The reticulon family has been found to induce apoptosis, inhibit axon regeneration and regulate protein trafficking. However, little is known about the mechanisms of how reticulon proteins are involved in neuronal death-promoting processes during ischemia. Here, we report that the expression of Reticulon Protein 1-C (RTN1-C) was associated with the progression of cerebral ischemia/reperfusion (I/R) injury. Using a combination of rat middle cerebral artery occlusion (MCAO) stroke and oxygen-glucose deprivation followed by reoxygenation (OGD/R) models, we determined that the expression of RTN1-C was significantly increased during cerebral ischemic/reperfusion. RTN1-C overexpression induced apoptosis and increased the cell vulnerability to ischemic injury, whereas RTN1-C knockdown reversed ischemia-induced apoptosis and attenuated the vulnerability of OGD/R-treated neural cells. Mechanistically, we demonstrated that RTN1-C mediated OGD/R-induced apoptosis through ER stress and mitochondria-associated pathways. RTN1-C interacted with Bcl-xL and increased its localization in the ER, thus reducing the anti-apoptotic activity of Bcl-xL. Most importantly, knockdown of Rtn1-c expression in vivo attenuated apoptosis in MCAO rats and reduced the extent of I/R-induced brain injury, as assessed by infarct volume and neurological score. Collectively, these data support for the first time that RTN1-C may represent a novel candidate for therapies against cerebral ischemia/reperfusion injury.

BackgroundThe neurotropic parasite T. gondii is widespread among mammalian hosts including humans. During the course of T. gondii infection, the central nervous system is the most commonly damaged of all invasive organs. The polymorphic rhoptry protein ROP18 has been identified as a key factor in the pathogenesis of T. gondii; however, the molecular mechanism by which this protein exerts neuropathogenesis remains elusive.MethodsImmunofluorescence staining was performed to detect neuropathogenesis of the mouse brain tissues. The apoptosis of neural cells and the expressions of related proteins in the endoplasmic reticulum stress (ER Stress)-mediated apoptosis pathway were detected by flow cytometry and Western blotting.ResultsImmunofluorescence staining reveals induction of the propidium iodide (PI) - positive neural cells in mouse cerebral cortex and hippocampus infected with ROP18 over-expressing transgenic tachyzoites. Western blotting analyses reveal that ROP18 increases the expressions of cleaved caspase-12, CHOP and cleaved caspase-3 when compared to the control groups. After the pretreatment of Z-ATAD-FMK (a specific caspase-12 inhibitor), the apoptotic level of neural cells had an apparent decline, and correspondingly, the expressions of those related proteins were notably decreased.ConclusionsOur findings here highlight that the virulence factor ROP18 in T. gondii may contribute to neuronal apoptosis through the ER stress-mediated apoptosis pathway, which may be a potential molecular mechanism responsible for neurological disorders of toxoplasmosis.

Traumatic brain injury (TBI) is a common cause of death and disability. Enhancing the midline-crossing of the contralateral corticospinal tract (CST) to the denervated side of spinal cord facilitates functional recovery after TBI. Activation of the gamma isoform of PKC (PKCγ) in contralateral CST implicates its roles in promoting CST remodeling after TBI. In this study, we deployed loss and gain of function strategies in N2a cells and primary cortical neurons in vitro, and demonstrated that PKCγ is not only important but necessary for neuronal differentiation, neurite outgrowth and axonal branching but not for axonal extension. Mechanically, through the phosphorylation of GSK3β, PKCγ stabilizes the expression of cytosolic β-catenin and increase GAP43 expression, thus promoting axonal outgrowth. Further, rAAV2/9-mediated delivery of constitutive PKCγ in the corticospinal tract after unilateral TBI in vivo additionally showed that specifically delivery of active PKCγ mutant to cortical neuron promotes midline crossing of corticospinal fibers from the uninjured side to the denervated cervical spinal cord. This PKCγ-mediated injury response promoted sensorimotor functional recovery. In conclusion, PKCγ mediates stability of β-catenin through the phosphorylation of GSK3β to facilitate neuronal differentiation, neurite outgrowth and axonal branching, and PKCγ maybe a novel therapeutic target for physiological and functional recovery after TBI.

Temozolomide (TMZ) resistance is a major clinical challenge for glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) mediated DNA damage repair is a key mechanism for TMZ resistance. However, MGMT-null GBM patients remain resistant to TMZ, and the process for resistance evolution is largely unknown. Here, we developed an acquired TMZ resistant xenograft model using serial implantation of MGMT-hypermethylated U87 cells, allowing the extraction of stable, TMZ resistant (TMZ-R) tumors and primary cells. The derived tumors and cells exhibited stable multidrug resistance both in vitro and in vivo. Functional experiments, as well as single-cell RNA sequencing (scRNA-seq), indicated that TMZ treatment induced cellular heterogeneity including quiescent cancer stem cells (CSCs) in TMZ-R tumors. A subset of these were labeled by NES+/SOX2+/CADM1+ and demonstrated significant advantages for drug resistance. Further study revealed that Epidermal Growth Factor Receptor (EGFR) deficiency and diminished downstream signaling may confer this triple positive CSCs subgroup’s quiescent phenotypes and chemoresistance. Continuous EGF treatment improved the chemosensitivity of TMZ-R cells both in vitro and in vivo, mechanically reversing cell cycle arrest and reduced drug uptake. Further, EGF treatment of TMZ-R tumors favorably normalized the response to TMZ in combination therapy. Here, we characterize a unique subgroup of CSCs in MGMT-null experimental glioblastoma, identifying EGF + TMZ therapy as a potential strategy to overcome cellular quiescence and TMZ resistance, likely endowed by deficient EGFR signaling.

