Unfolded Protein Response Sensor Research Articles

Dysregulation of ceramide metabolism causes phytoceramide-dependent induction of the unfolded protein response.

The unfolded protein response (UPR) detects and mitigates the harmful effects of dysregulated endoplasmic reticulum (ER) function. The UPR has been best characterized as a protein quality control response, and the sole UPR sensor in yeast, Ire1, is known to detect misfolded ER proteins. However, recent work suggests the UPR can also sense diverse defects within the ER membrane, including increased fatty acid saturation and altered phospholipid abundance. These and other lipid-related stimuli have been referred to as lipid bilayer stress and may be sensed independently through Ire1's transmembrane domain. Here, we show that the loss of Isc1, a phospholipase that catabolizes complex ceramides, causes UPR induction, even in the absence of exogenous stress. A series of chemical and genetic approaches identified a requirement for very long-chain fatty acid (VLCFA)-containing phytoceramides for UPR induction. In parallel, comprehensive lipidomics analyses identified large increases in the abundance of specific VLCFA-containing phytoceramides in the isc1Δ mutant. We failed to identify evidence of an accompanying defect in protein quality control or ER-associated protein degradation. These results extend our understanding of lipid bilayer stress in the UPR and provide a foundation for mechanistic investigation of this fascinating intersection between ceramide metabolism, membrane homeostasis, and the UPR.

The unfolded protein response sensor PERK mediates mechanical stress-induced maturation of focal adhesion complexes in glioblastoma cells.

Stiffening of the brain extracellular matrix (ECM) in glioblastoma promotes tumor progression. Previously, we discovered that protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) plays a role in glioblastoma stem cell (GSC) adaptation to matrix stiffness through PERK/FLNA-dependent F-actin remodeling. Here, we examined the involvement of PERK in detecting stiffness changes via focal adhesion complex (FAC) formation. Compared to control GSCs, PERK-deficient GSCs show decreased vinculin and tensin expression, while talin and integrin-β1 remain constant. Furthermore, vimentin was also reduced while tubulin increased, and a stiffness-dependent increase of the differentiation marker GFAP expression was absent in PERK-deficient GSCs. In conclusion, our study reveals a novel role for PERK in FAC formation during matrix stiffening, which is likely linked to its regulation of F-actin remodeling.

Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.

Endoplasmic reticulum: Monitoring and maintaining protein and membrane homeostasis in the endoplasmic reticulum by the unfolded protein response

The endoplasmic reticulum (ER) regulates essential cellular processes, including protein folding, lipid synthesis, and calcium homeostasis. The ER homeostasis is maintained by a conserved set of signaling cascades called the Unfolded Protein Response (UPR). How the UPR senses perturbations in ER homeostasis has been the subject of active research for decades. In metazoans, the UPR consists of three ER-membrane embedded sensors: IRE1, PERK and ATF6. These sensors detect the accumulation of misfolded proteins in the ER lumen and adjust protein folding capacity according to cellular needs. Early work revealed that the ER-resident chaperone BiP binds to all three UPR sensors in higher eukaryotes and BiP binding was suggested to regulate their activity. More recent data have shown that in higher eukaryotes the interaction of the UPR sensors with a complex network of chaperones and misfolded proteins modulates their activation and deactivation dynamics. Furthermore, emerging evidence suggests that the UPR monitors ER membrane integrity beyond protein folding defects. However, the mechanistic and structural basis of UPR activation by proteotoxic and lipid bilayer stress in higher eukaryotes remains only partially understood. Here, we review the current understanding of novel protein interaction networks and the contribution of the lipid membrane environment to UPR activation.

Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

Stearoyl-CoA desaturase 1 inhibition induces ER stress-mediated apoptosis in ovarian cancer cells

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis.

