The autophagy protein RUBCNL/PACER represses RIPK1 kinase-dependent apoptosis and necroptosis.

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs.

IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models.

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

The autophagy protein Def8 is altered in Alzheimer's disease and Aß42-expressing Drosophila brains.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by protein accumulation in the brain as a main neuropathological hallmark. Among them, Aß42 peptides tend to aggregate and create oligomers and plaques. Macroautophagy, a form of autophagy characterized by a double-membrane vesicle, plays a crucial role in maintaining neuronal homeostasis by degrading protein aggregates and dysfunctional organelles as a quality control process. Recently, DEF8, a relatively uncharacterized protein, has been proposed as a participant in vesicular traffic and autophagy pathways. We have reported increased DEF8 levels in lymphocytes from mild cognitive impairment (MCI) and early-stage AD patients and a neuronal profile in a murine transgenic AD model. Here, we analyzed DEF8 localization and levels in the postmortem frontal cortex of AD patients, finding increased levels compared to healthy controls. To evaluate the potential function of DEF8 in the nervous system, we performed an in silico assessment of its expression and network profiles, followed by an in vivo evaluation of a neuronal Def8 deficient model using a Drosophila melanogaster model of AD based on Aß42 expression. Our findings show that DEF8 is an essential protein for maintaining cellular homeostasis in the nervous system, and it is upregulated under stress conditions generated by Aß42 aggregation. This study suggests DEF8 as a novel actor in the physiopathology of AD, and its exploration may lead to new treatment avenues.

Neuronal Rubicon Represses Extracellular APP/Amyloid ß Deposition in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.

Calcium & ROS: Two orchestra directors for the requiem of death.

Tumor necrosis factor alpha (TNF) triggers regulated necrosis of mycobacterium-infected macrophages through of mitochondrial reactive oxygen species (mitoROS) production in a RIPK1/3-dependent manner. To explain that, Roca and colleagues describe an inter-orgallenar circuit which involves the lysosomal ceramide production, mitoROS, BAX activation and RyR Ca2+ efflux from the endoplasmic reticulum into the mitochondrion.

Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation.

RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice.

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.

MK2 phosphorylation of RIPK1 regulates TNF-mediated cell death.

TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders can, in certain conditions, be attributed to RIPK1 kinase-dependent cell death. Survival, however, is the default response of most cells to TNF stimulation, indicating that cell demise is normally actively repressed and that specific checkpoints must be turned off for cell death to proceed. We identified RIPK1 as a direct substrate of MK2 in the TNFR1 signalling pathway. Phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis. In line with these in vitro findings, MK2 inactivation greatly sensitizes mice to the cytotoxic effects of TNF in an acute model of sterile shock caused by RIPK1-dependent cell death. In conclusion, we identified MK2-mediated RIPK1 phosphorylation as an important molecular mechanism limiting the sensitivity of the cells to the cytotoxic effects of TNF.

When PERK inhibitors turn out to be new potent RIPK1 inhibitors: critical issues on the specificity and use of GSK2606414 and GSK2656157.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes a state of cellular stress known as ER stress. The cells respond to ER stress by activating the unfolded protein response (UPR), a signaling network emerging from the ER-anchored receptors IRE1α, PERK and ATF6. The UPR aims at restoring ER protein-folding homeostasis, but turns into a toxic signal when the stress is too severe or prolonged. Recent studies have demonstrated links between the UPR and inflammation. Consequently, small molecule inhibitors of IRE1α and PERK have become attractive tools for the potential therapeutic manipulation of the UPR in inflammatory conditions. TNF is a master pro-inflammatory cytokine that drives inflammation either directly by promoting gene activation, or indirectly by inducing RIPK1 kinase-dependent cell death, in the form of apoptosis or necroptosis. To evaluate the potential contribution of the UPR to TNF-induced cell death, we tested the effects of two commonly used PERK inhibitors, GSK2606414 and GSK2656157. Surprisingly, we observed that both compounds completely repressed TNF-mediated RIPK1 kinase-dependent death, but found that this effect was independent of PERK inactivation. Indeed, these two compounds turned out to be direct RIPK1 inhibitors, with comparable potency to the recently developed RIPK1 inhibitor GSK'963 (about 100 times more potent than NEC-1s). Importantly, these compounds completely inhibited TNF-mediated RIPK1-dependent cell death at a concentration that did not affect PERK activity in cells. In vivo, GSK2656157 administration protected mice from lethal doses of TNF independently of PERK inhibition and as efficiently as GSK'963. Together, our results not only report on new and very potent RIPK1 inhibitors but also highlight the risk of misinterpretation when using these two PERK inhibitors in the context of ER stress, cell death and inflammation.

Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 1. An overview.

Increased demand on the protein folding capacity of the endoplasmic reticulum (ER) engages an adaptive reaction known as the unfolded protein response (UPR). The UPR regulates protein translation and the expression of numerous target genes that contribute to restore ER homeostasis or induce apoptosis of irreversibly damaged cells. UPR signaling is highly regulated and dynamic and integrates information about the type, intensity, and duration of the stress stimuli, thereby determining cell fate. Recent advances highlight novel physiological outcomes of the UPR beyond specialized secretory cells, particularly in innate immunity, metabolism, and cell differentiation. Here we discuss studies on the fine-tuning of the UPR and its physiological role in diverse organs and diseases.

Pathogenic role of BECN1/Beclin 1 in the development of amyotrophic lateral sclerosis.

Pharmacological activation of autophagy is becoming an attractive strategy to induce the selective degradation of aggregate-prone proteins. Recent evidence also suggests that autophagy impairment may underlie the pathogenesis of several neurodegenerative diseases. Mutations in the gene encoding SOD1 (superoxide disumutase 1) trigger familial amyotrophic lateral sclerosis (ALS), inducing its misfolding and aggregation and the progressive loss of motoneurons. It is still under debate whether autophagy has a protective or detrimental role in ALS. Here we evaluate the impact of BECN1/Beclin 1, an essential autophagy regulator, in ALS. BECN1 levels were upregulated in both cells and animals expressing mutant SOD1. To evaluate the impact of BECN1 to the pathogenesis of ALS in vivo, we generated mutant SOD1 transgenic mice heterozygous for Becn1. We observed an unexpected increase in life span of mutant SOD1 transgenic mice haploinsufficient for Becn1 compared with littermate control animals. These effects were accompanied by enhanced accumulation of SQSTM1/p62 and reduced levels of LC3-II, and an altered equilibrium between monomeric and oligomeric mutant SOD1 species in the spinal cord. At the molecular level, we detected an abnormal interaction of mutant SOD1 with the BECN1-BCL2L1 complex that may impact autophagy stimulation. Our data support a dual role of BECN1 in ALS and depict a complex scenario in terms of predicting the effects of manipulating autophagy in a disease context.

Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis.

The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store-operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and, consequently, IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together, these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis.

When ER stress reaches a dead end.

Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways.

Bax Inhibitor-1-mediated Ca2+ leak is decreased by cytosolic acidosis.

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1's Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca(2+)-channel pore-dead mutant BI-1 (BI-1(D213R)) was developed. We determined whether BI-1 behaves as a bona fide H+/Ca2+ antiporter or as an ER Ca(2+)-leak channel by investigating the effect of pH on unidirectional Ca(2+)-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1(-/-) cells increased the ER Ca(2+)-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1(D231R) expression in BI-1(-/-), despite its ER localization, did not increase the ER Ca(2+)-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca2+ leak was blocked. Finally, a peptide representing the Ca(2+)-channel pore of BI-1 promoting Ca2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca(2+)-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.