  Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux.

• Lystad, Alf Håkon & Simonsen, Anne (2019). Mechanisms and Pathophysiological Roles of the ATG8 Conjugation Machinery. Cells. 8(9), p. 973–973. doi: 10.3390/cells8090973. Full text in Research Archive Show summary

  Since their initial discovery around two decades ago, the yeast autophagy-related (Atg)8 protein and its mammalian homologues of the light chain 3 (LC3) and γ-aminobutyric acid receptor associated proteins (GABARAP) families have been key for the tremendous expansion of our knowledge about autophagy, a process in which cytoplasmic material become targeted for lysosomal degradation. These proteins are ubiquitin-like proteins that become directly conjugated to a lipid in the autophagy membrane upon induction of autophagy, thus providing a marker of the pathway, allowing studies of autophagosome biogenesis and maturation. Moreover, the ATG8 proteins function to recruit components of the core autophagy machinery as well as cargo for selective degradation. Importantly, comprehensive structural and biochemical in vitro studies of the machinery required for ATG8 protein lipidation, as well as their genetic manipulation in various model organisms, have provided novel insight into the molecular mechanisms and pathophysiological roles of the mATG8 proteins. Recently, it has become evident that the ATG8 proteins and their conjugation machinery are also involved in intracellular pathways and processes not related to autophagy. This review focuses on the molecular functions of ATG8 proteins and their conjugation machinery in autophagy and other pathways, as well as their links to disease.

• Herzog, Laura K; Kevei, Eva; Marchante, Ricardo; Botther, Claudia; Bindesbøll, Christian & Lystad, Alf Håkon [Show all 12 contributors for this article] (2019). The Machado-Joseph disease deubiquitylase ataxin-3 interacts with LC3C/GABARAP and promotes autophagy. Aging Cell. ISSN 1474-9718. doi: 10.1111/acel.13051. Full text in Research Archive Show summary

  The pathology of spinocerebellar ataxia type 3, also known as Machado‐Joseph disease, is triggered by aggregation of toxic ataxin‐3 (ATXN3) variants containing expanded polyglutamine repeats. The physiological role of this deubiquitylase, however, remains largely unclear. Our recent work showed that ATX‐3, the nematode orthologue of ATXN3, together with the ubiquitin‐directed segregase CDC‐48, regulates longevity in Caenorhabditis elegans. Here, we demonstrate that the long‐lived cdc‐48.1; atx‐3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX‐3. Reducing the levels of the autophagy protein BEC‐1 sensitized worms to the effect of ATX‐3 deficiency, suggesting a role of ATX‐3 in autophagy. In support of this conclusion, the depletion of ATXN3 in human cells caused a reduction in autophagosomal degradation of proteins. Surprisingly, reduced degradation in ATXN3‐depleted cells coincided with an increase in the number of autophagosomes while levels of lipidated LC3 remained unaffected. We identified two conserved LIR domains in the catalytic Josephin domain of ATXN3 that directly interacted with the autophagy adaptors LC3C and GABARAP in vitro. While ATXN3 localized to early autophagosomes, it was not subject to lysosomal degradation, suggesting a transient regulatory interaction early in the autophagic pathway. We propose that the deubiquitylase ATX‐3/ATXN3 stimulates autophagic degradation by preventing superfluous initiation of autophagosomes, thereby promoting an efficient autophagic flux important to survive starvation.

• Rahman, Mohummad Aminur; Navarro, Andrea Gras; Brekke, Jorunn; Engelsen, Agnete; Bindesbøll, Christian & Sarowar, Shahin [Show all 16 contributors for this article] (2019). Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival. British Journal of Cancer. ISSN 0007-0920. 121, p. 545–555. doi: 10.1038/s41416-019-0551-1. Full text in Research Archive Show summary

  Background Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ. Methods Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan–Meier method. Results Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood–brain barrier, diminished proteasome activity and significantly prolonged animal survival. Conclusion BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

