Endoplasmic Reticulum Stress

Mitochondria have evolved a specialized mitochondrial unfolded protein response (UPRmt) to maintain proteostasis and promote recovery under stress. Studies in simple organisms have shown that UPRmt activation in glial cells supports proteostasis through beneficial non-cell-autonomous communication with neurons. However, the role of mitochondrial stress responses in the human brain remains unclear. To address this gap, we investigated the cell-type-specific effects of mitochondrial proteotoxic stress using human induced pluripotent stem cell-derived neuronal and glial cultures, as well as brain organoids. Here we show that mitochondrial proteotoxic stress induces metabolic rewiring in human microglia, marked by depletion of S-adenosylmethionine and lipid remodeling, ultimately leading to a senescent phenotype. Using human neuronal-glial tricultures and microglia-containing brain organoids, we identified the specific contributions of microglia to brain senescence and mitochondrial stress-driven neurodegenerative processes. UPRmt activation disrupts microglial communication with neighboring cells, triggering inflammatory signaling and impairing proteostasis. Together, these findings reveal how impaired mitochondrial proteostasis alters intercellular networks and identify a critical role for the UPRmt in neurodegenerative disease pathogenesis.

Background: Chronic exposure to free fatty acids (FFAs) and glucose may disrupt metabolic homeostasis and initiate pathological processes. This study investigated the effects of hyperglycemia and fat overload on renal endoplasmic reticulum (ER) stress, pyroptosis and fibrogenesis in mice and the underlying basis. We hypothesized that the combined insult would more severely induce Gα12-dependent ER stress and renal complications. Methods: Mice were subjected to either high fat diet (HFD)+streptozotocin (STZ), or STZ treatment, and AZ2 was used as an anti-diabetic agent. Blood sera were used for blood biochemistry, and tissues were employed for RNA sequencing, immunoblottings, TEM, histology and immunohistochemistry. HEK293 and other cells were used for high glucose (HG) and palmitate treatment, or Gα12 or siGα12 transfection. Results: The combined HFD and STZ treatment, showing enrichment of genes related to GPCR signaling, inflammasome, ER stress, and pyroptosis in the RNA-sequencing analysis, upregulated Gα12 in the kidney, alongside increased PGC1α and PPARα. IRE1α and ATF6 were elevated without an increase in GRP78. This was accompanied by elevated blood glucose, creatinine, and BUN levels. We also found increases of pro-IL-1β, IL-1β, caspase-1, and NLRP3, demonstrating pyroptosis. Immunoassays revealed increased fibrosis markers. AZ2 reversed these changes. STZ treatment alone exhibited mild complications in the absence of Gα12 induction despite severe hyperglycemia. In cell-based assays, HG+palmitate elicited IRE1 activation along with Gα12 overexpression although HG alone had a minimal effect. Overexpression of Gα12 facilitated the effect of HG+palmitate on ER stress, pyroptosis, and fibrosis, whereas Gα12 knockdown had the opposite effect, as corroborated by the outcomes obtained using STZ-treated Gα12-/-, Gα12+/-, and Gα13 liver-specific KO mice. Conclusion: These findings support the role of HG and lipid overload combination in driving renal pyroptosis and fibrogenesis through Gα12-mediated ER stress and inflammasome, delineating the mechanism underlying the conditions of diabetic renal complications and pharmacological intervention.

IntroductionLung ischemia-reperfusion injury (LIRI)-a fatal complication following major surgery, trauma, or shock- is characterized by pulmonary endothelial barrier (PEB) dysfunction. Although thioredoxin domain-containing protein 5 (TXNDC5) regulates endothelial homeostasis, its role in LIRI pathogenesis remains unknown.ObjectivesIn the present study, we investigate the role of TXNDC5 in LIRI-induced PEB disruption and explore its underlying mechanisms and therapeutic potential.MethodsWe established LIRI models in rats (left pulmonary hilum occlusion) and human umbilical vein endothelial cells (oxygen-glucose deprivation/reperfusion). TXNDC5 expression was modulated via AAV-shRNA knockdown, whereas endoplasmic reticulum (ER) stress was inhibited pharmacologically (4-PBA and Ceapin-A7). Structural and functional changes of the PEB, including HSP90/eNOS complex stability, were examined. Mechanistic studies included ChIP-seq identified ATF6 binding to the TXNDC5 promoter, followed by rescue experiments with ATF6 inhibition and TXNDC5 overexpression. Clinical validation was performed using plasma proteomics in patients with LIRI. A TXNDC5-based nomogram model for LIRI early-warning was constructed and internally validated. Incremental prediction value of TXNDC5 was evaluated using net reclassification improvement (NRI) and integrated discrimination improvement (IDI).ResultsSingle-cell RNA sequencing revealed TXNDC5 upregulation in pulmonary endothelial cells during LIRI, correlating with PEB disruption, apoptosis, ER stress, and NF-κB activation. TXNDC5 knockdown restored HSP90/eNOS stability and improved endothelial integrity in vitro/vivo. Ceapin-A7, or 4-PBA, attenuated TXNDC5-driven PEB injury, and TXNDC5 overexpression confirmed the causal role of the ATF6-TXNDC5 axis. Mechanistically, ER stress promoted TXNDC5 transcription via ATF6 binding to its promoter. Clinically, elevated plasma TXNDC5 levels predicted LIRI and correlated with the severity of the condition. Incorporating the TXNDC5 index into the clinical predictors model enhanced the forecast of LIRI, as demonstrated by the NRI and IDI.ConclusionOur findings demonstrate that ER stress-induced TXNDC5 upregulation exacerbates LIRI by impairing HSP90/eNOS-dependent endothelial function. ER stress-TXNDC5 signaling is a mechanistically distinct and potential target in LIRI.