KA-mediated excitotoxicity induces neuronal ferroptosis through activation of ferritinophagy.

OBJECTIVES: This study aims to elucidate the role of Fe2+ overload in kainic acid (KA)-induced excitotoxicity, investigate the involvement of ferritinophagy selective cargo receptor NCOA4 in the pathogenesis of excitotoxicity. METHODS: Western blotting was used to detect the expression of FTH1, NCOA4, Lamp2, TfR, FPN, and DMT1 after KA stereotaxic injection into the unilateral striatum of mice. Colocalization of Fe2+ with lysosomes in KA-treated primary cortical neurons was observed by using confocal microscopy. Desferrioxamine (DFO) was added to chelate free iron, a CCK8 kit was used to measure cell viability, and the Fe2+ levels were detected by FerroOrange. BODIPY C11 was used to determine intracellular lipid reactive oxygen species (ROS) levels, and the mRNA levels of PTGS2, a biomarker of ferroptosis, were measured by fluorescent quantitative PCR. 3-Methyladenine (3-MA) was employed to inhibit KA-induced activation of autophagy, and changes in ferritinophagy-related protein expression and the indicated biomarkers of ferroptosis were detected. Endogenous NCOA4 was knocked down by lentivirus transfection, and cell viability and intracellular Fe2+ levels were observed after KA treatment. RESULTS: Western blot results showed that the expression of NCOA4, DMT1, and Lamp2 was significantly upregulated, while FTH1 was downregulated, but there were no significant changes in TfR and FPN. The fluorescence results indicated that KA enhanced the colocalization of free Fe2+ with lysosomes in neurons. DFO intervention could effectively rescue cell damage, reduce intracellular lipid peroxidation, and decrease the increased transcript levels of PTGS2 caused by KA. Pretreatment with 3-MA effectively reversed KA-induced ferritinophagy and ferroptosis. Endogenous interference with NCOA4 significantly improved cell viability and reduced intracellular free Fe2+ levels in KA-treated cells. CONCLUSION: KA-induced excitotoxicity activates ferritinophagy, and targeting ferritinophagy effectively inhibits downstream ferroptosis. Interference with NCOA4 effectively attenuates KA-induced neuronal damage. This study provides a potential therapeutic target for excitotoxicity related disease conditions.

Regulation of FSP1 myristoylation by NADPH: A novel mechanism for ferroptosis inhibition.

Excitotoxicity is a prevalent pathological event in neurodegenerative diseases. The involvement of ferroptosis in the pathogenesis of excitotoxicity remains elusive. Transcriptome analysis has revealed that cytoplasmic reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels are associated with susceptibility to ferroptosis-inducing compounds. Here we show that exogenous NADPH, besides being reductant, interacts with N-myristoyltransferase 2 (NMT2) and upregulates the N-myristoylated ferroptosis suppressor protein 1 (FSP1). NADPH increases membrane-localized FSP1 and strengthens resistance to ferroptosis. Arg-291 of NMT2 is critical for the NADPH-NMT2-FSP1 axis-mediated suppression of ferroptosis. This study suggests that NMT2 plays a pivotal role by bridging NADPH levels and neuronal susceptibility to ferroptosis. We propose a mechanism by which the NADPH regulates N-myristoylation, which has important implications for ferroptosis and disease treatment.

Paraganglioma of the Urinary Bladder in a Dog.

A 3-year-old male Bichon Frise developed lethargy, anorexia and haematuria. B-scan ultrasonography examination revealed a small, irregular, soft-textured mass in the bladder. Histopathologically, there was an incomplete fibrous pseudocapsule around the tumour tissue and although there was clear demarcation from the surrounding tissue, there was invasion of the capsule. Tumour cells proliferated in nests or cords of variable size, separated by fibrovascular tissue. The neoplastic cells were immunopositive for chromogranin A, synaptophysin and neuron-specific enolase, and electron microscopy revealed that they contained cytoplasmic secretory granules. On the basis of these findings, the tumour was diagnosed as a primary paraganglioma of the urinary bladder.

Regulation of AR mRNA translation in response to acute AR pathway inhibition.

We report a new mechanism of androgen receptor (AR) mRNA regulation and cytoprotection in response to AR pathway inhibition (ARPI) stress in prostate cancer (PCA). AR mRNA translation is coordinately regulated by RNA binding proteins, YTHDF3 and G3BP1. Under ambient conditions m6A-modified AR mRNA is bound by YTHDF3 and translationally stimulated, while m6A-unmodified AR mRNA is bound by G3BP1 and translationally repressed. When AR-regulated PCA cell lines are subjected to ARPI stress, m6A-modified AR mRNA is recruited from actively translating polysomes (PSs) to RNA-protein stress granules (SGs), leading to reduced AR mRNA translation. After ARPI stress, m6A-modified AR mRNA liquid-liquid phase separated with YTHDF3, while m6A-unmodified AR mRNA phase separated with G3BP1. Accordingly, these AR mRNA messages form two distinct YTHDF3-enriched or G3BP1-enriched clusters in SGs. ARPI-induced SG formation is cell-protective, which when blocked by YTHDF3 or G3BP1 silencing increases PCA cell death in response to ARPI stress. Interestingly, AR mRNA silencing also delays ARPI stress-induced SG formation, highlighting its supportive role in triggering this stress response. Our results define a new mechanism for stress adaptive cell survival after ARPI stress involving SG-regulated translation of AR mRNA, mediated by m6A RNA modification and their respective regulatory proteins.