• Abudu, Yakubu Princely; Pankiv, Serhiy; Mathai, Benan John; Lamark, Trond; Johansen, Terje & Simonsen, Anne (2019). NIPSNAP1 and NIPSNAP2 act as “eat me” signals to allow sustained recruitment of autophagy receptors during mitophagy. Autophagy. ISSN 1554-8627. doi: 10.1080/15548627.2019.1637642. Full text in Research Archive
• Ballina, Laura Rodriguez de la; Munson, Michael Joseph & Simonsen, Anne (2019). Lipids and Lipid-Binding Proteins in Selective Autophagy. Journal of Molecular Biology (JMB). ISSN 0022-2836. p. 1–25. doi: 10.1016/j.jmb.2019.05.051. Full text in Research Archive Show summary

  Eukaryotic cells have the capacity to degrade intracellular components through a lysosomal degradation pathway called macroautophagy (henceforth referred to as autophagy) in which superfluous or damaged cytosolic entities are engulfed and separated from the rest of the cell constituents into double membraned vesicles known as autophagosomes. Autophagosomes then fuse with endosomes and lysosomes, where cargo is broken down into basic building blocks that are released to the cytoplasm for the cell to reuse. Autophagic degradation can target either cytoplasmic material in bulk (non-selective autophagy) or particular cargo in what is called selective autophagy. Proper autophagic turnover requires the orchestrated participation of several players that need to be tightly and temporally coordinated. Whereas a large number of autophagy-related (ATG) proteins have been identified and their functions and regulation are starting to be understood, there is substantially less knowledge regarding the specific lipids constituting the autophagic membranes as well as their role in initiating, enabling or regulating the autophagic process. This review focuses on lipids and their corresponding binding proteins that are crucial in the process of selective autophagy.

• Johannessen, Tor-Christian Aase; Hasan-Olive, Md Mahdi; Zhu, Huaiyang; Denisova, Oxana V.; Grudic, Amra & Latif, Md Abdul [Show all 20 contributors for this article] (2018). Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide. International Journal of Cancer. ISSN 0020-7136. 144(7), p. 1735–1745. doi: 10.1002/ijc.31912. Full text in Research Archive
• Kraft, Claudine; Boya, Patricia; Codogno, Patrice; Elazar, Zvulun; Eskelinen, Eeva-Liisa & Farres, Judith [Show all 14 contributors for this article] (2018). Driving next-generation autophagy researchers towards translation (DRIVE), an international PhD training program on autophagy. Autophagy. ISSN 1554-8627. doi: 10.1080/15548627.2018.1515532. Full text in Research Archive Show summary

  The European autophagy consortium Driving next-generation autophagy researchers towards translation (DRIVE) held its kick-off meeting in Groningen on the 14th and 15th of June 2018. This Marie Skłodowska-Curie Early Training Network was approved under the European Union’s Horizon 2020 Research and Innovation Program and is funded for 4 years. Within DRIVE, 14 European research teams from academia and industry will train 15 PhD students through applied, cross-disciplinary and collaborative macroautophagy/autophagy research. The goal of DRIVE is to stimulate applied approaches in autophagy research and provide training towards translation, while advancing our knowledge on autophagy in specific physiological and pathological states. The strong focus on translation will prepare the PhD students to be at the forefront to exploit autophagy for the development of therapies directly benefitting patients. Thereby, DRIVE will contribute to filling the educational gap that currently exists between academia and industry, and will prepare its PhD students for alternative and highly flexible professional paths.

