Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs

Gene encoding prolactin in cinnamon clownfish Amphiprion melanopus and its expression upon acclimation to low salinities

BACKGROUND: Prolactin (PRL) is a key hormone for osmoregulation in fish. Levels of PRL in the pituitary gland and plasma ion composition of clownfish seem to change to regulate their hydromineral balance during adaptation to waters of different salinities. In order to understand osmoregulatory mechanism and its association with growth performance and PRL in fish, the gene encoding PRL and its expression level in cinnamon clownfish Amphiprion melanopus upon acclimation to low salinity was analyzed. RESULTS: The PRL gene of A. melanopus encoded a protein of 212 amino acid residues comprised of a putative signal peptide of 24 amino acids and a mature protein of 188 amino acids. Analysis of growth performance under different salinities of 34, 25, 15, and 10 ppt indicated that cinnamon clownfish could survive under salinities as low as 10 ppt. A higher rate of growth was observed at the lower salinities as compared to that of 34 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased to reach the peak level until 24 h of acclimation at 15 ppt, but decreased back as adaptation continued to 144 h. In contrast, levels of plasma Na(+), Cl(-), and osmolality decreased at the initial stage (4–8 h) of acclimation at 15 pt but increased back as adaptation continued till 144 h. CONCLUSION: Cinnamon clownfish could survive under salinities as low as 10 ppt. Upon shifting the salinity of the surrounding water from 34 ppt to 15 ppt, the level of the PRL transcripts gradually increased during the initial stage of acclimation but decreased back to the normal level as adaptation continued. An opposite pattern of changes - decrease at the beginning followed by an increase - in the levels of plasma Na(+), Cl(-), and osmolality was found upon acclimation to low salinity. The results suggest an involvement of PRL in the processes of osmoregulation and homeostasis in A. melanopus

Self-Assembled Multi-Epitope Peptide Amphiphiles Enhance the Immune Response against Enterovirus 71

Subunit vaccines consist of non-genetic material, such as peptides or proteins. They are considered safe because they have fewer side effects; however, they have low immunogenicity when used alone. We aimed to enhance the immune response of peptide-based vaccines by using self-assembled multimeric peptide amphiphiles (PAs). We designed two epitope PAs by conjugating epitope peptides from Enterovirus 71 (EV71) virus particle (VP) 1 and VP3 capsid proteins with different fatty acid chain lengths (VP1PA and VP3PA). These PAs self-assembled into supramolecular structures at a physiological pH, and the resulting structures were characterized using atomic force microscopy. Multi-epitope PAs (m-PAs) consisted of a 1:1 mixture of VP1PA and VP3PA solutions. To evaluate immunogenicity, m-PA constructs were injected with adjuvant subcutaneously into female Balb/c mice. Levels of antigen-specific immunoglobulin G (IgG) and IgG1 in m-PA-injected mice serum samples were analyzed using ELISA and Western blotting. Additionally, cytokine production stimulated by each antigen was measured in splenocytes cultured from immunized mice groups. We found that m-PA showed improved humoral and cellular immune responses compared to the control and peptide groups. The sera from m-PA immunized mice group could neutralize EV71 infection and protect host cells. Thus, self-assembled m-PAs can promote a protective immune response and can be developed as a potential platform technology to produce peptide vaccines against infectious viral diseases

The Ca2+ -activated chloride channel anoctamin-2 mediates spike-frequency adaptation and regulates sensory transmission in thalamocortical neurons

Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+ -activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission. © The Author(s) 2016113131sciescopu

Electroconductive nanoscale topography for enhanced neuronal differentiation and electrophysiological maturation of human neural stem cells

Biophysical cues, such as topography, and electrical cues can provide external stimulation for the promotion of stem cell neurogenesis. Here, we demonstrate an electroconductive surface nanotopography for enhancing neuronal differentiation and the functional maturation of human neural stem cells (hNSCs). The electroconductive nanopatterned substrates were prepared by depositing a thin layer of titanium (Ti) with nanograting topographies (150 to 300 nm groove/ridge, the thickness of the groove -150 mu m) onto polymer surfaces. The Ti-coated nanopatterned substrate (TNS) induced cellular alignment along the groove pattern via contact guidance and promoted focal adhesion and cytoskeletal reorganization, which ultimately led to enhanced neuronal differentiation and maturation of hNSCs as indicated by significantly elevated neurite extension and the upregulated expression of the neuronal markers Tuj1 and NeuN compared with the Ti-coated flat substrate (TFS) and the nanopatterned substrate (NS) without Ti coating. Mechanosensitive cellular events, such as beta 1-integrin binding/clustering and myosin-actin interaction, and the Rho-associated protein kinase (ROCK) and mitogen-activated protein kinase/extracellular signal regulated kinase (MEK-ERK) pathways, were found to be associated with enhanced focal adhesion and neuronal differentiation of hNSCs by the TNS. Among the neuronal subtypes, differentiation into dopa-minergic and glutamatergic neurons was promoted on the TNS. Importantly, the TNS increased the induction rate of neuron-like cells exhibiting electrophysiological properties from hNSCs. Finally, the application of pulsed electrical stimulation to the TNS further enhanced neuronal differentiation of hNSCs due probably to calcium channel activation, indicating a combined effect of topographical and electrical cues on stem cell neurogenesis, which postulates the novelty of our current study. The present work suggests that an electroconductive nanopatterned substrate can serve as an effective culture platform for deriving highly mature, functional neuronal lineage cells from stem cells © The Royal Society of Chemistry 20171121sciescopu

The flexible neuronal conversion of malignant C6 glioma through the action of small molecules.