G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress.

Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.

TWIST1 upregulates miR-214 to promote epithelial-to-mesenchymal transition and metastasis in lung adenocarcinoma.

Epithelial-to-mesenchymal transition (EMT) is essential for the progression of non-invasive tumor cells into malignancy and metastasis. We found that miR-214 was increased in lung adenocarcinoma (LAD) and positively associated with metastasis, which was mediated by EMT. However, the mechanism whereby the overexpression of microRNAs (miRNAs), such as miR-214, promote EMT in LAD remains unclear. In this study, we found that TWIST1, an independent prognostic factor for overall survival, was increased in LAD and correlated positively with LAD recurrence and progression. We also found that TWIST1 contributes to the EMT process and metastasis of LAD cells. Most importantly, a positive correlation was found between the expression of miR-214 and TWIST1 in clinical LAD tissue. Additionally, miR-214 expression was decreased and its target gene suppressor of fused homolog (SUFU) was increased in LAD cells in response to the impairment of TWIST1 expression by shRNA. Overall, this study provides the first evidence to show that the high expression of TWIST1 increases the expression of miR-214 to promote the EMT process and metastasis in LAD. These findings contribute to clarify the mechanisms whereby miRNAs regulate the EMT process and implicate a new TWIST1-miR-214 pathway in the control of migration and invasion of LAD.

[The effect of vascular endothelial cells on the migration of periodontal ligament cells and gingival fibroblasts].

PURPOSE: To investigate the effect of human vascular endothelial cells (HUVEC) on the migration of human periodontal ligament cells (HPDLC) and human gingival fibroblasts (HGF) through a model mimicking periodontal wound healing or regeneration. METHODS: HUVEC, HPDLC and HGF were co-cultured using a Transwell system, while HPDLC or HGF was cultured independently as control. Chemotaxis assay was performed using cell culture inserts (0.8µm pore size) to assess the effect of HUVEC on HPDLC and HGF vertical migration at 24h. A scratch wound assay was performed to assess the effect of HUVEC on HPDLC and HGF horizontal migration at 0 h, 8 h, 16 h and 24 h. A round glass sheet assay was performed to assess the effect of HUVEC on HPDLC and HGF wound healing ability at 1d, 4d and 7d. The data was processed with SPSS 13.0 software package. RESULTS: At the round glass sheet assay, while HUVEC was present, the integrated option density (IOD) of HPDLCs and HGFs were significantly higher than each of the single culture (P<0.01). Results of chemotaxis assay and scratch wound assay showed that the cell number of HPDLCs and HGFs were significantly higher than each of the single culture while HUVEC was present (P<0.01), and the number of PHDLCs was significantly higher than HGFs on chemotaxis assay (P<0.01) but the result of scratch wound assay was on the contrary (P<0.01). CONCLUSIONS: The presence of HUVEC can promote migration of periodontal cells, and the effect is more prominent on HPDLC than on HGF in vertical migration and wound healing, while horizontal migration is more prominent on HGFs as contrast.

[The reliability of the salivary occult blood to test gingival inflammation].

PURPOSE: To evaluate the reliability of salivary occult blood test(SOBT) for gingival inflammation. METHODS: Forty-five pre- and post-stimulation saliva samples were tested for occult blood, with bleeding on probing (BOP) as an indicator of gingival inflammation. Subjects with BOP%<15% were defined as control group. The examinations were repeated 3 months after periodontal initial therapy. The relationship between the results of SOBT and gingival inflammation was analyzed. SAS6.12 software package was used for statistical analysis. RESULTS: The SOBT of pre-stimulation was higher in gingivitis group than in control group (P<0.05). BOP% and SOBT of post-therapy was significantly decreased (P<0.01), with 90% of pre- and 63% of post-stimulation SOBT turning negative. The sensitivity and specificity of pre- stimulation SOBT in screening gingivitis were 32.35% and 100%. The sensitivity and specificity of the post-stimulation SOBT were 88.24% and 81.82%. CONCLUSIONS: SOBT is correlated with gingival inflammation, which may be used as an objective parameter to evaluate gingival inflammation and the efficacy of periodontal therapy at individual level. It may offer a simple screening method for gingival inflammation applied in periodontal epidemiologic studies in large population.