Cytotoxicity of amine-modified polystyrene MPs and NPs on neural stem cells cultured from mouse subventricular zone.

Microplastics (MPs) and nanoplastics (NPs) are found in various environments such as aquatic, terrestrial, and aerial areas. Once ingested and inhaled, these tiny plastic debris damaged the digestive and respiratory organ systems in animals. In humans, the possible connection between MPs and various diseases such as lung diseases has been raised. Yet, the impact of MPs on the human nervous system has been unclear. Previous research using animals and cultured cells showed possible neurotoxicity of MPs and NPs. In this study, we used neural stem cells cultured from mouse subventricular zone to examine the effects of polystyrene (PS) NPs and MPs with sizes of 0.1 µm, 1 µm, and 2 µm on the cell proliferation and differentiation. We observed that only positively charged NPs and MPs, but not negatively charged ones, decreased cell viability and proliferation. These amine-modified NPs and MPs decreased both neurogenesis and oligodendrogenesis. Finally, fully differentiated neurons and oligodendrocytes were damaged and removed by the application of NPs and MPs. All these effects varied among different sizes of NPs and MPs, with the greatest effects from 1 µm and the least effects from 2 µm. These results clearly demonstrate the cytotoxicity and neurotoxicity of PS-NPs and MPs.

Long-Term Antioxidant Metal-Organic Frameworks.

Free radicals produced during metabolism induce effects, such as cell damage and cancer, because of their high reactivity. Although antioxidants in food products can eliminate free radicals, they are expelled within a relatively short period of time after serving their function. In this study, we investigated the possibility of using metal-organic frameworks (MOFs) with antioxidants as their ligands. Metal-organic frameworks are crystalline polymers with repetitively coordinated ligands and metal centers. We assume that once antioxidant-based MOFs are ingested, ligands are released on a long-term basis during the process of chemical and physical disintegration. To evaluate their eligibility, we established criteria for biocompatibility, particle size, and long-term antioxidant effects. For biocompatibility, we treated cells with various concentrations of MOFs and their precursors followed by a water-soluble tetrazolium 8 (WST-8) assay. The particle size distribution was analyzed using TEM and ImageJ software, and the antioxidant release was quantified using UV-vis spectroscopy. We concluded that Fe-based FeTHQ with the antioxidant tetrahydroxy-1,4-benzoquinone (THQ) as its ligand is the most effective long-term antioxidant with its effect lasting up to 7 days. Furthermore, microwave synthesis of FeTHQ was conducted to produce more suitable particles for in vivo antioxidant applications.

Effects of taxol on neuronal differentiation of postnatal neural stem cells cultured from mouse subventricular zone.

Taxol (paclitaxel), a chemotherapeutic agent for several cancers, can adversely affect the peripheral nervous system. Recently, its negative impact on cognitive function in cancer patients has become evident. In rodents, taxol impaired learning and memory, with other possible negative effects on the brain. In this study, we investigated the effects of taxol on cultured neural stem cells (NSCs) from the mouse neurogenic region, the subventricular zone (SVZ). Taxol significantly decreased both proliferation and neuronal differentiation of NSCs. Transient treatment with taxol for one day during a 4-day differentiation greatly decreased neurogenesis along with an abnormal cell cycle progression. Yet, taxol did not kill differentiated Tuj1+ neurons and those neurons had longer neurites than neurons under control conditions. For glial differentiation, taxol significantly reduced oligodendrogenesis as observed by immunostaining for Olig2 and O4. However, differentiation of astrocytes was not affected by taxol. In contrast, differentiated oligodendrocytes were extremely sensitive to taxol. Almost no Olig2-positive cells were observed after three days of treatment with taxol. Taxol has distinct effects on neurons and glial cells during their production through differentiation from NSCs as well as post-differentiation. Thus, we suggest that taxol might interfere with neurogenesis of NSCs possibly through a disturbance in the cell cycle and may eliminate differentiated oligodendrocytes.

The Ink4a/Arf locus is a barrier to direct neuronal transdifferentiation.

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16(Ink4a) and p19(Arf) indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo.

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression for the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Our data and workflow thus provide a uniquely coherent in vivo lncRNA analysis and form the foundation of a user-friendly online resource for the study of lncRNAs in development and disease.

Epigenetic regulation of the calcitonin gene-related peptide gene in trigeminal glia.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. METHODS: DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. RESULTS: A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. CONCLUSIONS: Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.

Control of the calcitonin gene-related peptide enhancer by upstream stimulatory factor in trigeminal ganglion neurons.

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening.

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.

A role for phosphatidylinositol 3-phosphate in abscisic acid-induced reactive oxygen species generation in guard cells.

Guard cells generate reactive oxygen species (ROS) in response to abscisic acid (ABA), which leads to stomatal closing. The upstream steps of the ABA-induced ROS generation pathway remain largely unknown. In animal cells, ROS generation in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P). Stomatal guard cells contain PI3P and PI 3-kinase activity. In this study, we tested whether PI3P has a role in ROS generation in guard cells exposed to ABA. We found that PI 3-kinase inhibitors wortmannin or LY294002 inhibited ABA-induced ROS generation and stomatal closing. Endosome-binding domain (of human EEA1), which specifically binds to PI3P, also inhibited ABA-induced ROS generation and stomatal closing when overexpressed in guard cells. Hydrogen peroxide partially reversed the effects of wortmannin or LY294002 on ABA-induced stomatal closing. These results support a role for PI3P in ABA-induced ROS generation and stomatal closing movement.