• Skah, Seham; Richartz, Nina; Duthil, Eva; Gilljam, Karin Margaretha; Bindesbøll, Christian & Naderi, Elin Hallan [Show all 11 contributors for this article] (2018). cAMP-mediated autophagy inhibits DNA damage-induced death of leukemia cells independent of p53. OncoTarget. 9(54), p. 30434–30449. doi: 10.18632/oncotarget.25758. Full text in Research Archive Show summary

  Autophagy is important in regulating the balance between cell death and survival, with the tumor suppressor p53 as one of the key components in this interplay. We have previously utilized an in vitro model of the most common form of childhood cancer, B cell precursor acute lymphoblastic leukemia (BCP-ALL), to show that activation of the cAMP signaling pathway inhibits p53-mediated apoptosis in response to DNA damage in both cell lines and primary leukemic cells. The present study reveals that cAMP-mediated survival of BCP-ALL cells exposed to DNA damaging agents, involves a critical and p53-independent enhancement of autophagy. Although autophagy generally is regarded as a survival mechanism, DNA damage-induced apoptosis has been linked both to enhanced and reduced levels of autophagy. Here we show that exposure of BCP-ALL cells to irradiation or cytotoxic drugs triggers autophagy and cell death in a p53-dependent manner. Stimulation of the cAMP signaling pathway further augments autophagy and inhibits the DNA damage-induced cell death concomitant with reduced nuclear levels of p53. Knocking-down the levels of p53 reduced the irradiation-induced autophagy and cell death, but had no effect on the cAMP-mediated autophagy. Moreover, prevention of autophagy by bafilomycin A1 or by the ULK-inhibitor MRT68921, diminished the protecting effect of cAMP signaling on DNA damage-induced cell death. Having previously proposed a role of the cAMP signaling pathway in development and treatment of BCP-ALLs, we here suggest that inhibitors of autophagy may improve current DNA damage-based therapy of BCP-ALL - independent of p53.

• Ogmundsdottir, MH; Fock, Valerie; Sooman, L; Pogenberg, V; Dilshat, R & Bindesbøll, Christian [Show all 10 contributors for this article] (2018). A short isoform of ATG7 fails to lipidate LC3/GABARAP. Scientific Reports. 8(1), p. 1–8. doi: 10.1038/s41598-018-32694-7. Full text in Research Archive Show summary

  Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.

• Søreng, Kristiane; Munson, Michael Joseph; Lamb, Christopher R.; Bjørndal, Gunnveig Toft; Pankiv, Serhiy & Carlsson, Sven R. [Show all 8 contributors for this article] (2018). SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin-2. EMBO Reports. ISSN 1469-221X. 19:e44837(4), p. 1–12. doi: 10.15252/embr.201744837. Full text in Research Archive Show summary

  Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.

• Søreng, Kristiane; Neufeld, Thomas P & Simonsen, Anne (2018). Membrane Trafficking in Autophagy. International Review of Cell and Molecular Biology. ISSN 1937-6448. 336, p. 1–92. doi: 10.1016/bs.ircmb.2017.07.001. Full text in Research Archive
• Aas, Aleksander; Isakson, Pauline; Bindesbøll, Christian; Alemu, Endalkachew Ashenafi; Klungland, Arne & Simonsen, Anne (2017). Nucleocytoplasmic shuttling of FTO does not affect starvation-induced autophagy. PLOS ONE. 12(3). doi: 10.1371/journal.pone.0168182. Full text in Research Archive Show summary

  Polymorphic variants of the FTO (fat mass and obesity) gene associate with body mass index in humans, but the underlying molecular mechanisms have not been firmly determined. FTO is linked to energy homeostasis via amino acid sensing and is thought to activate the mammalian target of rapamycin complex 1, a negative regulator of autophagy. FTO localises both to the nucleus and the cytoplasm, and in this study we identify a functional nuclear localisation signal (NLS) in the N-terminus of FTO, as well as nuclear localization information in its very C-terminus. Inhibition of FTO nuclear transport has no effect on autophagy and in contrast to a previously proposed role of FTO in autophagy, we find no difference in starvation-induced autophagy in control cells compared to a panel of cell types depleted of FTO. Future studies that further characterise the cellular functions of FTO will be important to understand why variants in FTO are associated with body weight.

• Schlafli, Anna M; Isakson, Pauline; Garattini, E; Simonsen, Anne & Tschan, Mario P (2017). The autophagy scaffold protein ALFY is critical for the granulocytic differentiation of AML cells. Scientific Reports. 7(1). doi: 10.1038/s41598-017-12734-4. Full text in Research Archive Show summary

  Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.

