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Senescent cells accumulate in various tissues and organs with aging altering surrounding tissue due to an active secretome, and at least in mice their elimination extends healthy lifespan and ameliorates several chronic diseases. Whether all cell types senesce, including post-mitotic cells, has been poorly described mainly because cellular senescence was defined as a permanent cell cycle arrest. Nevertheless, neurons with features of senescence have been described in old rodent and human brains. In this study we characterized an in vitro model useful to study the molecular basis of senescence of primary rat cortical cells that recapitulates senescent features described in brain aging. We found that in long-term cultures, rat primary cortical neurons displayed features of cellular senescence before glial cells did, and developed a functional senescence-associated secretory phenotype able to induce paracrine premature senescence of mouse embryonic fibroblasts but proliferation of rat glial cells. Functional autophagy seems to prevent neuronal senescence, as we observed an autophagic flux reduction in senescent neurons both in vitro and in vivo, and autophagy impairment induced cortical cell senescence while autophagy stimulation inhibited it. Our findings suggest that aging-associated dysfunctional autophagy contributes to senescence transition also in neuronal cells.

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and posttranslational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.

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242 25 Abstract 26 NR4A is a nuclear receptor protein family whose members act as sensors of cellular 27 environment and regulate multiple processes such as metabolism, proliferation, migration, 28 apoptosis, and autophagy. Since the ligand binding domains of these receptors have no 29 cavity for ligand interaction, their function is most likely regulated by protein abundance 30 and post-translational modifications. In particular, NR4A1 is regulated by protein 31 abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic 32 translocation), and acts both as a transcription factor and as a regulator of other 33 interacting proteins. SUMOylation is a post-translational modification that can affect protein 34 stability, transcriptional activity, alter protein-protein interactions and modify intracellular 35 localization of target proteins. In the present study we evaluated the role of SUMOylation 36 as a posttranslational modification that can regulate the activity of NR4A1 to induce 37 autophagy-dependent cell death. We focused on a model potentially relevant for neuronal 38 cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic 39 cell death. We observed that a triple mutant in SUMOylation sites has reduced 40 SUMOylation, increased transcriptional activity, altered intracellular distribution, and more 41 importantly, its ability to induce autophagic cell death is impaired. 42 43 Summary Statement 44 The modification of the nuclear receptor NR4A1 by SUMO regulates its transcriptional 45 activity, intracellular localization and is required to induce autophagic cell death.46 47 Short title: SUMOylated NR4A1 induces autosis 48 49 Keywords: 50 NR4A1, SUMO, autophagy, cell death, Substance P, NK 1 R3 51 52 Abbreviations list 53 NR4A Nuclear receptor group family A 54 NR4A1 Nuclear receptor group 4 family A member 1 (Nur77, NGF1B, TR3, etc.) 55 NR4A2 Nuclear receptor group 4 family A member 2 (Nurr1, NOT1, etc) 56 NR4A3 Nuclear receptor group 4 family A member 3 (Nor1, MINOR) 57 SUMO small ubiquitin-like modifier 58 SP Substance P 59 NK 1 R Neurokinin 1 Receptor 60 TAD Trans-Activation Domain 61 DBD DNA Binding Domain 62 LBD Ligand Binding Domain 63 PTM Post Translational Modifications 4 64 Introduction 65 Nuclear receptors are a superfamily of transcription factors involved in a vast number of 66 biological processes. They share three common structural domains: a N-terminal 67 transactivation domain (TAD), a central double zinc finger DNA binding domain (DBD) and 68 a C-terminal ligand binding domain (LBD). Among the known human nuclear receptors, 69 the ones belonging to the NR4A family act as sensors of the cellular environment and 70 contribute to cell fate decisions, such as cell proliferation, differentiation, migration, cell 71 death, etc. Physiologically, NR4A members influence the adaptive and innate immune 72 system, angiogenesis, metabolism and brain function. The NR4A family is comprised of 73 three members that bind the same DNA elements (1): NR4A1 (Nur77, TR3, ...