Abstract 6096: Targeting premalignant lung cancer to intercept progression to invasive disease

Abstract Increased implementation of low dose CT-guided screens in the clinic has led to the identification of premalignant lung nodules (ground glass opacity, GGO) which progress to invasive adenocarcinoma1,2. Using integrated genomic analysis of a cohort of >300 patients with premalignant lesions, we constructed the first high-resolution landscape of composition, lineage/functional states, developmental trajectories, and multicellular crosstalk networks, which revealed potential targets for therapeutic intervention. We uncovered that early lesions exhibit marked immune-suppressive phenotypes characterized by increased T lymphocyte exhaustion to cytotoxic scores, decrease in NKT cells, elevated Tregs, increased myeloid suppressor activity, accumulation of immunosuppressive myeloid cells activation of unfolded protein response (UPR) sensor IRE1α that activates the multitasking transcription factor XBP1 known to drive malignant progression. Targeting IRE1α endoribouclease with a small molecule drug limited disease progression with marked immunomodulation in our newly developed Kras and EGFR driven mouse models, which mirror progression of human precursor lesions to invasive disease. As an immunoprevention approach, we have generated LNP-mRNA vaccines targeting top two shared neoantigens in premalignant lesions, which have shown marked immunogenicity and efficacy in early disease. Our work provides the molecular foundation for precision oncology strategies to intercept transition of preinvasive to invasive adenocarcinoma. 1. Altorki, N.K., et al. Nat Rev Cancer, 2019. 2. Altorki, N.K., et al. Cell Rep, 2022. Citation Format: Mitchell Martin, Yongfeng He, Bhavneet Bhinder, Liron Yoffe, Sijin Luo Zhong, Arshdeep Singh, Olivier Elemento, Shaoyi Jiang, Nasser Altorki, Vivek Mittal. Targeting premalignant lung cancer to intercept progression to invasive disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6096.

IRE1 RNase controls CD95-mediated cell death

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response

The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

SPRTN is involved in hepatocellular carcinoma development through the ER stress response.

Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.

Bilayer tension-induced clustering of the UPR sensor IRE1

The endoplasmic reticulum acts as a protein quality control center where a range of chaperones and foldases facilitates protein folding. IRE1 is a sensory transmembrane protein that transduces signals of proteotoxic stress by forming clusters and activating a cellular program called the unfolded protein response (UPR). Recently, membrane thickness variation due to membrane compositional changes have been shown to drive IRE1 cluster formation, activating the UPR even in the absence of proteotoxic stress. Here, we demonstrate a direct relationship between bilayer tension and UPR activation based on IRE1 dimer stability. The stability of the IRE1 dimer in a (50%DOPC-50%POPC) membrane at different applied bilayer tensions was analyzed via molecular dynamics simulations. The potential of mean force for IRE1 dimerization predicts a higher concentration of IRE1 dimers for both tensed and compressed ER membranes. This study shows that IRE1 may be a mechanosensitive membrane protein and establishes a direct biophysical relationship between bilayer tension and UPR activation.

TMIC-80. PERK CONTRIBUTES TO GLIOBLASTOMA PROGRESSION BY REGULATING GLIOBLASTOMA STEM CELL ADAPTATION TO EXTRA CELLULAR MATRIX STIFFENING THROUGH F-ACTIN REMODELING AND FOCAL ADHESION COMPLEX REGULATION

Abstract The association between a stiffening extracellular matrix (ECM) and increased aggressiveness of gliomas is well established. Originally recognized as a protective mechanism against endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) is primarily responsible for maintaining cellular protein homeostasis and triggering cell death under severe stress conditions. However, emerging evidence indicates that the ER transmembrane UPR sensors, IRE1 and PERK, possess additional functions beyond proteostasis, such as modulating cancer stem cell differentiation, cell adhesion, and migration, which also involve more recently identified protein scaffold functions. In this study, we uncover a novel role of PERK in cellular adaptation to matrix stiffening. Glioblastoma stem cells (GSCs), when cultured on human blood plasma/alginate hydrogels with tuneable stiffness, exhibited stiffness-dependent cellular responses, including cell flattening, differentiation, and enhanced migration. Through genetic depletion and pharmacological inhibition strategies combined with immunofluorescence microscopy, we identified the involvement of a previously found PERK/FLNA signaling mechanism in the adaptive response to matrix stiffness, where F-actin polymerization emerged as a key mechanism. To explore the possible involvement of PERK in sensing ECM stiffness, the expression of various proteins that constitute the focal adhesion complex were studied. Remarkably, PERK also influenced the maturation of focal adhesion complexes components know to be involved in sensing ECM stiffness. Finally, comparative transcriptional profiling of PERK proficient and deficient GSCs uncovered significant alterations in the expression of genes associated with cell adhesion and extracellular matrix (ECM)-related processes. These findings reveal a novel function of PERK in stiffness-dependent cellular adaptations, with ongoing investigations exploring the underlying molecular mechanisms and therapeutic implications for glioblastoma progression

