Endoplasmic Reticulum Unfolded Protein Response Research Articles

Mid-late Gestation Leptin Infusion Induces Placental Mitochondrial and Endoplasmic Reticulum Unfolded Protein Responses in a Mouse Model of Preeclampsia

IntroductionPreeclamptic patients, both lean and obese, present with elevated leptin levels which are associated with the development of maternal endothelial dysfunction and adverse fetal outcomes, such as growth restriction, leading to low birth weight. Recent studies in pregnant mice demonstrate that mid-late gestation leptin infusion induces clinical characteristics of preeclampsia, including elevated maternal blood pressure, maternal endothelial dysfunction and fetal growth restriction. However, whether leptin triggers placental stress responses that contribute to adverse fetal outcomes as in preeclampsia is unknown. MethodsIn the current study we measured the expression of proteins involved in the endoplasmic reticulum (UPRer) and mitochondrial (UPRmt) unfolded protein responses in placentas of wild-type sham normal pregnant and leptin-infused preeclamptic mice. ResultsThe data show that mid-late gestation leptin infusion induced activation of indices of placental UPRer and UPRmt, while reducing placental repair mechanisms to UPRmt in preeclamptic mice. Mid-late gestation infusion with leptin upregulated markers of placental oxidative stress, reduced the placental expression levels of mitochondrial electron transport chain complexes I and II and increased the expression of placental endothelin-1 (ET-1) in preeclamptic mice. The leptin-induced activation of several placental UPRmt markers as well as ET-1 levels correlated with fetal growth restriction and impaired maternal endothelial function in preeclamptic mice. DiscussionCollectively, these data indicate that elevated levels of leptin in mid-late pregnancy in mice promote placental stress responses, akin to those in pregnant women with preeclampsia.

IGFBP6 Modulates Proteostasis by Activating ATF4 Targets and Reducing ER Retrotranslocon Expression.

Reduced expression of the IGFBP6 protein leads to an increase in the metastatic potential of breast cancer (BC) cells. The level of protein synthesis in tumor cells is increased, leading to a compensatory adjustment of proteostasis. One of the tools used to study proteostasis is protein toxins of the RIP-II family, which irreversibly inactivate ribosomes (particularly, viscumin). We investigated the effect of IGFBP6 gene knockdown on the proteostasis in the BC cell line MDA-MB-231. Ribosomes from MDA-MB-231IGFBP6 cells, knockdown for the IGFBP6 gene, are less efficiently modified by the toxin. This is probably due to the reduced transport of the viscumin catalytic subunit from the ER to the cytoplasm. MDA-MB-231IGFBP6 cells showed reduced expression of the retrotranslocon HRD1/Derlin subunit, which is a component of the ER-associated protein degradation system (ERAD). For ATF4 transcription factor, which is a part of the ER unfolded protein response (UPR) pathway, an increased expression of its targets was found.

Food grade titanium dioxide induced endoplasmic reticulum stress in colon cells: Comparison between normal and colorectal carcinoma cells

BackgroundFood-grade titanium dioxide (E171) has been under scrutiny in the last decade since its possible adverse effects; however, the cellular mechanisms underlying E171 toxicity have not been thoroughly described. AimWe aimed to compare the effects of E171 on endoplasmic reticulum (ER) homeostasis in normal and cancer colon cells. Experimental designWe exposed normal, carcinoma, and adenocarcinoma cells to 0.1, 1, 10, 50 and 100 μg/cm2 of E171 for 24, 48 and 72 h, and we evaluated ER stress, cell viability, titanium uptake, intracellular calcium concentration, and gene expression related to unfolded protein response (UPR) and chaperone pathways. ResultsCell viability decreased only after 72 h of exposure to 100 μg/cm2 of E171. Adenocarcinoma cells internalized higher titanium amounts than normal and carcinoma cells, but the effects in ER distribution, intracellular calcium concentration, and gene expression were similar among the three cell lines. The expression of UPR and chaperone pathways were downregulated at the lowest concentrations but upregulated at the highest concentrations in the three cell lines. ConclusionE171 induces ER stress through alterations in ER distribution, intracellular calcium, and UPR and chaperone protein pathways.