• Galluzzi, Lorenzo; Baehrecke, Eric H.; Ballabio, Andrea; Boya, Patricia; Pedro, José Manuel Bravo-San & Cecconi, Francesco [Show all 54 contributors for this article] (2017). Molecular definitions of autophagy and related processes. EMBO Journal. ISSN 0261-4189. 36(13), p. 1811–1836. doi: 10.15252/embj.201796697. Full text in Research Archive
• Kjos, Ingrid; Distefano, Marita Borg; Sætre, Frank Ove; Repnik, Urska; Holland, Petter & Jones, Arwyn T [Show all 10 contributors for this article] (2017). Rab7b modulates autophagic flux by interacting with Atg4B. EMBO Reports. ISSN 1469-221X. 18(10), p. 1727–1739. doi: 10.15252/embr.201744069. Full text in Research Archive Show summary

  Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.

• Søreng, Kristiane; Knævelsrud, Helene; Holland, Petter & Simonsen, Anne (2017). HS1BP3 inhibits autophagy by regulation of PLD1. Autophagy. ISSN 1554-8627. 13(5), p. 985–986. doi: 10.1080/15548627.2017.1291483. Full text in Research Archive Show summary

  Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization. This is an Accepted Manuscript of an article published by Taylor & Francis Group in Autophagy on 20 Mar 2017, available online: http://www.tandfonline.com/10.1080/12345678.1234.123456.

• Simonsen, Anne; Mathai, Benan John & Meijer, Annemarie (2017). Studying Autophagy in Zebrafish. Cells. 6(3), p. E21–E21. doi: 10.3390/cells6030021. Full text in Research Archive Show summary

  Autophagy is an evolutionarily conserved catabolic process which allows lysosomal degradation of complex cytoplasmic components into basic biomolecules that are recycled for further cellular use. Autophagy is critical for cellular homeostasis and for degradation of misfolded proteins and damaged organelles as well as intracellular pathogens. The role of autophagy in protection against age-related diseases and a plethora of other diseases is now coming to light; assisted by several divergent eukaryotic model systems ranging from yeast to mice. We here give an overview of different methods used to analyse autophagy in zebrafish—a relatively new model for studying autophagy—and briefly discuss what has been done so far and possible future directions.

• Mamińska, Agnieszka; Bartosik, Anna; Banach-Orlowska, Magdalena; Pilecka, Iwona; Jastrzębski, Kamil & Zdżalik-Bielecka, Daria [Show all 19 contributors for this article] (2016). ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors. Science Signaling. ISSN 1945-0877. 9(411). doi: 10.1126/scisignal.aad0848. Full text in Research Archive
• Holland, Petter; Knævelsrud, Helene; Søreng, Kristiane; Mathai, Benan John; Lystad, Alf Håkon & Pankiv, Serhiy [Show all 16 contributors for this article] (2016). HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels. Nature Communications. 7. doi: 10.1038/ncomms13889. Full text in Research Archive Show summary

  A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

• Shatz, Oren; Holland, Petter; Elazar, Zuvlun & Simonsen, Anne (2016). Complex relations between phospholipids, autophagy, and neutral lipids. TIBS -Trends in Biochemical Sciences. Regular ed.. ISSN 0968-0004. 41(11), p. 907–923. doi: 10.1016/j.tibs.2016.08.001. Full text in Research Archive
• Lystad, Alf Håkon & Simonsen, Anne (2016). Phosphoinositide-binding proteins in autophagy. FEBS Letters. ISSN 0014-5793. p. 2454–2468. doi: 10.1002/1873-3468.12286. Full text in Research Archive
• Andersen, Aram Nikolai; Landsverk, Ole J. B.; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre & Øynebråten, Inger (2016). Coupling of HIV-1 antigen to the selective autophagy receptor SQSTM1/p62 promotes T-cell-mediated immunity. Frontiers in Immunology. 7. doi: 10.3389/fimmu.2016.00167. Full text in Research Archive Show summary

  Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans.

