Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca2+/NFATc3 Signaling.

The quiescence, activation, and subsequent neurogenesis of neural stem cells (NSCs) play essential roles in the physiological homeostasis and pathological repair of the central nervous system. Previous studies indicate that transmembrane protein Ttyh1 is required for the stemness of NSCs, whereas the exact functions in vivo and precise mechanisms are still waiting to be elucidated. By constructing Ttyh1-promoter driven reporter mice, we determined the specific expression of Ttyh1 in quiescent NSCs and niche astrocytes. Further evaluations on Ttyh1 knockout mice revealed that Ttyh1 ablation leads to activated neurogenesis and enhanced spatial learning and memory in adult mice (6-8 weeks). Correspondingly, Ttyh1 deficiency results in accelerated exhaustion of NSC pool and impaired neurogenesis in aged mice (12 months). By RNA-sequencing, bioinformatics and molecular biological analysis, we found that Ttyh1 is involved in the regulation of calcium signaling in NSCs, and transcription factor NFATc3 is a critical effector in quiescence versus cell cycle entry regulated by Ttyh1. Our research uncovered new endogenous mechanisms that regulate quiescence versus activation of NSCs, therefore provide novel targets for the intervention to activate quiescent NSCs to participate in injury repair during pathology and aging.

Tweety-Homolog 1 Facilitates Pain via Enhancement of Nociceptor Excitability and Spinal Synaptic Transmission.

Tweety-homolog 1 (Ttyh1) is expressed in neural tissue and has been implicated in the generation of several brain diseases. However, its functional significance in pain processing is not understood. By disrupting the gene encoding Ttyh1, we found a loss of Ttyh1 in nociceptors and their central terminals in Ttyh1-deficient mice, along with a reduction in nociceptor excitability and synaptic transmission at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) in the basal state. More importantly, the peripheral inflammation-evoked nociceptor hyperexcitability and spinal synaptic potentiation recorded in spinal-PAG projection neurons were compromised in Ttyh1-deficient mice. Analysis of the paired-pulse ratio and miniature excitatory postsynaptic currents indicated a role of presynaptic Ttyh1 from spinal nociceptor terminals in the regulation of neurotransmitter release. Interfering with Ttyh1 specifically in nociceptors produces a comparable pain relief. Thus, in this study we demonstrated that Ttyh1 is a critical determinant of acute nociception and pain sensitization caused by peripheral inflammation.

Neurons can upregulate Cav-1 to increase intake of endothelial cells-derived extracellular vesicles that attenuate apoptosis via miR-1290.

Extracellular vesicles (EVs) including exosomes can serve as mediators of cell-cell communication under physiological and pathological conditions. However, cargo molecules carried by EVs to exert their functions, as well as mechanisms for their regulated release and intake, have been poorly understood. In this study, we examined the effects of endothelial cells-derived EVs on neurons suffering from oxygen-glucose deprivation (OGD), which mimics neuronal ischemia-reperfusion injury in human diseases. In a human umbilical endothelial cell (HUVEC)-neuron coculture assay, we found that HUVECs reduced apoptosis of neurons under OGD, and this effect was compromised by GW4869, a blocker of exosome release. Purified EVs could be internalized by neurons and alleviate neuronal apoptosis under OGD. A miRNA, miR-1290, was highly enriched in HUVECs-derived EVs and was responsible for EV-mediated neuronal protection under OGD. Interestingly, we found that OGD enhanced intake of EVs by neurons cultured in vitro. We examined the expression of several potential receptors for EV intake and found that caveolin-1 (Cav-1) was upregulated in OGD-treated neurons and mice suffering from middle cerebral artery occlusion (MCAO). Knock-down of Cav-1 in neurons reduced EV intake, and canceled EV-mediated neuronal protection under OGD. HUVEC-derived EVs alleviated MCAO-induced neuronal apoptosis in vivo. These findings suggested that ischemia likely upregulates Cav-1 expression in neurons to increase EV intake, which protects neurons by attenuating apoptosis via miR-1290.

Deficiency of Ttyh1 downstream to Notch signaling results in precocious differentiation of neural stem cells.