MONITTR allows real-time imaging of transcription and endogenous proteins in C. elegans.

Maximizing cell survival under stress requires rapid and transient adjustments of RNA and protein synthesis. However, capturing these dynamic changes at both single-cell level and across an organism has been challenging. Here, we developed a system named MONITTR (MS2-embedded mCherry-based monitoring of transcription) for real-time simultaneous measurement of nascent transcripts and endogenous protein levels in C. elegans. Utilizing this system, we monitored the transcriptional bursting of fasting-induced genes and found that the epidermis responds to fasting by modulating the proportion of actively transcribing nuclei and transcriptional kinetics of individual alleles. Additionally, our findings revealed the essential roles of the transcription factors NHR-49 and HLH-30 in governing the transcriptional kinetics of fasting-induced genes under fasting. Furthermore, we tracked transcriptional dynamics during heat-shock response and ER unfolded protein response and observed rapid changes in the level of nascent transcripts under stress conditions. Collectively, our study provides a foundation for quantitatively investigating how animals spatiotemporally modulate transcription in various physiological and pathological conditions.

The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APCCdc20.

Aberrant regulation of unfolded protein response (UPR)/endoplasmic reticulum (ER) stress pathway is associated with cancer development, metastasis, and relapse, and the UPR signal transducer ATF6 has been proposed as a diagnostic and prognostic marker for many cancers. However, a causal molecular link between ATF6 activation and carcinogenesis is not established. Here, it is found that tumor protein D52 (TPD52) integrates ER stress and UPR signaling with the chaperone machinery by promoting S2P-mediated cleavage of ATF6. Although TPD52 has been generally considered as an oncogene, TPD52 is identified as a novel tumor suppressor in bladder cancer. Significantly, attenuation of the ER stress via depletion of TPD52 facilitated tumorigenesis in a subset of human carcinomas. Furthermore, the APCCdc20 E3 ligase is validated as the upstream regulator marking TPD52 for polyubiquitination-mediated proteolysis. In addition, inactivation of Cdc20 sensitized cancer cells to treatment with the ER stress inducer in a TPD52-dependent manner. Thus, the study suggests that TPD52 is a novel Cdc20 substrate that may modulate ER stress to prevent tumorigenesis.

Olfactory dysfunction as an early pathogenic indicator in C. elegans models of Alzheimer's and polyglutamine diseases.

Neurodegenerative diseases such as Alzheimer's disease and polyglutamine diseases are characterized by abnormal accumulation of misfolded proteins, leading to neuronal dysfunction and subsequent neuron death. However, there is a lack of studies that integrate molecular, morphological, and functional analyses in neurodegenerative models to fully characterize these time-dependent processes. In this study, we used C. elegans models expressing Aβ1-42 and polyglutamine to investigate early neuronal pathogenic features in olfactory neurons. Both models demonstrated significant reductions in odor sensitivity in AWB and AWC chemosensory neurons as early as day 1 of adulthood, while AWA chemosensory neurons showed no such decline, suggesting cell-type-specific early neuronal dysfunction. At the molecular level, Aβ1-42 or Q40 expression caused age-dependent protein aggregation and morphological changes in neurons. By day 6, both models displayed prominent protein aggregates in neuronal cell bodies and neurites. Notably, AWB neurons in both models showed significantly shortened cilia and increased instances of enlarged cilia as early as day 1 of adulthood. Furthermore, AWC neurons expressing Aβ1-42 displayed calcium signaling defects, with significantly reduced responses to odor stimuli on day 1, further supporting early behavioral dysfunction. In contrast, AWA neuron did not exhibit reduced calcium responses, consistent with the absence of detectable decreases in olfactory sensitivity in these neurons. These findings suggest that decreased calcium signaling and dysfunction in specific sensory neuron subtypes are early indicators of neurodegeneration in C. elegans, occurring prior to the formation of visible protein aggregates. We found that the ER unfolded protein response (UPR) is significantly activated in worms expressing Aβ1-42. Activation of the AMPK pathway alleviates olfactory defects and reduces fibrillar Aβ in these worms. This study underscores the use of C. elegans olfactory neurons as a model to elucidate mechanisms of proteostasis in neurodegenerative diseases and highlights the importance of integrated approaches.