The Unfolded Protein Response Sensor IRE1 Regulates Activation of In Vitro Differentiated Type 1 Conventional DCs with Viral Stimuli.

Type 1 conventional dendritic cells (cDC1s) are leukocytes competent to coordinate antiviral immunity, and thus, the intracellular mechanisms controlling cDC1 function are a matter of intense research. The unfolded protein response (UPR) sensor IRE1 and its associated transcription factor XBP1s control relevant functional aspects in cDC1s including antigen cross-presentation and survival. However, most studies connecting IRE1 and cDC1 function are undertaken in vivo. Thus, the aim of this work is to elucidate whether IRE1 RNase activity can also be modeled in cDC1s differentiated in vitro and reveal the functional consequences of such activation in cells stimulated with viral components. Our data show that cultures of optimally differentiated cDC1s recapitulate several features of IRE1 activation noticed in in vivo counterparts and identify the viral analog Poly(I:C) as a potent UPR inducer in the lineage. In vitro differentiated cDC1s display constitutive IRE1 RNase activity and hyperactivate IRE1 RNase upon genetic deletion of XBP1s, which regulates production of the proinflammatory cytokines IL-12p40, TNF-α and IL-6, Ifna and Ifnb upon Poly(I:C) stimulation. Our results show that a strict regulation of the IRE1/XBP1s axis regulates cDC1 activation to viral agonists, expanding the scope of this UPR branch in potential DC-based therapies.

Therapeutic potential of endoplasmic reticulum stress inhibitors in the treatment of diabetic peripheral neuropathy.

Endoplasmic stress response, the unfolded protein response (UPR), is a homeostatic signaling pathway comprising transmembrane sensors that get activated upon alterations in ER luminal environment. Studies suggest a relation between activated UPR pathways and several disease states such as Parkinson, Alzheimer, inflammatory bowel disease, tumor growth, and metabolic syndrome. Diabetic peripheral neuropathy (DPN), a common microvascular complication of diabetes-related chronic hyperglycemia, causes chronic pain, loss of sensation, foot ulcers, amputations, allodynia, hyperalgesia, paresthesia, and spontaneous pain. Factors like disrupted calcium signaling, dyslipidemia, hyperglycemia, inflammation, insulin signaling, and oxidative stress disturb the UPR sensor levels manifesting as DPN. We discuss new effective therapeutic alternatives for DPN that can be developed by targeting UPR pathways like synthetic ER stress inhibitors like 4-PhenylButyric acid (4-PBA), Sephin 1, Salubrinal and natural ER stress inhibitors like Tauroursodeoxycholic acid (TUDCA), Cordycepin, Proanthocyanidins, Crocin, Purple Rice extract and cyanidin and Caffeic Acid Phenethyl Ester (CAPE).

