Fetuin-A alleviates neuroinflammation against traumatic brain injury-induced microglial necroptosis by regulating Nrf-2/HO-1 pathway.

BACKGROUND: The microglia-mediated inflammatory response is a vital mechanism of secondary damage following traumatic brain injury (TBI), but the underlying mechanism of microglial activation is unclear. METHODS: Controlled cortical impact (CCI) was induced in adult male C57BL/6J mice, and glutamate was used to construct a classical in vitro injury model in the primary microglia. Microglial activation was determined by western blot and immunostaining. The inflammatory factors were measured by enzyme-linked immunosorbent assay. The oxidative stress marker and mitochondrial reactive oxygen species (ROS) were measured by immunoblotting and MitoSox Red staining. Transmission electron microscopy was used to observe the typical morphology of necroptotic cells. RESULTS: Our quantitative proteomics identified 2499 proteins; 157 were significantly differentially expressed in brain tissue between the 6 h after CCI (CCI6h) group and sham group, and 109 were significantly differentially expressed between the CCI24h and sham groups. Moreover, compared with the sham group, the terms "acute-phase response", "inflammation", and "protein binding" were significantly enriched in CCI groups. Fetuin-A, a liver-secreted acute-phase glycoprotein, was involved in these biological processes. Using an experimental TBI model, we found that the Fetuin-A level peaked at 6 h and then decreased gradually. Importantly, we showed that administration of Fetuin-A reduced the cortical lesion volume and edema area and inhibited the inflammatory response, which was associated with suppressing microglial necroptosis, thus decreasing microglial activation. Furthermore, administration of Fetuin-A attenuated mitochondrial oxidative stress in glutamate-treated microglial cells, which is a critical mechanism of necroptosis suppression. In addition, we demonstrated that Fetuin-A treatment promoted translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm to the nucleus in vivo; however, the Nrf-2 inhibitor ML385 and si-heme oxygenase-1 (si-HO-1) disrupted the regulation of oxidative stress by Fetuin-A and induced increased ROS levels and necroptosis in glutamate-treated microglial cells. Fetuin-A also protected neurons from adverse factors in vivo and in vitro. CONCLUSIONS: Our results demonstrated that Fetuin-A activated Nrf-2/HO-1, suppressed oxidative stress and necroptosis levels, and thereby attenuates the abnormal inflammatory response following TBI. The findings suggest a potential therapeutic strategy for TBI treatment.

Hsp90 induces Acsl4-dependent glioma ferroptosis via dephosphorylating Ser637 at Drp1.

Ferroptosis is a newly identified form of regulated cell death (RCD) characterized by the iron-dependent lipid reactive oxygen species (ROS) accumulation, but its mechanism in gliomas remains elusive. Acyl-coenzyme A (CoA) synthetase long-chain family member 4 (Acsl4), a pivotal enzyme in the regulation of lipid biosynthesis, benefits the initiation of ferroptosis, but its role in gliomas needs further clarification. Erastin, a classic inducer of ferroptosis, has recently been found to regulate lipid peroxidation by regulating Acsl4 other than glutathione peroxidase 4 (GPX4) in ferroptosis. In this study, we demonstrated that heat shock protein 90 (Hsp90) and dynamin-related protein 1 (Drp1) actively regulated and stabilized Acsl4 expression in erastin-induced ferroptosis in gliomas. Hsp90 overexpression and calcineurin (CN)-mediated Drp1 dephosphorylation at serine 637 (Ser637) promoted ferroptosis by altering mitochondrial morphology and increasing Acsl4-mediated lipid peroxidation. Importantly, promotion of the Hsp90-Acsl4 pathway augmented anticancer activity of erastin in vitro and in vivo. Our discovery reveals a novel and efficient approach to ferroptosis-mediated glioma therapy.

Loss of deubiquitylase USP2 triggers development of glioblastoma via TGF-ß signaling.

Glioblastoma (GBM) is the most aggressive primary brain tumor as one of the deadliest cancers. The TGF-ß signaling acts as an oncogenic factor in GBM, and plays vital roles in development of GBM. SMAD7 is a major inhibitor of TGF-ß signaling, while the deubiquitination of SMAD7 has been poorly studied in GBM. Here, we found USP2 as a new prominent candidate that could regulate SMAD7 stability. USP2 was lost in GBM, leading to the poor prognosis in patients. Moreover, aberrant DNA methylation mediated by DNMT3A induced the low expression of USP2 in GBM. USP2 depletion induced TGF-ß signaling and progression of GBM. In contrast, overexpressed USP2 suppressed TGF-ß signaling and GBM development. Specifically, USP2 interacted with SMAD7 and prevented SMAD7 ubiquitination. USP2 directly cleaved Lys27- and Lys48-linked poly-ubiquitin chains of SMAD7, and Lys27-linked poly-ubiquitin chains of SMAD7 K185 mediated the recruitment of SMAD7 to HERC3, which regulated Lys63-linked poly-ubiquitination of SMAD7. Moreover, we demonstrated that the DNMT3A inhibitor SGI-1027 induced USP2, suppressed TGF-ß signaling and GBM development. Thus, USP2 repressed development of GBM by inhibition TGF-ß signaling pathway via the deubiquitination of SMAD7.