Insulin-degrading enzyme inhibition increases the unfolded protein response and favours lipid accumulation in the liver.

Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin-degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR. We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and lipid accumulation in the liver. We found that IDE-deficient mice display higher hepatic triglyceride content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis. These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.

The Involvement of Endoplasmic Reticulum Stress during the Interaction between Calcium Oxalate Crystals and Renal Tubular Epithelial Cells.

Our study aimed to elucidate the mechanisms behind the interaction between calcium oxalate (CaOx) crystals and renal tubular epithelial cells through transcriptome sequencing analysis. HK-2 cells were stimulated with or without CaOx monohydrate crystals and subjected to RNA-seq to assess the effects of CaOx crystals on gene expression changes, key pathways, and molecular players during this interaction. A total of 629 differentially expressed genes (DEGs) were identified between the control group and experimental group, with 491 genes up-regulated and 138 down-regulated. Functional enrichment analysis indicated that the DEGs were significantly associated with endoplasmic reticulum stress (ERS) and unfolded protein response. To validate our findings, we compared our results with the public dataset GSE73680 and confirmed the increased expression of two ERS-related DEGs, CHAC1 and FGF21, in renal papillary tissues from patients with CaOx stones. Collectively, these findings suggest that ERS plays a crucial role in the crystal-cell interaction and highlight the potential for developing therapeutic strategies aimed at reducing CaOx stone formation by targeting ERS-related molecules and pathways.

Temperature-dependent lifespan extension is achieved in miR-80-deleted Caenorhabditis elegans by NLP-45 to modulate endoplasmic reticulum unfolded protein responses.

MicroRNA plays a crucial role in post-transcriptional gene regulation and has recently emerged as a factor linked to aging, but the underlying regulatory mechanisms remain incompletely understood. In this study, we observed lifespan-extending effects in miR-80-deficient Caenorhabditis elegans at 20°C but not 25°C. At 20°C, miR-80 deletion leads to NLP-45 upregulation, which positively correlates to increased abu transcripts and extended lifespan. Supportively, we identified miR-80 binding regions in the 5' and 3' UTR of nlp-45. As the temperature rises to 25°C, wildtype increases miR-80 levels, but removal of miR-80 is accompanied by decreased nlp-45 expression, suggesting intervention from other temperature-sensitive mechanisms. These findings support the concept that microRNAs and neuropeptide-like proteins can form molecular regulatory networks involving downstream molecules to regulate lifespan, and such regulatory effects vary on environmental conditions. This study unveils the role of an axis of miR-80/NLP-45/UPRER components in regulating longevity, offering new insights on strategies of aging attenuation and health span prolongation.

Post-myocardial infarction heart failure and long-term high-fat diet: Cardiac endoplasmic reticulum stress and unfolded protein response in Sprague Dawley rat model.

