Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation.

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

Size-Dependent Effects of Suspended Graphene Oxide Nanoparticles on the Cellular Fate of Mouse Neural Stem Cells.

PURPOSE: In this study, we aim to explore the effects of graphene oxide (GO), a derivative of graphene, nanoparticles of four different sizes on the cellular fate of mouse neural stem cells (mNSCs). METHODS: GO NPs were characterized with transmission electron microscopy (TEM), scanning electron micrography (SEM), atomic force microscopy (AFM) and Raman Spectra analysis. The cytotoxic effects of the GO NPs of different sizes on the mNSCs were determined using CCK-8 assay, Annexin V-APC/ 7-AAD staining and EdU staining assays. We investigated the biological and the mechanisms of GO NPs on cells using immunofluorescence analysis and quantitative real-time PCR (qPCR). RESULTS: The average hydrodynamic sizes of the GO NPs were 417 nm, 663 nm, 1047 nm, and 4651 nm, with a thickness of approximately 22.5 nm, 17.7 nm, 22.4 nm, and 13.4 nm, respectively. GO NPs of all sizes showed low cytotoxicity at a concentration of 20 µg/mL on the mNSCs. Immunostaining demonstrated that treatment with GO NPs, especially the 663 nm ones, enhanced the self-renewal ability of mNSCs in the absence of EGF and bFGF. Under differentiation medium conditions that are free of mitogenic factors, all the GO NPs, particularly the 4651 nm ones, increased the expression level of Tuj1 and GFAP. With regards to the migration ability, we found that 417 nm GO-NP-treated mNSCs migrated over a longer distance than the control group obviously. In addition, higher expression of Rap1, Vinculin and Paxillin was observed in the GO NP-treated groups compared to the control group. mRNA-Sequence analysis and Western blotting results suggested that the 4651 nm GO NPs triggered positive neuronal differentiation through phosphorylation of ERK1/2 by the downregulating of TRPC2. CONCLUSION: GO NPs play an important role in the applications of inducing self-renewal and differentiation of mNSC, and are promising in the future for further studies.

Suspended graphene oxide nanosheets maintain the self-renewal of mouse embryonic stem cells via down-regulating the expression of Vinculin.

Graphene oxide (GO), with good hydrophilicity and biocompatibility, is widely explored as a carrier for various factors in the field of stem cell differentiation. However, its function of sustaining the stemness of mouse embryonic stem cells (mESCs) and the underlying mechanisms of this process remains undiscovered. Herein, we explored the biofunction of GO on mESCs and revealed the involved signaling pathways and key gene. The alkaline phosphatase activity detection, pluripotency genes quantification and the teratomas formation in vivo confirmed that GO nanosheets could sustain the self-renewal ability of mESCs instead of influencing its pluripotency. The underlying signaling pathways were uncovered by RNA-seq that integrin signaling pathway was involved in the biofunction of GO on mESCs and Vinculin turned to be a key gene for the effect of GO. Further experiments confirmed that the downregulation of Vinculin influenced the fate of mESCs through decreasing the expression of MEK1. Altogether, the study demonstrated for the first time that GOs hold the potential in sustaining the self-renewal of mESCs and clarified the mechanism of this function, which make it play a new role in stem cell research and regenerative medicine.