Nuclear receptor coactivator 4-mediated ferritinophagy contributes to cerebral ischemia-induced ferroptosis in ischemic stroke.

Ischemic stroke poses a significant health risk due to its high rate of disability and mortality. To address this problem, several therapeutic approaches have been proposed, including interruption targeting programmed cell death (PCD). Ferroptosis is a newly defined PCD characterized by iron-dependent accumulation of lipid peroxidation, and is becoming a promising target for treating numerous diseases. To explore the underlying mechanisms of the initiation and execution of ferroptosis in ischemic stroke, we established stroke models in vivo and in vitro simulating ischemia/reperfusion (I/R) neuronal injury. Different from previous reports on stroke, we tested ferroptosis by measuring the levels of core proteins, such as ACSL4, 15-LOX2, Ferritin and GPX4. In addition, I/R injury induces excessive degradation of ferritin via the autophagy pathway and subsequent increase of free iron in neurons. This phenomenon has recently been termed ferritinophagy and reported to be regulated by nuclear receptor coactivator 4 (NCOA4) in some cell lines. Increased NCOA4 in cytoplasm was detected in our study and then silenced by shRNA to investigate its function. Both in vivo and in vitro, NCOA4 deletion notably abrogated ferritinophagy caused by I/R injury and thus inhibited ferroptosis. Furthermore, we found that NCOA4 was upregulated by ubiquitin specific peptidase 14 (USP14) via a deubiquitination process in damaged neurons, and we found evidence of pharmacological inhibition of USP14 effectively reducing NCOA4 levels to protect neurons from ferritinophagy-mediated ferroptosis. These findings suggest a novel and effective target for treating ischemic stroke.

Prokineticin-2 prevents neuronal cell deaths in a model of traumatic brain injury.

Prokineticin-2 (Prok2) is an important secreted protein likely involved in the pathogenesis of several acute and chronic neurological diseases through currently unidentified regulatory mechanisms. The initial mechanical injury of neurons by traumatic brain injury triggers multiple secondary responses including various cell death programs. One of these is ferroptosis, which is associated with dysregulation of iron and thiols and culminates in fatal lipid peroxidation. Here, we explore the regulatory role of Prok2 in neuronal ferroptosis in vitro and in vivo. We show that Prok2 prevents neuronal cell death by suppressing the biosynthesis of lipid peroxidation substrates, arachidonic acid-phospholipids, via accelerated F-box only protein 10 (Fbxo10)-driven ubiquitination, degradation of long-chain-fatty-acid-CoA ligase 4 (Acsl4), and inhibition of lipid peroxidation. Mice injected with adeno-associated virus-Prok2 before controlled cortical impact injury show reduced neuronal degeneration and improved motor and cognitive functions, which could be inhibited by Fbxo10 knockdown. Our study shows that Prok2 mediates neuronal cell deaths in traumatic brain injury via ferroptosis.

Curcumin alleviates neuroinflammation, enhances hippocampal neurogenesis, and improves spatial memory after traumatic brain injury.

Cognitive decline is one of the most obvious symptoms of traumatic brain injury (TBI). Previous studies have demonstrated that cognitive decline is related to substantially increased neuroinflammation and decreased neurogenesis in the hippocampus in a rat model of TBI. Using this model, we explored the role of curcumin (Cur) in ameliorating TBI-impaired spatial memory because Cur has been shown to exhibit anti-chronic-neuroinflammatory, neurogenesis-promoting, and memory-improving properties. Animals received daily Cur or vehicle treatment for 28 days after TBI and also received 50-bromodeoxyuridine(BrdU) for the first 7 days of the treatment for assaying neurogenesis. An optimal Cur dose of 30 mg/kg, selected from a range of 10-50 mg/kg, was used for the present study. Neuroinflammation was evaluated by astrocyte hypertrophy, activated microglia, and inflammatory factors in the hippocampus. Behavioral water-maze studies were conducted for 5 days, starting at 35-day post-TBI. The tropomyosin receptor kinase B (Trkb) inhibitor, ANA-12, was used to test the role of the brain-derived neurotrophic factor (BDNF)/ TrkB/Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in regulating inflammation and neurogenesis in the hippocampus. Treatment with Cur ameliorated the spatial memory of TBI rats, reduced TBI-induced chronic inflammation, typified by diminished astrocyte hypertrophy, reduction in activated microglia, declined inflammatory factors, and increased neurogenesis in the hippocampus. We also found that BDNF/Trkb/PI3K/Akt signaling was involved in the effects of Cur in TBI rats. Thus, Cur treatment can ameliorate the spatial memory in a murine model of TBI, which may be attributable to decreased chronic neuroinflammation, increased hippocampal neurogenesis, and/or BDNF/Trkb/PI3K/Akt signaling.