Myocardial infarction (MI) significantly contributes to the global mortality rate, often leading to heart failure (HF) due to left ventricular remodeling. Key factors in the pathomechanism of HF include nitrosative/oxidative stress, inflammation, and endoplasmic reticulum (ER) stress. Furthermore, while a high-fat diet (HFD) is known to exacerbate post-MI cardiac remodeling, its impact on these critical factors in the context of HF is not as well understood. This study aimed to assess the impact of post-MI HF and HFD on inflammation, nitro-oxidative stress, ER stress, and unfolded protein response (UPR). The study was performed on fragments of the left ventricle harvested from 30 male adult Sprague Dawley rats, which were divided into four groups based on diet (normal-fat vs. high-fat) and surgical procedure (sham operation vs. coronary artery ligation to induce MI). We assessed body weight, NT-proBNP levels, protein levels related to nitrosative/oxidative stress, ER stress, UPR, apoptosis, and nitric oxide synthases, through Western Blot and ELISA. HFD and MI significantly influenced body weight and NT-proBNP concentrations. HFD elevated 3-nitrotyrosine and myeloperoxidase levels and altered nitric oxide synthase levels. HFD and MI significantly affected ER stress markers and activated or inhibited UPR pathways. The study demonstrates significant impacts of post-MI HF and dietary fat content on cardiac function and stress markers in a rat model. The interaction between HFD and MI on UPR activation suggests the importance of dietary management in post-MI recovery and HF prevention.

African swine fever virus modulates the endoplasmic reticulum stress-ATF6-calcium axis to facilitate viral replication

ABSTRACT African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development

Disruption of endoplasmic reticulum (ER) homeostasis, i.e., ER stress, is intrinsically linked with lipid metabolism disorders in dairy cows. Caveolin 1 (CAV1) is a ubiquitously-expressed membrane-associated scaffolding protein involved in regulating the secretory pathway within the ER. Whether inhibiting the activity of CAV1 affects the ER and its potential role in hepatic lipid deposition in dairy cows is unknown. Biopsies of liver tissue from Holstein cows (days in milk: median = 13 d, range = 5 to 21) diagnosed as healthy (n = 6, hepatic TAG levels < 1%, milk production: median = 38.9 kg/day, Interquartile range = 38.0 and 40.8) or suffering from fatty liver (n = 6, hepatic TAG levels > 5%, milk production: median = 36.6 kg/day, Interquartile range = 35.7 and 38.1) revealed that fatty liver was associated with lower abundance of CAV1 gene and protein, higher phosphorylation (p) levels of PERK and IRE1α, and increased abundance of ATF6, GRP78, CHOP protein, and several unfolded protein response (UPR) genes (ATF4, sXBP1, and GRP78). Proteins related to de novo fatty acid synthesis, including ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN) also had greater abundance. This in vivo analysis highlighted a mechanistic link between CAV1 protein abundance, ER stress, and lipid metabolism during fatty liver. A mechanistic study was then performed in vitro with primary hepatocytes isolated from 5 healthy calves (weight, 40-45 kg; 1 d old). Initially, hepatocytes were treated with FFA (1.2 mM) for 1, 3, 6, or 12 h. FFA treatment reduced CAV1 protein abundance linearly while it reduced abundance of ER stress-related proteins, p-IRE1α, p-PERK, GRP78, ATF6, and CHOP. Proteins related to de novo fatty acid synthesis (ACC1, SREBP-1c, PPARγ) also increased linearly, and lipid droplets accumulated progressively over time following FFA treatment. Subsequently, to assess the role of CAV1 in FFA-induced ER stress and de novo fatty acid synthesis, hepatocytes were transfected with pCMV-CAV1 (cattle)-3 × FLAG-Neo (pc-CAV1) plasmid to overexpress CAV1 or with siRNA to silence CAV1 (siCAV1) transcription. Overexpression of CAV1 alleviated ER stress by reducing levels of p-PERK and p-IRE1α, as well as the protein abundance of ATF6, GRP78, CHOP, and several UPR genes (GRP78, ATF4, and sXBP1). Similarly, CAV1 overexpression decreased protein abundance of ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN). Conversely, silencing CAV1 exacerbated FFA-induced ER stress and de novo fatty acid synthesis. Considering the negative role of FFA-induced ER stress on lipid accumulation in hepatocytes, a second in vitro experiment involved hepatocytes treated with 0.5 μg/mL tunicamycin (TM, a typical ER stress inducer) for 24 h with or without overexpressing CAV1 (pc-CAV1). Overexpressing caveolin 1 reversed TM-induced increases in mRNA and protein associated with ER stress and de novo fatty acid synthesis. Furthermore, use of hepatocytes transfected with pc-CAV1 for 48 h and subjected to co-immunoprecipitation revealed that CAV1 interacts with IRE1α and ATF6. Overall, the data suggest that CAV1 may help reduce hepatic ER stress and mitigate fatty acid synthesis by binding to and inhibiting IRE1α and ATF6 signaling.

