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Fas apoptotic inhibitory molecule 2: a novel regulator of autophagy by facilitating autophagosome-lysosome fusion

Fas apoptotic inhibitory molecule 2: a novel regulator of autophagy by facilitating autophagosome-lysosome fusion
Jeeyeon Hong
DGIST Authors
Hong, Jeeyeon; Kim, Seong Who; Yu, Seong-Woon
Seong Who Kim
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adult neurogenesis, autolysosome, autophagosome maturation, FAIM2, LC3, LIR, xbx-6
In the adult brain, programmed cell death (PCD) is a critical process to maintain an adequate pool of self-renewing neural stem cells (NSCs) and newly generated cells in the brain. To date, the studies of key players regulating PCD have been extensively centered around apoptosis. Therefore, I sought to expand the knowledge on underlying mechanisms of autophagic cell death. Based on the gene expression profiling of the model of autophagic cell death in NSCs, I discovered Fas apoptotic inhibitory molecule 2 (FAIM2) as one of the marker genes. FAIM2 is a seven-transmembrane domain-containing protein that plays a neuroprotective role via antagonizing the extrinsic apoptosis signaling. However, whether FAIM2 plays a role in other forms of molecular signaling has been unknown. Here I show that FAIM2 localizes to the lysosomes at basal state and facilitates autophagy through interaction with LC3 in human neuroblastoma SH-SY5Y cells. FAIM2 overexpression increased autophagy flux, while autophagy flux was impaired in shRNAmediated knockdown (shFAIM2) cells, and the impairment was more evident in the presence of rapamycin. In shFAIM2 cells, autophagosome maturation through fusion with lysosomes was impaired, leading to accumulation of autophagosomes. A functional LC3- interacting region motif within FAIM2 was essential for the interaction with LC3 and the rescue of autophagy flux in shFAIM2 cells, while LC3-binding property of FAIM2 was dispensable for the anti-apoptotic function in response to Fas receptor-mediated apoptosis. Suppression of autophagosome maturation was also observed in a null mutant of Caenorhabditis elegans lacking xbx-6, the ortholog of FAIM2. Current study suggests that FAIM2 is a novel regulator of autophagy mediating autophagosome maturation through the interaction with LC3.
Table Of Contents
CHAPTER 1: General Introduction 1 1.1 Programmed Cell Death: The Classifications 2 1.1.1 Autophagic cell death 2 1.1.2 Apoptosis 3 1.1.3 Necrosis 3 1.2 Regulation of Programmed Cell Death and Adult Neurogenesis 7 1.2.1 Insulin on NSCs 7 1.2.2 IGF-1 on NSCs 7 1.2.3 Steroid hormones on NSCs 8 1.2.4 Stress hormones on NSCs 9 1.3 Fas apoptotic inhibitory molecule 2 11 1.4 Aim and Objectives 13 CHAPTER 2: Identification of FAIM2 in Adult Hippocampal NSCs 14 2.1 Introduction 14 2.2 Materials and Methods 14 2.2.1 Cell culture and reagents 14 2.2.2 Quantitative real-time RT-PCR 15 2.2.3 Statistical analysis 16 2.3 Results 17 2.3.1 FAIM2 is expressed in the adult brain and in hippocampal NSCs 17 2.3.2 Differential dynamics of FAIM2 following insulin withdrawal 17 2.3.3 Faim2 knockdown sensitizes NSCs to Fas ligand 22 2.3.4 Faim2 knockdown sensitizes NSCs to autophagic cell Death 22 CHAPTER 3: Fas-apoptotic inhibitory molecule 2 localizes to the lysosome and facilitates autophagosome-lysosome fusion through the LC3 interaction region motif-dependent interaction with LC3 3.1 Introduction 24 3.2 Materials and Methods 27 3.2.1 Cell culture and shRNA knockdown 29 3.2.2 C. elegans strains and quantification of autophagic vesicles 29 3.2.3 Antibodies and reagents 30 3.2.4 Immunocytochemistry and confocal microscopy 32 3.2.5 Immunoblotting 32 3.2.6 Immunoprecipitation (IP) 33 3.2.7 Live-cell Imaging 34 3.2.8 In situ Proximity Ligation Assay (PLA) 34 3.2.9 Flow cytometry 35 3.2.10 LC3-interacting region (LIR) motif prediction 35 3.2.11 Cell viability assay 36 3.2.12 Apoptotic cell death assay 36 3.2.13 Image and statistical analysis 37 3.3. Results 38 3.3.1 FAIM2 overexpression elevates basal and rapamycin-activated autophagy in SH-SY5Y cells 38 3.3.2 FAIM2 knockdown impairs autophagic flux by blocking autophagosome maturation 41 3.3.4 FAIM2 localization to MAP1LC3B- and LAMP1-positive organelles is robustly increased under rapamycin-induced autophagy 46 3.3.4 FAIM2 plays an integral role in the biogenesis of acidified lysosomes 50 3.3.5 FAIM2 localizes to lysosomes and is required for autophagosome maturation 53 3.3.6 FAIM2 directly binds to the lipidated form of LC3 via its N-terminal LIR motif 57 3.3.7 XBX-6, the FAIM2 ortholog in C. elegans, mediates autolysosome formation 66 3.3.8 LIR motif of FAIM2 is not required for anti-apoptotic function 69 3.4 Discussion 72 REFERENCES 77 Abstract in Korean 94
Brain and Cognitive Sciences
Related Researcher
  • Author Yu, Seong-Woon Laboratory of Neuronal Cell Death
  • Research Interests Molecular mechanisms of neuronal cell death and neurodegeneration
Department of Brain and Cognitive SciencesThesesPh.D.

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