NMDA Receptor-Dependent Synaptic Potentiation via APPL1 Signaling Is Required for the Accessibility of a Prefrontal Neuronal Assembly in Retrieving Fear Extinction.

BACKGROUND: The ventromedial prefrontal cortex has been viewed as a locus for storage and recall of extinction memory. However, the synaptic and cellular mechanisms underlying these processes remain elusive. METHODS: We combined transgenic mice, electrophysiological recording, activity-dependent cell labeling, and chemogenetic manipulation to analyze the role of adaptor protein APPL1 in the ventromedial prefrontal cortex in fear extinction retrieval. RESULTS: We found that both constitutive and conditional APPL1 knockout decreased NMDA receptor (NMDAR) function in the ventromedial prefrontal cortex and impaired fear extinction retrieval. Moreover, APPL1 undergoes nuclear translocation during extinction retrieval. Blocking APPL1 nucleocytoplasmic translocation reduced NMDAR currents and disrupted extinction retrieval. We also identified a prefrontal neuronal ensemble that is both necessary and sufficient for the storage of extinction memory. Inducible APPL1 knockout in this ensemble abolished NMDAR-dependent synaptic potentiation and disrupted extinction retrieval, while chemogenetic activation of this ensemble simultaneously rescued the impaired behaviors. CONCLUSIONS: Our results indicate that a prefrontal neuronal ensemble stores extinction memory, and APPL1 signaling supports these neurons in retrieving extinction memory by controlling NMDAR-dependent potentiation.

Identification of core genes as potential biomarkers for predicting progression and prognosis in glioblastoma.

Background: Glioblastoma is a common malignant neuroepithelial neoplasm with poor clinical outcomes and limited treatment options. It is extremely important to search and confirm diverse hub genes that are effective in the advance and prediction of glioblastoma. Methods: We analyzed GSE50161, GSE4290, and GSE68848, the three microarray datasets retrieved from the GEO database. GO function and KEGG pathway enrichment analyses for differentially expressed genes (DEGs) were performed using DAVID. The PPI network of the DEGs was analyzed using the Search Tool for the Retrieval of Interacting Genes database and visualized by Cytoscape software. Hub genes were identified through the PPI network and a robust rank aggregation method. The Cancer Genome Atlas (TCGA) and the Oncomine database were used to validate the hub genes. In addition, a survival curve analysis was conducted to verify the correlation between the expression of hub genes and patient prognosis. Human glioblastoma cells and normal cells were collected, and then RT-PCR, Western blot, and immunofluorescence were conducted to validate the expression of the NDC80 gene. A cell proliferation assay was used to detect the proliferation of glioma cells. The effects of NDC80 expression on migration and invasion of GBM cell lines were evaluated by conducting scratch and transwell assays. Results: A total of 716 DEGs were common to all three microarray datasets, which included 188 upregulated DEGs and 528 downregulated DEGs. Furthermore, we found that among the common DEGs, 10 hub genes showed a high degree of connectivity. The expression of the 10 hub genes in TCGA and the Oncomine database was significantly overexpressed in glioblastoma compared with normal genes. Additionally, the survival analysis showed that the patients with low expression of six genes (BIR5C, CDC20, NDC80, CDK1, TOP2A, and MELK) had a significantly favorable prognosis (p < 0.01). We discovered that NDC80, which has been shown to be important in other cancers, also has an important role in malignant gliomas. The RT-PCR, Western blot, and immunofluorescence results showed that the expression level of NDC80 was significantly higher in human glioblastoma cells than in normal cells. Moreover, we identified that NDC80 increased the proliferation and invasion abilities of human glioblastoma cells. Conclusion: The six genes identified here may be utilized to form a panel of disease progression and predictive biomarkers of glioblastoma for clinical purposes. NDC80, one of the six genes, was discovered to have a potentially important role in GBM, a finding that needs to be further studied.

Adaptor protein APPL1 links neuronal activity to chromatin remodeling in cultured hippocampal neurons.