Unfolded protein response factor ATF6 augments T helper cell responses and promotes mixed granulocytic airway inflammation

The unfolded protein response (UPR) is associated with the risk of asthma, including treatment-refractory severe asthma. Recent studies demonstrated a pathogenic role of activating transcription factor 6a (ATF6a or ATF6), an essential UPR sensor, in airway structural cells. However, its role in T helper (TH) cells has not been well examined. In this study, we found that ATF6 was selectively induced by signal transducer and activator of transcription6 (STAT6) and STAT3 in TH2 and TH17 cells, respectively. ATF6 upregulated UPR genes and promoted the differentiation and cytokine secretion of TH2 and TH17 cells. T cell-specific Atf6-deficiency impaired TH2 and TH17 responses in vitro and in vivo and attenuated mixed granulocytic experimental asthma. ATF6 inhibitor Ceapin A7 suppressed the expression of ATF6 downstream genes and TH cell cytokines by both murine and human memory clusters of differentiation 4 (CD4)+ T cells. At the chronic stage of asthma, administration of Ceapin A7 lessened TH2 and TH17 responses, leading to alleviation of both airway neutrophilia and eosinophilia. Thus, our results demonstrate a critical role of ATF6 in TH2 and TH17 cell-driven mixed granulocytic airway disease, suggesting a novel option to combat steroid-resistant mixed and even T2-low endotypes of asthma by targeting ATF6.

Ac-SDKP inhibits Tunicamycin-induced ER stress UPR's sensors and subsequently inflammation and fibrosis in human cardiac fibroblasts

Background: Endoplasmic Reticulum (ER) stress, chronic inflammation and cardiac fibrosis are very common pathophysiologic processes found in hypertension and other cardiovascular diseases. Prolonged ER stress disturbs ER functional capacity, resulting from accumulated unfolded or misfolded proteins in ER lumen. Thereby, cells activate an adaptive mechanism ‘unfolded protein response (UPR)’ to restore ER normal function. Consequently, a series of downstream signaling pathways are activated. N-acetyl-seryl-aspartyl-proline (Ac-SDKP) is an endogenous tetrapeptide released from its precursor thymosin-β4 and found in circulation and organs including heart and kidneys. Previously, our in-vivo studies showed that Ac-SDKP inhibits inflammation and fibrosis through the mechanism that remains elusive. Methods and Results: Quiescent human Cardiac Fibroblast (hCFB) cells were treated with tunicamycin (TM; 0.25 μg/mL) in absence or presence of Ac-SDKP (10 nM) and Tauroursodeoxycholic acid (TUDCA; 500 μg/mL). Ac-SDKP inhibited TM-induced expression of ER stress and downstream mediated UPRs sensors IRE1ɑ, XBP1, ATF4 and CHOP encoding molecular chaperons and protein processing enzymes, at the transcriptional and translational levels [ IRE1α; Ctl (1.0 ± 0.39)***, TM (2.99 ± 0.34), Ac-SDKP +TM (1.9 ± 0.10)*** Tudca +TM (2.5 ± 0.20)***. XBP1; Ctl (1.0 ± 0.36)***, TM (8.56 ± 0.77), Ac-SDKP +TM (4.57 ± 0.4)***, Tudca +TM (5.1 ± 0.6)***. ATF4; Ctl (1.0 ± 0.17)***, TM (5.35 ± 0.63), Ac-SDKP +TM (3.0 ± 0.31)**, Tudca +TM (2.9 ± 0.36)**. CHOP; Ctl (1.0 ± 0.17)***, TM (3.1 ± 0.21), Ac-SDKP +TM (1.75 ± 0.24)***, Tudca +TM (2.0 ± 0.18)**]. The inhibition of ER and UPRs sensors subsequently prevented proinflammatory mediator NF-kB activation, cytokine IL-6 and fibrotic Collagen-I (Coll I) expression [ NF-κB; Ctl (1.0 ± 0.10)*, TM (1.6 ± 0.13), Ac-SDKP +TM (1.08 ± 0.11)†, Tudca +TM (1.09 ± 0.14)†. IL-6; Ctl (1.0 ± 0.18)***, TM (6.8 ± 1.4), Ac-SDKP +TM (1.24 ± 0.23***), Tudca +TM (1.3 ± 0.35)***. Coll I; Ctl (1.0 ± 0.02)*** TM (2.5 ± 0.43), Ac-SDKP +TM (1.13 ± 0.13)***, Tudca +TM (1.22 ± 0.18)***]. TM also induced collagen production (Hydroxyproline assay), which was significantly inhibited by Ac-SDKP. The common ER stress inhibitor, TUDCA, exhibited similar inhibitory profile as Ac-SDKP. Conclusion: Ac-SDKP prevents inflammation and fibrosis in hCFB, likely by blocking the overexpressed detrimental ER stress UPRs sensors and its downstream molecular chaperon. Notes: Data are shown a mean ± SEM, p values were calculated using one-way ANOVA compared to CTL. All the experiments are representative of n = 5 – 8.***:p ≤ 0.0001 vs TM; **:p ≤ 0.001 vs TM; *: p ≤ 0.05 vs TM; †:p ≤ 0.05 vs TM. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ursodeoxycholic Acid Binds PERK and Ameliorates Neurite Atrophy in a Cellular Model of GM2 Gangliosidosis.