463. ISOFORM SWITCHING EVENTS IN ESOPHAGEAL ADENOCARCINOMA MAY INFORM NEW THERAPEUTIC TARGETS THROUGH IDENTIFYING MOLECULAR MECHANISMS CONTRIBUTING TO POOR PATIENT OUTCOMES

Abstract Background Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis due to late-stage diagnosis coupled with therapeutic resistance. Moreover, EAC is a cancer with a high mutational burden but lacks highly prevalent oncogenic drivers that can be successfully targeted. Herein, we investigated whether newly identified isoform switching events, defined as differential usage of gene transcripts between conditions, may reveal new therapeutic targets and inform the molecular mechanisms contributing to EAC resistance. Methods We conducted RNA-sequencing followed by isoform switching analysis using IsoformSwitchAnalyzeR on Barrett’s low-grade dysplasia (BE.LGD) compared to Barrett’s with high-grade dysplasia (BE.HGD)+EAC tissues alone or in combination with TP53 mutation. Patients were stratified into tertiles based on isoform fraction levels, followed by overall and cancer-free survival analysis using the Cox proportional-hazards model. To assess whether mortality-linked isoforms influence cancer cell growth, viability, migration, and response to chemotherapy (Paclitaxel and Carboplatin). Isoform-specific siRNA knockdown experiments targeting HM13 were performed in OE19 and OE33 EAC cell lines, with STRING protein prediction interaction analysis and western blots conducted to assess pathway and protein alterations. Results Twenty-one isoform switched genes were significantly linked with all-cause mortality in BE.LGD versus BE.HGD+EAC and similarly 20 were significantly associated with cancer-specific patient mortality with 14 shared between the two groups. With inclusion of TP53 mutation status, 13 isoform switched genes were significantly linked with cancer-specific mortality. HM13, DANT2, CFDP1, KRAS, SIMM6 were among the most significantly altered. Knockdown of the pro-cancer isoform of HM13 significantly inhibited EAC cell migration and induced EAC cell death and knockdown of HM13 combined with chemotherapeutic treatment exhibited synergistic effects in EAC cell lines. Protein interaction analysis supported that HM13 is linked to ER-stress associated degradation, cellular response to stress, and ER unfolded protein response which were validated using a panel of relevant proteins (i.e., PERK, IRE-1a, ATF4, BIP, P-JNK). Conclusions Our research identified isoform switching events significantly linked to all-cause mortality as well as cancer specific mortality among EAC patients with and without TP53 mutations. Knockdown of the pro-cancer isoform of HM13 isoform significantly inhibited EAC cell migration and induced EAC cell death with synergistic effects observed with chemotherapeutic agent treatment supporting a role for specific isoform switches in therapeutic sensitization and in this case via the ERAD and ER unfolded protein response pathways. Modulation of specific proteins with established roles in ERAD and ER unfolded responses following isoform specific HM13 knockdown lends further support to an important role for this isoform, especially in TP53 mutant EAC. In conclusion, isoform switches may inform molecular mechanisms contributing to poor patient outcomes and reveal new potential therapeutic targets for EAC.