<p>(A and B) Representative images of C6 glioma before neural induction. (C) Representative fluorescence image of Tuj1-positive cells 7 days after neural induction of glioma with CHIR99021 (2 μM) and forskolin (10 μM). (D) Representative image of Tuj1/DAPI staining at 7 days after treatment of mouse embryonic fibroblasts with a combination of CHIR99021 (2 μM) and forskolin (10 μM). (E) Representative image of Tuj1-positive cells 7 days after treatment of glioma with a variety of concentrations of CHIR99021 and forskolin. Scale bars represent 500 μm (A), 50 μm (B), 100 μm (C), 100 μm (D), and 100 μm (E).</p

Gene expression profile of a malignant glioma and SMiN.

<p>(A) Heat map of significant gene in C6 glioma and SMiNs. (B) Up- or down-regulated gene ontology term in SMiNs. (C) Comparison of neuronal differentiation-related genes in C6 glioma and SMiNs. (D) Comparison of cell cycle-related genes in C6 glioma and SMiNs. (E) Comparison of ECM-related genes in C6 glioma and SMiNs. (F) Comparison of vessel development-related genes in C6 glioma and SMiNs. (G) Enriched gene list in C6 glioma and SMiNs.</p

Growth suppression effect of a combination of CHIR99021 and forskolin in C6 glioma.

<p>(A) The proliferation rate of normal C6 glioma in neural induction media without small molecules. Cells were transferred to a new large dish at day 7. (B and C) Quantitative results of cell density at day 7 after neural induction. (D) Quantitative results of cell density at day 7 after neural induction. C, CHIR99021 (20 μM); F, forskolin (100 μM). (E) Quantitative results of cell density at days 7 and 14 days after neural induction with CHIR99021 (20 μM) and forskolin (100 μM). (F and G) Representative image and percentages of Ki67-positive cells in C6 glioma and SMiN cultures 35 days after neural induction with CHIR99021 (20 μM) and forskolin (100 μM). (H) Quantitative results of cell density at 7 days post-treatment. C, CHIR99021 (20 μM); F, forskolin (100 μM); T, temozolomide (50 μM). * indicates <i>p < 0</i>.<i>05</i>. Data are presented as the mean ± S.E.M. Scale bars represent 100 μm (F and G).</p

Direct reprogramming of C6 glioma to neurons using two small molecules.

<p>(A) Representative fluorescence images of small molecule-induced neurons (SMiNs) 35 days after neural induction with CHIR99021 (20 μM) and forskolin (100 μM). (B) Quantitative result of Tuj1, MAP2, GFAP, PDGFR, and NG2 staining in SMiNs 35 days after neural induction. (C) Representative image of Tuj1- or MAP2-positive SMiNs 35 days after neural induction with CHIR99021 (20 μM) and forskolin (100 μM). (D) Quantitative result of morphological complexity of Tuj1-positive SMiNs 35 days after neural induction. (E) Representative band image of SSEA1 (a cancer stem cell marker) and C-Myc (an oncogene marker) 35 days after neural induction with CHIR99021 (20 μM) and forskolin (100 μM). (F) Representative fluorescence images of SMiNs 7 days after neural induction with lithium carbonate (3 mM) and forskolin (100 μM). (G) Representative fluorescence images of SMiNs 7 days after neural induction with CHIR99021 (2 μM) and dbcAMP (0.5 mM). (H) Representative images of patch-clamp in SMiNs. (I) Representative traces of sodium current of SMiN 84 days after neural induction before (left) and after (right) 0.5μM TTX bath application. (J) Representative traces of action potential spike of SMiN 84 days after neural induction before (left) and after (right) 0.5μM TTX bath application. (K) Representative traces of sodium current (left) and action potential spike (right) of undifferentiated C6 glioma in proliferation media. (L) Representative traces of sodium current (left) and action potential spike (right) of undifferentiated C6 glioma in neural differentiation media without small molecules. Data are presented as the mean ± S.E.M. Scale bars represent 50 μm (A), 20 μm (C), 100 μm (F), and 100 μm (G).</p

Anti-cancer effects of combined CHIR99021 and forskolin in a severe IMSCT model.

<p>(A) Neurologic function tests. (B) Representative image of dissected spinal cord at 16 days post-transplantation with RFP-expressing C6 glioma. (C) Representative optical imaging of dissected spinal cord at 16 days post-transplantation with RFP-expressing C6 glioma. (D) Representative image of sagittal sectioned-spinal cord at 16 days post-transplantation with RFP-expressing C6 glioma. (E) Quantitative result of ROI at 16 days post-transplantation with RFP-expressing C6 glioma. (F) Quantitative result of tumor area at 16 days post-transplantation with RFP-expressing C6 glioma. C, CHIR99021 (12.5 mg/kg); F, forskolin (10 mg/kg). * indicates <i>p</i> < 0.05. Asterisk means a significant difference to control. Data are presented as the mean ± S.E.M. Control (n = 11), CF (n = 7). Scale bars represent 1 cm (B) and 2 mm (D).</p