Sustained activation of signal transducer and activator of transcription 3 (STAT3) is a critical contributor in tumorigenesis and chemoresistance, thus making it an attractive cancer therapeutic target. Here, SH2 domain‐containing adapter protein F (SHF) is identified as a tumor suppressor in glioblastoma Multiforme (GBM) and its negative regulation of STAT3 activity is characterized. Mechanically, SHF selectively binds and inhibits acetylated STAT3 dimerization without affecting STAT3 phosphorylation or acetylation. Additionally, by blocking STAT3‐DNMT1 (DNA Methyltransferase 1) interaction, SHF relieves methylation of tumor suppressor genes. The SH2 domain is documented to be essential for SHF's actions on STAT3, and almost entirely replaces the functions of SHF on STAT3 independently. Moreover, the peptide C16 a peptide derived from the STAT3‐binding sites of SHF inhibits STAT3 dimerization and STAT3/DNMT1 interaction, and achieves remarkable growth inhibition in GBM cells in vitro and in vivo. These findings strongly identify targeting of the SHF/STAT3 interaction as a promising strategy for developing an optimal STAT3 inhibitor and provide early evidence of the potential clinical efficacy of STAT3 inhibitors such as C16 in GBM.

Previous studies have demonstrated that glucocorticoid receptor β (GRβ) functions as an oncoprotein, regulating the malignant phenotypes and stem-like cells maintaining in human glioblastoma (GBM). Of the GR isoforms, GRβ and GRα are highly homologous, though the mechanism underlying the distinct functions of these two isoforms in GBM has not been clarified.Here by establishing a C-terminal deletion mutant, we determined that GRβ can be ubiquitinated.We also found that its C-terminal is essential for this ubiquitination. The mutation of a lysine to arginine at residue 733 (K733R) blocked the ubiquitination of GRβ, indicating that K733 is a key site for ubiquitination. Using K733R to establish non-ubiquitinated GRβ, we demonstrated that ubiquitination not only regulates the stability and nuclear translocation of GRβ, but is also a vital mechanism for its oncogenic functions in vitro and in vivo. Protein interaction assay further indicated that ubiquitin-specific protease 49 (USP49) is a GRβ-binding protein and the interaction depends on GRβ ubiquitination. USP49 knockdown resulted in a decrease of cell proliferation, invasion, and an increase of cell apoptosis. More importantly, USP49 knockdown increased ubiquitination and amplified the oncogenic effects of GRβ, confirming the decisive role of ubiquitination on GRβ carcinogenicity. Taken together, these findings established that ubiquitination is a vial process for GRβ the execution of oncogenic functions in GBM and that the K733 site is crucial for ubiquitination of GRβ.Implications: This work is the first identify of the activation GRβ by a single lysine point-mediated ubiquitination and proteasome degradation, which determines its oncogenic functions in GBM.

Background NF‐κB signaling is widely linked to the pathogenesis and treatment resistance in cancers. Increasing attention has been paid to its anti‐oncogenic roles, due to its key functions in cellular senescence and the senescence‐associated secretory phenotype (SASP). Therefore, thoroughly understanding the function and regulation of NF‐κB in cancers is necessary prior to the application of NF‐κB inhibitors. Methods We established glioblastoma (GBM) cell lines expressing ectopic TCF4N, an isoform of the β‐catenin interacting transcription factor TCF7L2, and evaluated its functions in GBM tumorigenesis and chemotherapy in vitro and in vivo. In p65 knock‐out or phosphorylation mimic (S536D) cell lines, the dual role and correlation of TCF4N and NF‐κB signaling in promoting tumorigenesis and chemosensitivity was investigated by in vitro and in vivo functional experiments. RNA‐seq and computational analysis, immunoprecipitation and ubiquitination assay, minigene splicing assay and luciferase reporter assay were performed to identify the underlying mechanism of positive feedback regulation loop between TCF4N and the p65 subunit of NF‐κB. A eukaryotic cell‐penetrating peptide targeting TCF4N, 4N, was used to confirm the therapeutic significance. Results Our results indicated that p65 subunit phosphorylation at Ser 536 (S536) and nuclear accumulation was a promising prognostic marker for GBM, and endowed the dual functions of NF‐κB in promoting tumorigenesis and chemosensitivity. p65 S536 phosphorylation and nuclear stability in GBM was regulated by TCF4N. TCF4N bound p65, induced p65 phosphorylation and nuclear translocation, inhibited its ubiquitination/degradation, and subsequently promoted NF‐κB activity. p65 S536 phosphorylation was essential for TCF4N‐led senescence‐independent SASP, GBM tumorigenesis, tumor stem‐like cell differentiation and chemosensitivity. Activation of p65 was closely connected to alterative splicing of TCF4N, a likely positive feedback regulation loop between TCF4N and p65 in GBM. 4N increased chemosensitivity, highlighting a novel anti‐cancer strategy. Conclusion Our study defined key roles of TCF4N as a novel regulator of NF‐κB through mutual regulation with p65 and provided a new avenue for GBM inhibition.

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