Efficient VEGF targeting delivery of DOX using Bevacizumab conjugated SiO2@LDH for anti-neuroblastoma therapy.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and is highly expressed in carcinoma, which make it an important target for tumor targeting therapy. Neuroblastoma is the main cause for cancer-related death in children. Like most solid tumors, it is also accompanied with the overexpression of VEGF. Doxorubicin Hydrochloride (DOX), a typical chemotherapeutic agent, exhibits efficient anticancer activities for various cancers. However, DOX, without targeting ability, usually causes severe damage to normal tissues. To overcome the shortages, we designed a novel nano-composite, which is Bevacizumab (Bev) modified SiO2@LDH nanoparticles (SiO2@LDH-Bev), loading with DOX to achieve targeting ability and curative efficiency. SiO2@LDH-DOX and SiO2@LDH-Bev-DOX nanoparticles were synthesized and the physicochemical properties were characterized by TEM detection, Zeta potential analysis, FTIR, Raman and XPS analysis. Then in vitro and in vivo anti-neuroblastoma efficiency, targeting ability and mechanisms of anti-carcinoma and anti-angiogenesis of SiO2@LDH-Bev-DOX were explored. Our results indicated that we obtained the core-shell structure SiO2@LDH-Bev with an average diameter of 253±10nm and the amount of conjugated Bev was 4.59±0.38µg/mg SiO2@LDH-Bev. SiO2@LDH-Bev-DOX could improve the cellular uptake and the targeting effect of DOX to brain and tumor, enhance the anti-neuroblastoma and anti-angiogenesis efficiency both in vitro and in vivo, and alleviate side effects of DOX sharply, especially hepatic injury. In addition, we also demonstrated that angiogenesis inhibitory effect was mediated by DOX and VEGF triggered signal pathways, including PI3K/Akt, Raf/MEK/ERK, and adhesion related pathways. In summary, SiO2@LDH-Bev could be a potential VEGF targeting nanocarrier applied in VEGF positive cancer therapy. STATEMENT OF SIGNIFICANCE: This paper explored that a novel core-shell structure nanomaterial SiO2@LDH and modified SiO2@LDH with Bevacizumab (Bev) to form a new tumor vasculature targeting nanocarrier SiO2@LDH-Bev as vector of DOX, which was not reported before. The results indicated that SiO2@LDH-Bev could improve the VEGF targeting ability, anti-neuroblastoma and anti-angiogenesis efficiency of DOX. At the same time, SiO2@LDH-Bev-DOX could erase the cardiac toxicity and hepatic injury coming from DOX. Tube formation showed SiO2@LDH-Bev-DOX had the strongest effect on inhibiting angiogenesis among all the four formulations. SiO2@LDH-Bev-DOX could downregulate expression of p-VEGFR and inhibit activation of the Raf/MEK/ERK, p38MAPK, PI3K/Akt and FAK signaling pathways to achieve the goal of anti-angiogenesis. This work provides a novel system for the safe and efficient use of Bev and DOX on Neuroblastoma and explores the mechanism of the function of nano carrier in cancer therapy both in vitro and in vivo.

Targeting the Endocannabinoid/CB1 Receptor System For Treating Major Depression Through Antidepressant Activities of Curcumin and Dexanabinol-Loaded Solid Lipid Nanoparticles.

BACKGROUND/AIMS: This study investigated the underlying mechanisms of the antidepressant effects of curcumin and dexanabinol-loaded solid lipid nanoparticles in corticosterone-induced cell and mice depression models. METHODS: Curcumin and dexanabinol-loaded solid lipid nanoparticles (Cur/SLNs-HU-211) were synthesized via an emulsifcation and low-temperature solidification method. Antidepressant activities of nanoparticles in a corticosterone-induced major depression model were investigated by MTT assay, cellular uptake by flow cytometry, behaviour by Forced Swimming Test and rotarod test, neurotransmitters by High Performance Liquid Chromatography, Western blotting, qPCR and immunofluorescence. RESULTS: Treatment with Cur/SLNs-HU-211 induced greater dopamine (DA)/5-hydroxytryptamine (5-HT) release with reduced corticosterone-induced apoptotic cell death in PC12 cells. Additionally, in vivo Cur/SLNs-HU-211 significantly induced recovery from depressive behaviour with increased DA/5-HT levels, CB1 mRNA levels and CB1, p-MEK1 and p-ERK1/2 protein expression levels in the hippocampus and striatum. Cur/SLNs-HU-211 improved CB1 expression and inspired the proliferation of astrocytes in the hippocampus and striatum, exerted neuroprotective effects by preventing corticosterone -induced BDNF/NeuN expression reduction. CONCLUSION: Our study implies that Cur/SLNs-HU-211 may be a useful approach for treatment of major depression.