Local signaling events at synapses or axon terminals are communicated to the nucleus to elicit transcriptional responses, and thereby translate information about the external environment into internal neuronal representations. This retrograde signaling is critical to dendritic growth, synapse development, and neuronal plasticity. Here, we demonstrate that neuronal activity induces retrograde translocation and nuclear accumulation of endosomal adaptor APPL1. Disrupting the interaction of APPL1 with Importin α1 abolishes nuclear accumulation of APPL1, which in turn decreases the levels of histone acetylation. We further demonstrate that retrograde translocation of APPL1 is required for the regulation of gene transcription and then maintenance of hippocampal late-phase long-term potentiation. Thus, these results illustrate an APPL1-mediated pathway that contributes to the modulation of synaptic plasticity via coupling neuronal activity with chromatin remodeling.

Identification of the role of TRPM8 in glioblastoma and its effect on proliferation, apoptosis and invasion of the U251 human glioblastoma cell line.

Glioblastoma multiforme (GBM) is the most commonly occurring brain cancer, and is characterized by its poor patient outcomes. The present study examined the mRNA expression levels of the transient receptor potential melastatin (TRPM) family in various types of cancer using the ONCOMINE database, along with their corresponding expression profiles in an array of cancer cell lines based on the Cancer Cell Line Encyclopedia (CCLE) datasets. Kaplan­Meier plotter survival analysis via the Chinese Glioma Genome Atlas (CGGA) database was also used to evaluate the prognostic value of transient receptor potential melastatin 8 (TRPM8). For the activity test on the TRPM8 channel, patch­clamp recordings and Ca2+ measurements by fluorescence imaging of Fluo­4am were performed. Short hairpin RNA (shRNA) targeting TRPM8 was designed, synthesized and then transfected into the U251 cells via Lipofectamine 2000. The expression of extracellular singnal­regulated kinase (ERK), cyclin D1 and Bcl­2 were detected by performing western blotting and immunofluorescence. The apoptosis, proliferation and invasion of glioma cells were detected by using flow cytometry, and CCK­8 and Transwell invasion assays. In the present study, TRPM8 was distinctively upregulated in GBM cell lines. TRPM8 is functional and has the characteristic of outward rectification, which was verified via electrophysiology and Ca2+ fluorescence imaging in U251 cells. The western blot and immunofluorescence results revealed that the expression of ERK, cyclin D1 and Bcl­2 were decreased in the shRNA interference group. The CCK­8 assay demonstrated that the proliferation ability of U251 cells in the U251/TRPM8 group was higher than that in the U251 group and U251/Con group (P<0.05). The result of the Transwell invasion assay indicated that the invasion of human glioblastoma U251 cells was positively correlated with the expression level of TRPM8. Collectively, the results of the present study indicated that Ca2+­permeable TRPM8 nonselective cation channels contribute to survival, proliferation, apoptosis, and local tumor invasion of glioblastoma. Therefore, TRPM8 is a promising biomarker for aggressiveness of GBM, and a potential target in future anti­glioblastoma therapies.

Subchronic perfluorooctanesulfonate (PFOS) exposure induces elevated mutant frequency in an in vivo λ transgenic medaka mutation assay.

Perfluorooctanesulfonate (PFOS) has been widely detected in the environment, wildlife and humans, but few studies have ever examined its mutagenic effect in vivo. In the present study, we use a transgenic fish model, the λ transgenic medaka, to evaluate the potential mutagenicity of PFOS in vivo following a subchronic exposure of 30 days. The mutant frequency of cII target gene was 3.46 × 10-5 in liver tissue from control fish, which increased by 1.4-fold to 4.86 × 10-5 in fish exposed to 6.7 µg/L PFOS, 1.55-fold to 5.36 × 10-5 in fish exposed to 27.6 µg/L PFOS, and 2.02-fold to 6.99 × 10-5 in fish exposed to 87.6 µg/L PFOS. This dose-dependent increase of mutant frequency was also accompanied with mutational spectrum changes associated with PFOS exposure. In particular, PFOS-induced mutation was characterized by +1 frameshift mutations, which increased from 0% in control fish to 13.2% in fish exposed to 27.6 µg/L PFOS and 14.6% in fish exposed to 87.6 µg/L PFOS. Our findings provide the first evidence of PFOS's mutagenicity in an aquatic model system. Given the fact that most conventional mutagenic assays were negative for PFOS, we propose that PFOS-induced mutation in liver tissue of λ transgenic medaka may be mediated through compromised liver function.