Mammalian neural stem cells (NSCs) are not only responsible for normal development of the central nervous system (CNS), but also participate in brain homeostasis and repair, thus hold promising clinical potentials in the treatment of neurodegenerative diseases and trauma. However the molecular networks regulating the stemness and differentiation of NSCs have not been fully understood. In this study, we show that Tweety-homolog 1 (Ttyh1), a five-pass transmembrane protein specifically expressed in mouse brain, is involved in maintaining stemness of murine NSCs. Blocking or activating Notch signal led to downregulation and upregulation of Ttyh1 in cultured NSCs, respectively, suggesting that Ttyh1 is under the control of Notch signaling. Knockdown of Ttyh1 in cultured NSCs resulted in a transient increase in the number and size of neurospheres, followed by a decrease of stemness as manifested by compromised neurosphere formation, downregulated stem cell markers, and increased neuronal differentiation. We generated Ttyh1 knockout mice by deleting its exon 4 using the CRISPR-Cas9 technology. Surprisingly, in contrast to a previous report, Ttyh1 knockout did not result in embryonic lethality. NSCs derived from Ttyh1 knockout mice phenocopied NSCs transfected with Ttyh1 siRNA. Immunofluorescence showed that loss of Ttyh1 leads to the increase of neurogenesis in adult mice. Taken together, these findings indicate that Ttyh1, which is likely downstream to Notch signaling, plays an important role in regulating NSCs.

[Mining and discovery for active components of "Coptidis Rhizome-Magnoliae Officinalis Cortex" based on network pharmacology].

To mine and discover the active components of " Coptidis Rhizome-Magnoliae Officinalis Cortex( C&M) " based on the network pharmacology,integrate and analyze the potential targets and mechanisms. The TCMSP database was used to screen active ingredients. TTD and Drug Bank databases were used to predict the potential targets by referring to relevant literature,and the pathway annotation technology was used to enrich and analyze the active ingredients and potential targets of " C&M". A total of 29 potential target active ingredients were screened from " C&M",including 12 alkaloids components such as( R)-canadine,berberine,coptisine,and palmatine; 3 lignans consisting of magnolol,honokiol and obovatol; 6 volatile oils consisting of α-eudesmol,ß-eudesmol,eucalyptol and so on,and flavonoids including quercetin and neohesperidin. Corresponding 199 predicted targets were screened out,mainly including PTGS2,PTGS1,NCOA2,Hsp90 AB1,and so on. 72 signaling pathways were involved,8 of which were related to cancer,such as prostate cancer,bladder cancer,and pancreatic cancer; 9 of which were related to endocrine,including oxytocin signaling pathway,insulin signaling pathway,thyroid hormone signaling pathway and so on,as well as inflammation-related pathway. This study has preliminarily mined and discovered the main active components and potential targets of " C&M",providing material source for the study on the preparation of structural components of traditional Chinese medicine.

Stem cells: a promising candidate to treat neurological disorders.

Neurologic impairments are usually irreversible as a result of limited regeneration in the central nervous system. Therefore, based on the regenerative capacity of stem cells, transplantation therapies of various stem cells have been tested in basic research and preclinical trials, and some have shown great prospects. This manuscript overviews the cellular and molecular characteristics of embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal stem/progenitor cells, mesenchymal stem/stromal cells, and their derivatives in vivo and in vitro as sources for regenerative therapy. These cells have all been considered as candidates to treat several major neurological disorders and diseases, owing to their self-renewal capacity, multi-directional differentiation, neurotrophic properties, and immune modulation effects. We also review representative basic research and recent clinical trials using stem cells for neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and age-related macular degeneration, as well as traumatic brain injury and glioblastoma. In spite of a few unsuccessful cases, risks of tumorigenicity, and ethical concerns, most results of animal experiments and clinical trials demonstrate efficacious therapeutic effects of stem cells in the treatment of nervous system disease. In summary, these emerging findings in regenerative medicine are likely to contribute to breakthroughs in the treatment of neurological disorders. Thus, stem cells are a promising candidate for the treatment of nervous system diseases.

[Effective components and signaling pathways of Epimedium brevicornumbased on network pharmacology].

The aim of this paper was to find out the active components of Epimedium brevicornum using network pharmacology, and find the potential targets and mechanisms. The TCMSP database was used to screen the active ingredients, and TTD and DrugBank databases were used to predict the potential targets with the literature mining. The pathway annotation was used to enrich and analyze the active ingredients and potential targets of E. brevicornum. The results showed that E. brevicornum had34 potential target active ingredients, including 21 flavones components, such as icariin, epimedin A, epimedin B, epimedin C, Yinyanghuo A, Yinyanghuo C and so on, 2 lignans involved in (+)-cycloolivil and olivil, 3 sterols consisting of sitosterol, 24-epicampesterol and poriferast-5-en-3beta-ol. The main predicted targets included Ptgs2, NCOA6, RANK, OPG, WNT9B, PTH1R, BMPs, SMAD4A and so on. There were 88 signaling pathways involved in 10 signaling pathways which was related to inflammation, such as NF-kappa B signaling pathway, T cell receptor signaling pathway, IL-17 signaling pathway and 10 pathways which was related to cancer included breast cancer, bladder cancer, pancreatic cancer and so on, and estrogen related signaling pathways included estrogen signaling pathway. This laid the foundation for the discovery of the active components of Epimedium and the study on its mechanism of action.