Astaxanthin inhibits apoptosis in a cell model of tauopathy by attenuating endoplasmic reticulum stress and unfolded protein response

The accumulation of misfolded proteins is a common pathological characteristic shared by many neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. The disruption of proteostasis triggers endoplasmic reticulum (ER) stress, during which the unfolded protein response (UPR) is initiated by the activation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). These three branches of UPR signals act in concert to reduce the levels of abnormal proteins and restore ER homeostasis. However, the overactivation of UPR impairs cell function and induces apoptosis, which has been implicated in neurodegeneration. Astaxanthin is a xanthophyll carotenoid which has been shown to have neuroprotective effects in both cell and animal models; however, its effects on ER stress and UPR induced by disrupted proteostasis remain unclear. In this study, the effects of astaxanthin on ER stress and cytotoxicity were investigated in N2a cells stably expressing the pro-aggregant tau repeat domain carrying FTDP-17 mutation ΔK280 (Tau4RDΔK280). The results demonstrated that astaxanthin significantly inhibited Tau4RDΔK280-induced loss of cell viability and apoptosis, attenuating Tau4RDΔK280-induced caspase-3 activation and decrease of Bcl-2. Further studies revealed that astaxanthin treatment alleviated Tau4RDΔK280-induced ER stress and suppressed the activation of PERK, IRE1 and ATF6 signaling pathways. These findings suggested that astaxanthin might inhibit Tau4RDΔK280-induced cytotoxicity by attenuating UPR and ER stress. In addition, astaxanthin treatment resulted in a great reduction in the production of intracellular reactive oxygen species and a significant decrease in calcium influx induced by Tau4RDΔK280, which also contributed to the protective effects of astaxanthin against Tau4RDΔK280-induced cytotoxicity.

Could the Propionic Acid Treatment in Combination with Metformin be Safe for the Small Intestine of Diabetic Rats?

Effects of propionic acid (PA) on the cellular and molecular processes in the small intestine under type 2 diabetes mellitus (T2DM)-induced endoplasmic reticulum (ER) stress remain incompletely studied. The aim of the study was to assess the state of unfolded protein response (UPR) system in the small intestine of diabetic rats and to explore PA's influence on metformin treatment. Male Wistar rats were divided into 1) control and 2) T2DM groups, and groups receiving (14 days, orally) 3) metformin (60 mg/kg), 4) PA (60 mg/kg), and 5) PA+metformin. Western blotting, RT-PCR, and transmission electron microscopy were performed. We found that T2DM induced elevation of ER intermembrane space and UPR overactivation based on increased GRP78, ATF6 and PERK levels in small intestine. Metformin treatment led to a further UPR activation. PA supplementation partially restored enterocytes functioning via normalization of ATF6 and PERK content, while IRE1 level reached the maximum value, compared to all groups. The most pronounced effect of adaptation to the T2DMinduced ER stress was observed after combined metformin and PA action. In particular, decreased ER intermembrane space in enterocytes was detected compared to separate metformin and PA administration, which was accompanied by restored GRP78, PERK and IRE1 levels. Our study proves the safety of additional therapy with propionic acid in combination with metformin for the functional state of small intestine. Due to its ability to modulate UPR signaling, PA may be considered a safe and perspective candidate for supportive therapy in T2DM, especially for neuroprotection.

Endoplasmic reticulum unfolded protein response transcriptional targets of XBP-1s mediate rescue from tauopathy

Pathological tau disrupts protein homeostasis (proteostasis) within neurons in Alzheimer’s disease (AD) and related disorders. We previously showed constitutive activation of the endoplasmic reticulum unfolded protein response (UPRER) transcription factor XBP-1s rescues tauopathy-related proteostatic disruption in a tau transgenic Caenorhabditis elegans (C. elegans) model of human tauopathy. XBP-1s promotes clearance of pathological tau, and loss of function of the ATF-6 branch of the UPRER prevents XBP-1s rescue of tauopathy in C. elegans. We conducted transcriptomic analysis of tau transgenic and xbp-1s transgenic C. elegans and found 116 putative target genes significantly upregulated by constitutively active XBP-1s. Among these were five candidate XBP-1s target genes with human orthologs and a previously known association with ATF6 (csp-1, dnj-28, hsp-4, ckb-2, and lipl-3). We examined the functional involvement of these targets in XBP-1s-mediated tauopathy suppression and found loss of function in any one of these genes completely disrupts XBP-1s suppression of tauopathy. Further, we demonstrate upregulation of HSP-4, C. elegans BiP, partially rescues tauopathy independent of other changes in the transcriptional network. Understanding how the UPRER modulates pathological tau accumulation will inform neurodegenerative disease mechanisms and direct further study in mammalian systems with the long-term goal of identifying therapeutic targets in human tauopathies.

