Overexpression of p62/SQSTM1 promotes the degradations of abnormally accumulated PrP mutants in cytoplasm and relieves the associated cytotoxicities via autophagy-lysosome-dependent way.

The protein of p62/sequestosome 1 (SQSTM1), a key cargo adaptor protein involved in autophagy-lysosome degradation, exhibits inclusion bodies structure in cytoplasm and plays a protective role in some models of neurodegenerative diseases. Some PrP mutants, such as PrP-CYTO and PrP-PG14, also form cytosolic inclusion bodies and trigger neuronal apoptosis either in cultured cells or in transgenic mice. Here, we demonstrated that the cellular p62/SQSTM1 incorporated into the inclusion bodies formed by expressing the abnormal PrP mutants, PrP-CYTO and PrP-PG14, in human embryonic kidney 293 cells. Overexpression of p62/SQSTM1 efficiently relieved the cytosolic aggregations and cell apoptosis induced by the abnormal PrPs. Autophagy-lysosome inhibitors instead of proteasome inhibitor sufficiently blocked the p62/SQSTM1-mediated degradations of abnormal PrPs. Overexpression of p62/SQSTM1 did not alter the levels of light chain 3 (LC3) in the cells expressing various PrPs. However, more complexes of p62/SQSTM1 with LC3 were detected in the cells expressing the misfolded PrPs. These data imply that p62/SQSTM1 plays an important role in the homeostasis of abnormal PrPs via autophagy-lysosome-dependent way.

Silencing lncRNA FOXD2-AS1 inhibits proliferation, migration, invasion and drug resistance of drug-resistant glioma cells and promotes their apoptosis via microRNA-98-5p/CPEB4 axis.

OBJECTIVE: This study was conducted to elucidate the long non-coding RNA FOXD2-AS1 (lncRNA FOXD2-AS1) expression in glioma and its mechanism on the biological features of glioma cells and the drug resistance of temozolomide (TMZ). RESULTS: Highly expressed FOXD2-AS1 was found in glioma. There was more powerful chemotherapeutic resistance of TMZ resistant cell lines than that of the parent cell lines. Silence of FOXD2-AS1 suppressed proliferation and drug resistance and promoted apoptosis of drug-resistant glioma cells. Overexpressed FOXD2-AS1 presented an opposite trend. FOXD2-AS1 could be used as a competing endogenous RNA to adsorb miR-98-5p, thereby up-regulating CPEB4. CONCLUSION: Our study suggests that down-regulated FOXD2-AS1 repressed invasion, proliferation, migration and drug resistance of drug-resistant glioma cells while stimulating their apoptosis via increasing miR-98-5p and inhibiting CPEB4 expression. METHODS: FOXD2-AS1, microRNA-98-5p (miR-98-5p) and cytoplasmic polyadenylation element binding (CPEB4) expression in glioma tissues were tested. Expression of E-cadherin, N-cadherin and Vimentin in glioma cells were explored. A series of assays were conducted to detect the function of FOXD2-AS1 in migration, proliferation, apoptosis, and invasion of glioma cells. Changes in drug-resistance of cells under TMZ treatment were examined, and tumor formation in nude mice was performed to test the changes of drug resistance in vivo.

The Effects of Intelectin-1 on Antioxidant and Angiogenesis in HUVECs Exposed to Oxygen Glucose Deprivation.

Objective: Ischemic stroke leads to cellular death and tissue damage by depriving the areas of glucose and oxygen supplies. The effective treatment of stroke remains a challenge for modern medicine. This study used an oxygen-glucose deprivation (OGD) model of human umbilical vein endothelial cells (HUVECs) to mimic ischemic injuries and explored the role and mechanism of intelectin-1. Methods: Intelectin-1 was transduced into the HUVECs using a lentiviral vector. The PI3K/Akt signaling was examined in intelectin-induced eNOS phosphorylation. The PI3K inhibitor LY294002 was dealed in HUVECs. Results: Our results demonstrated an increase in capillary density, decrease in apoptotic cells, and increase in HIF-1α protein expression following intelectin-1 treatment. Real-time PCR and Western blotting revealed the increased intelectin-1 expression alongside eNOS and Akt phosphorylation with enhanced bcl-2 expression under OGD. Capillary density decreased significantly after LY294002 treatment. Conclusion: These results suggest intelectin-1 promotes angiogenesis, inhibits oxidative stress and reduces apoptosis by stimulating the Akt-eNOS signaling pathway in response to ischemia in vitro.

miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1.