Exosomes derived from human umbilical vein endothelial cells promote neural stem cell expansion while maintain their stemness in culture.

The neural stem cell (NSC) niche in subventricular zone (SVZ) of adult mammalian brain contains dense vascular plexus, where endothelial cells (ECs) regulate NSCs by releasing plenty of angiocrine factors. However, the role of ECs-derived exosomes, a novel type of mediators of intercellular communications, in the regulation of NSCs remains unclear. In the current study, primary NSCs isolated from embryonic mouse brains form more neurospheres when cultured in the presence of human umbilical vein endothelial cells (HUVECs). The supportive role of ECs in the coculture was significantly attenuated when GW4869, a blocker of exosome formation, was included, suggesting that HUVECs-derived exosomes played a significant role in supporting NSCs. In order to investigate the role of ECs-derived exosomes on NSCs, we collected exosomes from HUVECs. We found that HUVECs-derived exosomes could significantly promote the formation of neurospheres by primary murine NSCs. EdU incorporation and TUNEL assays indicated that the proliferation of NSCs increased while apoptosis decreased when cultured in the presence of HUVECs-derived exosomes. NSCs incubated with the HUVECs-derived exosomes maintained their potential of multi-lineage differentiation potentials. The expression of stemness-related genes was up-regulated. These data suggested that ECs-derived exosomes could play an importantly role in NSC niche, and they might be used as a reagent for ex vivo NSC amplification for medical application.

[Speciation and bioavailability of heavy metals in paddy soil irrigated by acid mine drainage].

Based on field investigation and laboratory analysis, the speciation and bioavailability of the heavy metals (Cd, Cu, Pb, and Zn) was studied. These metals were tested from 16 soil samples taken from paddy soils irrigated by acid mine drainage in the lower stream of Dabaoshan mine area, Guangdong Province, China. The toxicity characteristic leaching procedure (TCLP) and three-step community bureau of reference (BCR) sequential extraction procedure methods were used to analyse the bioavailability of heavy metals. TCLP is currently recognized as an international method for evaluation of heavy metal pollution in soils. Two methods were compared in bioavailability by stepwise multiple linear regression analysis. Cd was dominated mainly by the acid-extractable and residual fractions, and followed by the order of acid-extractable > residual > oxidisable > reducible. Cu was dominated mainly by the residual fraction, and decreased in the following order: residual > reducible > acid-extractable > oxidisable. Pb was dominantly released in the reducible and residual fractions, they account for 86.54% of the total contents, and decreased in the following order: reducible > residual > acid-extractable > oxidisable. Zn was dominated mainly by the residual fraction, it takes more than 50% of the total contents, and followed by the order of residual > acid-extractable > reducible > oxidisable. The levels of Cd, Cu, Pb and Zn extracted by TCLP were 0.17-0.89, 8.12-70.33, 3.16-90.33 and 10.24-106.85 mg x kg(-1), respectively, and the average concentrations of Cd, Cu, Pb and Zn were 0.41, 36.60, 15.97 and 50.78 mg x kg(-1), respectively. The concentrations of Cd, Cu, Ph and Zn in rice seeds were 0.183-0.947, 3.542-5.997, 0.285-1.532 and 17.54-41.10 mg x kg(-1), respectively. Comparing to the national standards on heavy metals in foods by the Chinese Ministry of Health, the contents of Cu and Zn in rice seeds didn't exceed the upper limit, while the contents of Cd and Pb in rice seeds exceed the upper limit by 87.50% and 81.25%, respectively. Results of the stepwise multiple linear regression analysis revealed that contents of Cd and Zn in seeds of rice were significantly influenced by contents of acid-extractable Cd and Zn, that contents of Cu in seeds of rice were significantly influenced by contents of acid-extractable and oxidisable Cu, and the contents of Pb in seeds of rice were significantly influenced by contents of reducible Pb and organic matter. Contents of four metals in seeds of rice were significantly influenced by contents of metals extracted by TCLP, and the influences of total content, pH and organic matter were not significant. In short, TCLP method was fast, simple and suitable for evaluation of bioavailability of heavy metals in sewage-irrigated paddy soils by acid mine drainage.