The effect of overexpression of CyPA on gene expression in human umbilical vein endothelial cells.

The aim of this study is to screen the differentially expressed genes and genes with alternative splicing in PPIA overexpressing cells by transcriptome sequencing. Transcriptome sequencing was performed to identify differentially expressed genes and genes with altered alternative splicing in PPIA overexpressing cells and results were validated by real-time quantitative polymerase chain reaction. The biological function and pathways of those genes were further explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses. A total of 157 significantly upregulated genes and 171 significantly downregulated genes were identified in PPIA overexpressing cells, and the splicing pattern of LHPP, APH1A, BRD1, and ORAI3 was found to be altered. GO analyses showed that the most enriched GO terms of the 157 upregulated genes included extracellular region, protein binding, and metal ion, and the most enriched GO terms of the 171 downregulated genes included binding neuron projection, protein binding, and endoplasmic reticulum unfolded protein response. Kyoto Encyclopedia of Genes and Genomes analyses showed that the 157 upregulated genes were mainly enriched in gastric acid secretion, Mitogen-activated protein kinase signaling pathway, etc, and the 171 downregulated genes were mainly enriched in transcriptional misregulation in cancer, Tumor necrosis factor signaling pathway, etc. The overexpression of PPIA in human umbilical vein endothelial cells causes changes in the expression of downstream genes and induces alternative splicing in multiple genes. PPIA alters the expression or the alternative splicing pattern of downstream genes, leading to pathogenesis of vascular endothelial injury by high glucose mediated through CyPA.

Network pharmacology and molecular dynamics study of the effect of the Astragalus-Coptis drug pair on diabetic kidney disease.

Diabetic kidney disease (DKD) is the primary cause of end-stage renal disease. The Astragalus-Coptis drug pair is frequently employed in the management of DKD. However, the precise molecular mechanism underlying its therapeutic effect remains elusive. To investigate the synergistic effects of multiple active ingredients in the Astragalus-Coptis drug pair on DKD through multiple targets and pathways. The ingredients of the Astragalus-Coptis drug pair were collected and screened using the TCMSP database and the SwissADME platform. The targets were predicted using the SwissTargetPrediction database, while the DKD differential gene expression analysis was obtained from the Gene Expression Omnibus database. DKD targets were acquired from the GeneCards, Online Mendelian Inheritance in Man database, and DisGeNET databases, with common targets identified through the Venny platform. The protein-protein interaction network and the "disease-active ingredient-target" network of the common targets were constructed utilizing the STRING database and Cytoscape software, followed by the analysis of the interaction relationships and further screening of key targets and core active ingredients. Gene Ontology (GO) function and Kyoto Ency-clopedia of Genes and Genomes (KEGG) pathway enrichments were performed using the DAVID database. The tissue and organ distributions of key targets were evaluated. PyMOL and AutoDock software validate the molecular docking between the core ingredients and key targets. Finally, molecular dynamics (MD) simulations were conducted to simulate the optimal complex formed by interactions between core ingredients and key target proteins. A total of 27 active ingredients and 512 potential targets of the Astragalus-Coptis drug pair were identified. There were 273 common targets between DKD and the Astragalus-Coptis drug pair. Through protein-protein interaction network topology analysis, we identified 9 core active ingredients and 10 key targets. GO and KEGG pathway enrichment analyses revealed that Astragalus-Coptis drug pair treatment for DKD involves various biological processes, including protein phosphorylation, negative regulation of apoptosis, inflammatory response, and endoplasmic reticulum unfolded protein response. These pathways are mainly associated with the advanced glycation end products (AGE)-receptor for AGE products signaling pathway in diabetic complications, as well as the Lipid and atherosclerosis. Molecular docking and MD simulations demonstrated high affinity and stability between the core active ingredients and key targets. Notably, the quercetin-AKT serine/threonine kinase 1 (AKT1) and quercetin-tumor necrosis factor (TNF) protein complexes exhibited exceptional stability. This study demonstrated that DKD treatment with the Astragalus-Coptis drug pair involves multiple ingredients, targets, and signaling pathways. We propose a novel approach for investigating the molecular mechanism underlying the therapeutic effects of the Astragalus-Coptis drug pair on DKD. Furthermore, we suggest that quercetin is the most potent active ingredient and specifically targets AKT1 and TNF, providing a theoretical foundation for further exploration of pharmacologically active ingredients and elucidating their molecular mechanisms in DKD treatment.