OBJECTIVE: To investigate the role of FOXO1 and miR-183-96-182 clusters in ox-LDL induced endothelial cell apoptosis. METHODS: FOXO1 overexpression (OE) and knockdown (KD) as well as AKT1 OE in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were achieved by lentiviral transduction. Upregulation of miR-183-5p, miR-182-5p or miR-96-5p was mimicked by agomir treatment. FOXO1 gene transcription was monitored by FOXO1 promotor reporter assay. Cell apoptosis in culture was monitored by TiterTACS in situ detection. Regulation of FOXO1 gene expression by an miRNA targeting mechanism was monitored by AGO2-RNA immunoprecipitation assay. RESULTS: FOXO1 mRNA and protein expression levels in ox-LDL treated HUVECs or HAECs were significantly upregulated due to transcriptional and miRNA targeting mechanisms. MiR-183-5p, miR-182-5p and miR-96-5p expression levels in HUVECs or HAECs were significantly reduced by ox-LDL treatment, the overexpression of which by agomir treatment partially reduced the FOXO1 mRNA/protein expression levels and cell apoptosis which was upregulated by ox-LDL treatment. FOXO1 overexpression antagonized the effect of the agomir treatment indicated above. MiR-183-5p, miR-182-5p and miR-96-5p agomir treatment partially rescued the FOXO1 pSer256/total FOXO1 protein ratio and the AKT1 pSer473 level that were reduced by ox-LDL treatment in the HUVECs or HAECs. AKT1 overexpression significantly reduced FOXO1 protein expression, increased miR-182-5p and miR-183-5p expression, and partially alleviated ox-LDL induced HUVEC or HAEC apoptosis in an miR-183-5p and miR-182-5p-dependent manner. CONCLUSION: miR-183-96-182 clusters could partially alleviate ox-LDL-induced apoptosis in HUVECs or HAECs by targeting FOXO1.

FBXW7-Induced MTOR Degradation Forces Autophagy to Counteract Persistent Prion Infection.

Autophagy is an important protein degradation pathway and a part of the innate immune system that is activated in the brain tissue during animal and human prion diseases. However, the possible mechanism by which prion infection triggers autophagy and the significance of activated autophagy on prion accumulation remain unknown. Here, we demonstrated that autophagic flux was enhanced in the persistent prion-infected cell line, SMB-S15. Knockdown of ATG5 and the presence of three autophagic inhibitors resulted in a significant increase of PrP(Sc). The mammalian target of rapamycin (MTOR) levels in SMB-S15 cells were also markedly decreased, in direct relation to PrP(Sc) accumulation. F-box and WD repeat domain containing 7 (FBXW7) levels in SMB-S15 cells and in the brains of scrapie-agent 263K-infected hamsters were upregulated at the early stage of infection, leading to active ubiquitination and degradation of MTOR. Knockdown of FBXW7 in SMB-S15 cells remarkably inhibited autophagic flux and increased PrP(Sc) accumulation. Thus, we conclude that prion infection induced the expression of FBXW7, which mediated MTOR ubiquitination and degradation, further altering phosphorylation status through cross talk between MTORC1 and AMPK and increasing autophagic flux. Autophagy may serve as innate immunity to degrade PrP(Sc) and maintain prion homeostasis.

Significant reduction of the GLUT3 level, but not GLUT1 level, was observed in the brain tissues of several scrapie experimental animals and scrapie-infected cell lines.