Up-regulation of CHMP4B alleviates microglial necroptosis induced by traumatic brain injury.

Microglial cells are key component of central nervous system (CNS) and mediate the immune response of the brain under physiological or pathological conditions. It tends to activate into a pro-inflammatory M1 phenotype after traumatic brain injury (TBI) and promote secondary brain damage. Recently, necroptosis was found to promote microglial activation and neuroinflammation after TBI. However, the mechanism and specific interventions of microglial necroptosis after TBI remain poorly investigated. Here, we reported that overexpress the charged multivesicular body protein 4b (CHMP4B) which is a core member of the endosomal sorting required for transport complex III (ESCRT-III) significantly decreased the level of necroptosis in microglia, improved neurological function recovery and protected against cell death after TBI. Further investigation showed that forkhead transcription factor O1 (FOXO1) was a crucial transcription factor that increased CHMP4B transcription by binding to the promoter region, thereby inhibiting necroptosis in microglia. Collectively, our findings demonstrated that CHMP4B relieved microglial necroptosis and neuroinflammation after TBI, and promote the recovery of nerve function. FOXO1 is an important factor in promoting CHMP4B expression. This study provides the novel viewpoint for TBI prevention and treatment.

Smoothened Promotes Glioblastoma Radiation Resistance Via Activating USP3-Mediated Claspin Deubiquitination.

PURPOSE: Glioblastoma (GBM) is one of the most aggressive and lethal cancer types in humans. The standard treatment approach is surgery followed by chemoradiation. However, the molecular mechanisms of innate tumor radioresistance remain poorly understood. EXPERIMENTAL DESIGN: We tested the expression of Smoothened (Smo) in primary and recurrent GBM tissues and cells. Then, we determined radiation effectiveness against primary and recurrent GBM cells. Lastly, the functional role of Smo in GBM radioresistance was further confirmed by in vitro and in vivo experiments. RESULTS: We reported that Smo was significantly upregulated in recurrent GBM cell lines and tumor tissues following radiation treatment. Higher Smo expression indicated poor prognosis of GBM patients after radiation treatment. Smo had radioresistance effects in both GBM cells and human tumor xenografts. The mechanisms underlying these effects involved the attenuation of DNA damage repair caused by IR. Importantly, we found that the effect of Smo on radioresistance was mediated by Claspin polyubiquitination and proteasomal degradation, leading to the regulation of ATR-Chk1 signaling. Moreover, we found that Smo reduced Claspin polyubiquitination and proteasomal degradation by promoting USP3 transcription. Furthermore, we demonstrated that the Smo inhibitor GDC-0449 induced radiosensitivity to GBM. CONCLUSIONS: These data suggest that Smo confers radiation resistance in GBM by promoting USP3 transcription, leading to the activation of Claspin-dependent ATR-Chk1 signaling. These findings identify a potential mechanism of GBM resistance to radiation and suggest a potential therapeutic target for radiation resistance in GBM.

S100A11 functions as novel oncogene in glioblastoma via S100A11/ANXA2/NF-κB positive feedback loop.

Glioblastoma (GBM) is the most universal type of primary brain malignant tumour, and the prognosis of patients with GBM is poor. S100A11 plays an essential role in tumour. However, the role and molecular mechanism of S100A11 in GBM are not clear. Here, we found that S100A11 was up-regulated in GBM tissues and higher S100A11 expression indicated poor prognosis of GBM patients. Overexpression of S100A11 promoted GBM cell growth, epithelial-mesenchymal transition (EMT), migration, invasion and generation of glioma stem cells (GSCs), whereas its knockdown inhibited these activities. More importantly, S100A11 interacted with ANXA2 and regulated NF-κB signalling pathway through decreasing ubiquitination and degradation of ANXA2. Additionally, NF-κB regulated S100A11 at transcriptional level as a positive feedback. We also demonstrated the S100A11 on tumour growth in GBM using an orthotopic tumour xenografting. These data demonstrate that S100A11/ANXA2/NF-κB positive feedback loop in GBM cells that promote the progression of GBM.