Identifying transcriptomic profiles in ovine spleen after repetitive vaccination.

Aluminum hydroxide has long been employed as a vaccine adjuvant for its safety profile, although its precise mechanism of action remains elusive. In this study, we investigated the transcriptomic responses in sheep spleen following repetitive vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. Notably, this work represents the first exploration of the sheep spleen transcriptome in such conditions. Animals were splitted in 3 treatment groups: vaccine group, adjuvant alone group and control group. A total of 18 high-depth RNA-seq libraries were sequenced, resulting in a rich dataset which also allowed isoform-level analysis. The comparisons between vaccine-treated and control groups (V vs C) as well as between vaccine-treated and adjuvant-alone groups (V vs A) revealed significant alterations in gene expression profiles, including protein coding genes and long non-coding RNAs. Among the differentially expressed genes, many were associated with processes such as endoplasmic reticulum (ER) stress, immune response and cell cycle. The analysis of co-expression modules further indicated a correlation between vaccine treatment and genes related to ER stress and unfolded protein response. Surprisingly, adjuvant-alone treatment had little impact on the spleen transcriptome. Additionally, the role of alternative splicing in the immune response was explored. We identified isoform switches in genes associated with immune regulation and inflammation, potentially influencing protein function. In conclusion, this study provides valuable insights into the transcriptomic changes in sheep spleen following vaccination with aluminum adjuvanted vaccines and aluminum hydroxide alone. These findings shed light on the molecular mechanisms underlying vaccine-induced immune responses and emphasize the significance of antigenic components in aluminum adjuvant mechanism of action. Furthermore, the analysis of alternative splicing revealed an additional layer of complexity in the immune response to vaccination in a livestock species.

Blood Brain Barrier-Crossing Delivery of Felodipine Nanodrug Ameliorates Anxiety-Like Behavior and Cognitive Impairment in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder leading to cognitive decline. Excessive cytosolic calcium (Ca2+) accumulation plays a critical role in the pathogenesis of AD since it activates the NOD-like receptor family, pyrin domain containing 3 (NLRP3), switches the endoplasmic reticulum (ER) unfolded protein response (UPR) toward proapoptotic signaling and promotes Aβ seeding. Herein, a liposomal nanodrug (felodipine@LND) is developed incorporating a calcium channel antagonist felodipine for Alzheimer's disease treatment through a low-intensity pulse ultrasound (LIPUS) irradiation-assisted blood brain barrier (BBB)-crossing drug delivery. The multifunctional felodipine@LND is effectively delivered to diseased brain through applying a LIPUS irradiation to the skull, which resulted in a series of positive effects against AD. Markedly, the nanodrug treatment switched the ER UPR toward antioxidant signaling, prevented the surface translocation of ER calreticulin (CALR) in microglia, and inhibited the NLRP3 activation and Aβ seeding. In addition, it promoted the degradation of damaged mitochondria via mitophagy, thereby inhibiting the neuronal apoptosis. Therefore, the anxiety-like behavior and cognitive impairment of 5xFAD mice with AD is significantly ameliorated, which manifested the potential of LIPUS - assisted BBB-crossing delivery of felodipine@LND to serve as a paradigm for AD therapy based on the well-recognized clinically available felodipine.