Regulation of neuronal Dense Core Vesicle (DCV) Biogenesis and Exocytosis through Rab GTPases.

• Applicant: Professor Dr. Stefan Eimer
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2017 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 384170970

• Final Report Year: 2023

Final Report Abstract

The main goal of the funded project was to determine the molecular mechanisms how Rab GTPases regulate the biogenesis and release of neuropeptide containing dense core vesicles (DCVs) in the nematode Caenorhabditis elegans, as a multicellular model organism. Neuropeptides released from DCVs act as long-range signal to modulate neuronal function and activity, in contrast to chemical neurotransmitters, which mediate neuronal communication via local signaling at synapses. As neuropeptides can globally modulate neuronal activity, an understanding of the molecular mechanism that regulate their release from DCVs would enable us to us the neuropeptide release machinery for future therapeutic strategies for neurodegenerative diseases or psychiatric disorders. In previous work, we had identified two Rab GTPases RAB-2 and RAB-10, which are required for DCV biogenesis and secretion, respectively. Through subsequent yeast two-hybrid screens, we had isolated several novel RAB-2 and RAB-10 interacting proteins, some of which were linked to membrane signaling. Phosphatidyl-inositols are phospholipids organizing membrane mirco-domains, which are used to regulate membrane trafficking, sorting and signaling events. Thus, phosphatidyl-inositols are attractive factors regulating DCV biogenesis and release. To analyze whether DCVs are controlled by phosphatidyl-inositols, we conducted an RNAi screen inactivating phosphatidyl-inositols converting kinase and phosphatases in C. elegans. We could show that indeed DCV release is strongly controlled by phosphatidyl-inositol-4,5-bis-phosphate (PI(4,5)P2) as well as phosphatidyl-inositol-3,4,5-tris-phosphate (PI(3,4,5)P3). Furthermore, we could demonstrate that high levels of PI(4,5)P2 at intracellular membranes lead to an accumulation of stable f-actin patches through inactivation of the actin remodeling protein UNC-60A/cofilin, which is required for DCV secretion. This work established that DCV release depends on active actin remodeling trough UNC-60A/cofilin. In addition, we could also demonstrate that DCV secretion is also positively controlled by the insulinsignaling pathway via the action of PI(3,4,5)P3. We identified the insulin-pathway downstream kinase SGK-1 to function as a gate to integrate upstream metabolic inputs via insulin and TORC2 signaling as well as PI(3)P, to allow high levels of DCV release only if all upstream signals are present. This proposes an interesting model of how diabetes might globally influence neuronal activity. In a last set of experiments, we also could show that the newly identified RAB-2 interacting protein PDZD-8 leads to DCV hyper-secretion when inactivated, suggesting that PDZD-8 dependent neuronal membrane contact site between the endoplasmic reticulum and mitochondria and/or lysosomes also impact DCV release. Overall, we found interesting new insights about the molecular mechanisms how neuropeptide release from DCV is regulated through phosphatidyl-inositol signaling.

Publications

• Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy. Blood, 133(2), 168-179.
  Nguyen, The Duy; Shaid, Shabnam; Vakhrusheva, Olesya; Koschade, Sebastian E.; Klann, Kevin; Thölken, Marlyn; Baker, Fatima; Zhang, Jing; Oellerich, Thomas; Sürün, Duran; Derlet, Anja; Haberbosch, Isabella; Eimer, Stefan; Osiewacz, Heinz D.; Behrends, Christian; Münch, Christian; Dikic, Ivan & Brandts, Christian H.
  
• Aromaticity at position 39 in α‐synuclein: A modulator of amyloid fibril assembly and membrane‐bound conformations. Protein Science, 31(7).
  Buratti, Fiamma A.; Boeffinger, Nicola; Garro, Hugo A.; Flores, Jesica S.; Hita, Francisco J.; Gonçalves, Phelippe do Carmo; Copello, Federico dos Reis; Lizarraga, Leonardo; Rossetti, Giulia; Carloni, Paolo; Zweckstetter, Markus; Outeiro, Tiago F.; Eimer, Stefan; Griesinger, Christian & Fernández, Claudio O.
  
• Disruption of the MICOS complex leads to an aberrant cristae structure and an unexpected, pronounced lifespan extension in Podospora anserina. Journal of Cellular Biochemistry, 123(8), 1306-1326.
  Warnsmann, Verena; Marschall, Lisa‐Marie; Meeßen, Anja C.; Wolters, Maike; Schürmanns, Lea; Basoglu, Marion; Eimer, Stefan & Osiewacz, Heinz D.
  
• Lipid and protein content profiling of isolated native autophagic vesicles. EMBO reports, 23(12).
  Schmitt, Daniel; Bozkurt, Süleyman; Henning‐Domres, Pascale; Huesmann, Heike; Eimer, Stefan; Bindila, Laura; Behrends, Christian; Boyle, Emily; Wilfling, Florian; Tascher, Georg; Münch, Christian; Behl, Christian & Kern, Andreas
  
• Adaptive responses of neuronal cells to chronic endoplasmic reticulum (ER) stress. Redox Biology, 67, 102943.
  Pham, Thu Nguyen Minh; Perumal, Natarajan; Manicam, Caroline; Basoglu, Marion; Eimer, Stefan; Fuhrmann, Dominik C.; Pietrzik, Claus U.; Clement, Albrecht M.; Körschgen, Hagen; Schepers, Jana & Behl, Christian
  
• Deciphering the mitophagy receptor network identifies a crucial role for OPTN (optineurin) in acute myeloid leukemia. Autophagy, 19(11), 2982-2996.
  Meyer, Laura M.; Koschade, Sebastian E.; Vischedyk, Jonas B.; Thoelken, Marlyn; Gubas, Andrea; Wegner, Martin; Basoglu, Marion; Knapp, Stefan; Kaulich, Manuel; Eimer, Stefan; Shaid, Shabnam & Brandts, Christian H.
  
• ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy. Journal of Cell Science, 136(3).
  Sánchez-Martín, Pablo; Kriegenburg, Franziska; Alves, Ludovico; Adam, Julius; Elsaesser, Jana; Babic, Riccardo; Mancilla, Hector; Licheva, Mariya; Tascher, Georg; Münch, Christian; Eimer, Stefan & Kraft, Claudine