• Dragich, Joanna M; Kuwajima, Takaaki; Hirose-Ikeda, Megumi; Yoon, Michael S; Eenjes, Evelien & Bosco, Joan R [Show all 18 contributors for this article] (2016). Autophagy linked FYVE (Alfy/WDFY3) is required for establishing neuronal connectivity in the mammalian brain. eLIFE. 5(September). doi: 10.7554/eLife.14810. Full text in Research Archive
• Holland, Petter & Simonsen, Anne (2015). Actin shapes the autophagosome. Nature Cell Biology. ISSN 1465-7392. 17(9), p. 1094–1096. doi: 10.1038/ncb3224. Full text in Research Archive
• Galluzzi, Lorenzo; Pietrocola, Federico; San-Pedro, Jose Manuel; Amaravadi, Ravi; Baehreck, Eric H & Cecconi, Francesco [Show all 24 contributors for this article] (2015). Autophagy in malignant transformation and cancer progression. EMBO Journal. ISSN 0261-4189. 34(7), p. 856–880. doi: 10.15252/embj.201490784. Full text in Research Archive
• Carlsson, Sven R & Simonsen, Anne (2015). Membrane dynamics in autophagosome biogenesis. Journal of Cell Science. ISSN 0021-9533. 128(2), p. 193–205. doi: 10.1242/jcs.141036. Full text in Research Archive
• Eriksen, Agnete Bratsberg; Torgersen, Maria Lyngaas; Holm, Kristine Lillebø; Abrahamsen, Greger; Spurkland, Anne & Moskaug, Jan Øivind [Show all 8 contributors for this article] (2015). Retinoic acid-induced IgG Production in TLR-activated human primary B cells involves ULK1-mediated autophagy. Autophagy. ISSN 1554-8627. 11(3), p. 460–471. doi: 10.1080/15548627.2015.1009797. Full text in Research Archive
• Hieke, Nora; Löffler, Antje S.; Kaizuka, Takeshi; Berleth, Niklas; Böhler, Philip & Drießen, Stefan [Show all 18 contributors for this article] (2015). Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells. Autophagy. ISSN 1554-8627. 11(9), p. 1471–1483. doi: 10.1080/15548627.2015.1068488. Full text in Research Archive
• Yla-Anttila, P; Mikkonen, E; Happonen, KE; Holland, Petter; Ueno, T & Simonsen, Anne [Show all 7 contributors for this article] (2015). RAB24 facilitates clearance of autophagic compartments during basal conditions. Autophagy. ISSN 1554-8627. 11(10), p. 1833–1848. doi: 10.1080/15548627.2015.1086522. Full text in Research Archive
• Drießen, Stefan; Berleth, Niklas; Friesen, Olena; Löffler, Antje S.; Böhler, Philip & Hieke, Nora [Show all 15 contributors for this article] (2015). Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy. Autophagy. ISSN 1554-8627. 11(9), p. 1458–1470. doi: 10.1080/15548627.2015.1067359. Full text in Research Archive
• Lystad, Alf Håkon & Simonsen, Anne (2015). Assays to monitor aggrephagy. Methods. ISSN 1046-2023. 75, p. 112–119. doi: 10.1016/j.ymeth.2014.12.019. Full text in Research Archive
• Holland, Petter; Torgersen, Maria Lyngaas; Sandvig, Kirsten & Simonsen, Anne (2014). LYST affects lysosome size and quantity, but not trafficking or degradation through autophagy or endocytosis. Traffic: The moving front of cell biology. ISSN 1398-9219. 15(12), p. 1390–1405. doi: 10.1111/tra.12227. Full text in Research Archive
• Lystad, Alf Håkon; Ichimura, Yoshinobu; Takagi, K; Yang, Yinjie; Pankiv, Serhiy & Kanegae, Yumi [Show all 12 contributors for this article] (2014). Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures. EMBO Reports. ISSN 1469-221X. 15(5), p. 557–565. doi: 10.1002/embr.201338003. Full text in Research Archive
• Mandell, Michael A.; Jain, Ashish; Arko-Mensah, John; Chauhan, Santosh; Kimura, Tomonori & Dinkins, Christina [Show all 14 contributors for this article] (2014). TRIM Proteins Regulate Autophagy and Can Target Autophagic Substrates by Direct Recognition. Developmental Cell. ISSN 1534-5807. 30(4), p. 394–409. doi: 10.1016/j.devcel.2014.06.013. Full text in Research Archive
• Isakson, Pauline; Holland, Petter & Simonsen, Anne (2013). The role of ALFY in selective autophagy. Cell Death and Differentiation. ISSN 1350-9047. 20(1), p. 12–20. doi: 10.1038/cdd.2012.66. Full text in Research Archive
• Knævelsrud, Helene; Søreng, Kristiane; Raiborg, Camilla; Håberg, Karin; Rasmuson, Fredrik & Brech, Andreas [Show all 12 contributors for this article] (2013). Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. Journal of Cell Biology. ISSN 0021-9525. 202(2), p. 331–349. doi: 10.1083/jcb.201205129. Full text in Research Archive
• Engedal, Kim Nikolai; Torgersen, Maria Lyngaas; Guldvik, Ingrid Jenny; Barfeld, Stefan; Bakula, D & Sætre, Frank Ove [Show all 12 contributors for this article] (2013). Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy. ISSN 1554-8627. 9(10), p. 1475–1490. doi: 10.4161/auto.25900. Full text in Research Archive
• Torgersen, Maria Lyngaas; Engedal, Kim Nikolai; Bøe, Stig Ove; Hokland, Peter & Simonsen, Anne (2013). Targeting autophagy potentiates the apoptotic effect of histone deacetylase inhibitors in t(8;21) AML cells. Blood. ISSN 0006-4971. 122(14), p. 2467–2476. doi: 10.1182/blood-2013-05-500629. Full text in Research Archive
• Sengupta, Tanima; Torgersen, Maria Lyngaas; Kassahun, Henok; Vellai, Tibor; Simonsen, Anne & Nilsen, Hilde (2013). Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy. Nature Communications. 4. doi: 10.1038/ncomms3674. Full text in Research Archive