The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization.

Normal tissue homeostasis and impairment of selective inflammatory responses in dendritic cells deficient for ATF6α.

The initiation of adaptive immunity relies on the performance of dendritic cells (DCs), which are specialized leukocytes with professional antigen presenting capabilities. As such, the molecular mechanisms safeguarding DC homeostasis are matter of intense research. Sensors of the unfolded protein response (UPR) of the endoplasmic reticulum, a three-pronged signaling pathway that maintains the fidelity of the cellular proteome, have emerged as regulators of DC biology. The archetypical example is the IRE1/XBP1s axis, which supports DC development and survival of the conventional type 1 DC (cDC1) subtype. However, the role of additional UPR sensors in DC biology, such as the ATF6α branch, has not been clearly elucidated. Even though Xbp1 is transcriptionally induced by ATF6α under ER stress, it is unclear if cDCs also co-opt the ATF6α branch in tissues. Here, we examine the role of ATF6α in cDC homeostasis in vivo and upon innate stimulation in vitro. In steady state, animals lacking ATF6α in CD11c+ cells (Itgax Cre x Atf6 fl/fl mice) display normal cDC frequencies in spleen, intestine, liver, and lung. Also, ATF6α deficient cDCs express normal levels of Xbp1 mRNA and additional UPR components. However, a reduction of lung monocytes is observed in Itgax Cre x Atf6 fl/fl conditional deficient animals suggesting that ATF6α may play a role in the biology of monocyte subsets. Notably, in settings of DC activation, ATF6α contributes to the production of IL-12 and IL-6 to inflammatory stimuli. Thus, although ATF6α may be dispensable for tissue cDC homeostasis in steady state, the transcription factor plays a role in the acquisition of selective immunogenic features by activated DCs.

Runx1/3-driven adaptive endoplasmic reticulum stress pathways contribute to neurofibromagenesis.

Neurofibromatosis type 1 (NF1) patients are predisposed to develop plexiform neurofibromas (PNFs). Three endoplasmic reticulum (ER) stress response pathways restore cellular homeostasis. The unfolded protein response (UPR) sensors contribute to tumor initiation in many cancers. We found that all three UPR pathways were activated in mouse and human PNFs, with protein kinase RNA [PKR]-like ER kinase (PERK) the most highly expressed. We tested if neurofibroma cells adapt to ER stress, leading to their growth. Pharmacological or genetic inhibition of PERK reduced mouse neurofibroma-sphere number, and genetic inhibition in PERK in Schwann cell precursors (SCPs) decreased tumor-like lesion numbers in a cell transplantation model. Further, in a PNF mouse model, deletion of PERK in Schwann cells (SCs) and SCPs reduced tumor size, number, and increased survival. Mechanistically, loss of Nf1 activated PERK-eIF2α-ATF4 signaling and increased ATF4 downstream target gene p21 translocation from nucleus to cytoplasm. This nucleus-cytoplasm translocation was mediated by exportin-1. Runx transcriptionally activated ribosome gene expression and increased protein synthesis to allow SCs to adapt to ER stress and tumor formation. We propose that targeting proteostasis might provide cytotoxic and/or potentially durable novel therapy for PNFs.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic β cells invitro and in models of neurodegeneration and diabetes invivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons invitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.