Glucose transporters 1 (GLUT1) and 3 (GLUT3) belong to the solute carrier family 2 (SLC2, facilitated glucose transporter) and are the two most important glucose transporters (GLUTs) in brain tissue, and between them, GLUT3 is the primary one for neurons, which is responsible for glucose uptake. To obtain insights into the possible alterations of GLUT1 and GLUT3 in transmissible spongiform encephalopathies (TSEs), the protein levels of GLUT1 and GLUT3 in the brain tissues of agents 263K- and 139A-infected hamsters, as well as agents 139A- and ME7-infected mice, were evaluated. Western blots, immunofluorescent assay (IFA), and immunohistochemical (IHC) assays revealed that at the terminal stages of the infection, GLUT3 level in the brain tissues of scrapie-infected rodents was significantly downregulated, while GLUT1 level remained almost unchanged. The decline of GLUT3 level was closely related with prolonged incubation time. In line with these results in vivo, the GLUT3 level in a prion persistently infected cell line SMB-S15 was also lower than that of its normal cell line SMB-PS. Moreover, the level of hypoxia-inducible factor-1 alpha (HIF-1α), which positively regulated the expressions of GLUTs, was also markedly downregulated in the brains of several scrapie-infected animals. In vitro glucose uptake assays illustrated a markedly decreased 2-[N-(7-nitrobenze-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose uptake activity in SMB-S15 cells. Our data indicate that the reduction of GLUT3 is a common phenomenon in prion diseases, which occurs much earlier than the appearance of clinical symptoms. Defect in glucose uptake and metabolism of neurons, like in other neurodegenerative diseases, for example, Alzheimer's disease (AD), may be one of the essential processes in the pathogenesis of prion diseases.

[Analysis of the expressions of alphaB-crystallin in the brain tissues of agent 263K-infected hamsters at terminal stage].

alphaB-crystallin is a member of the sHSP (Small heat shock protein) family, which plays an impor tant role in multiple neurodegeneration diseases. To give insight into the possible alternation and the role of aB-crystallin in prion disease, the alphaB-crystallin levels in the brain tissues of agent 263K-infected hamsters were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, the levels of alphaB-crystallin were increased up to 3-fold in the brain tissues of scrapie infected 263K hamsters compared with normal controls. Immunofluorescent assays revealed that the up-regulated alphaB-crystallin was mainly observed in astrocytes, but not in neurons. The co-localization between alphaB-crystallin and abnormal deposition of PrPsc in the brain tissues of the scrapie infected hamsters was not observed. The study may provide a foundation for further revealing the potential role of alphaB-crystallin in prion disease.

Abnormally upregulated αB-crystallin was highly coincidental with the astrogliosis in the brains of scrapie-infected hamsters and human patients with prion diseases.

αB-crystallin is a member of the small heat shock protein family constitutively presenting in brains at a relatively low level. To address the alteration of αB-crystallin in prion disease, the αB-crystallin levels in the brains of scrapie agent 263 K-infected hamsters were analyzed. The levels of αB-crystallin were remarkably increased in the brains of 263 K-infected hamsters, showing a time-dependent manner along with incubation time. Immunohistochemical (IHC) and immunofluorescent (IFA) assays illustrated more αB-crystallin-positive signals in the regions of the cortex and thalamus containing severe astrogliosis. Double-stained IFA verified that the αB-crystallin signals colocalized with the enlarged glial fibrillary acidic protein-positive astrocytes, but not with neuronal nuclei-positive cells. IHC and IFA of the serial brain sections of infected hamsters showed no colocalization and correlation between PrP(Sc) deposits and αB-crystallin increase. Moreover, increased αB-crystallin deposits were observed in the brain sections of parietal lobe of a sporadic Creutzfeldt-Jakob disease (sCJD) case, parietal lobe and thalamus of a G114V genetic CJD case, and thalamus of a fatal family insomnia (FFI) case, but not in a parietal lobe of FFI where only very mild astrogliosis was addressed. Additionally, the molecular interaction between αB-crystallin and PrP was only observed in the reactions of recombinant proteins purified from Escherichia coli, but not either in that of brain homogenates or in that of the cultured cell lysates expressing human PrP and αB-crystallin. Our data indicate that brain αB-crystallin is abnormally upregulated in various prion diseases, which is coincidental with astrogliosis. Direct interaction between αB-crystallin and PrP seems not to be essential during the pathogenesis of prion infection.

[Study on the correlation between membrane protein Flotillin-1 and PrPc endocytosis].

OBJECTIVE: To explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc. METHODS: The expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method. RESULTS: (1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time. CONCLUSION: The membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.