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• Wang, Heling; Siow, Richard; Schmauck-Medina, Tomás Alejandro; Zhang, Jianying; Sandset, Per Morten & Filshie, Clare [Show all 38 contributors for this article] (2024). Meeting Summary of The NYO3 5th NO-Age/AD Meeting and the 1st Norway-UK Joint Meeting on Aging and Dementia: Recent Progress on the Mechanisms and Interventional Strategies. The journals of gerontology. Series A, Biological sciences and medical sciences. ISSN 1079-5006. 79(4), p. 1–10. doi: 10.1093/gerona/glae029. Full text in Research Archive Show summary

  Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a “Money Follows Cooperation agreement” between the 2 nations. The inaugural Norway–UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging.

• Simonsen, Anne Gjøen (2024). Regulation of hypoxia-induced mitophagy by lipid-binding proteins. Full text in Research Archive
• Simonsen, Anne Gjøen (2024). Role of Mitophagy in Regulation of Cellular Bioenergetics. Full text in Research Archive
• Simonsen, Anne Gjøen (2023). Regulation of mitophagy and cellular bioenergetics by lipid-binding proteins. Full text in Research Archive
• Simonsen, Anne Gjøen (2023). Regulation of autophagy and cellular bioenergetics by lipid-binding proteins. Full text in Research Archive
• Mizushima, Noboru & Simonsen, Anne Gjøen (2023). Editorial overview: Autophagy editorial COPHYS special Issue. Current Opinion in Physiology. ISSN 2468-8681. 32. doi: 10.1016/j.cophys.2023.100646. Full text in Research Archive
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