Search Results (20)

The presence of pesticides in surface waters has been widely reported worldwide and represents a significant problem that raises concerns on local, regional, national, and international scales. Among these, metolachlor is one of the most widely used herbicides to control annual grasses and broadleaf weeds in various crops. Despite the existing research, data on the effects of metolachlor on the nervous system of fishes, remain limited. The present study aims to investigate the impact of metolachlor during embryonic development on the formation of the nervous system and the subsequent inflammatory response in zebrafish (Danio rerio), focusing specifically on larvae at 24 h post-fertilization (hpf). To achieve this, transgenic zebrafish lines marking neuronal populations Tg(Hu:GFP), glial cells Tg(gfap:GFP), and circulating macrophages Tg(mpeg:GFP) were employed. Following exposure to sub-lethal doses of metolachlor, we observed a significant decrease in GPF-positive cells marking the neuronal population, accompanied by an increase in apoptotic cells within the brain region. Additionally, treated embryos exhibited a marked neuroinflammatory response, characterized by astrogliosis and the specific accumulation of microglia/macrophage-positive cells in the head region. In situ hybridization and real-time PCR analyses revealed a significant downregulation of the neurogenin-1 (ngn1) transcript and a noticeable upregulation of the pro-inflammatory cytokine interleukin-1 beta (il1b). Our findings contribute to the growing body of evidence suggesting that metolachlor, even at early developmental stages, can have detrimental effects on both the formation of the nervous system and the regulation of immune responses. Full article

Bisphenol A diglycidyl ether (BADGE) is the main component of epoxy resin and is used for the inner coating of canned foods and plastic food containers. BADGE can easily migrate from containers and result in food contamination; the compound is known as an endocrine-disrupting chemical. We previously reported that maternal exposure to bisphenol A bis (2,3-dihydroxypropyl) ether (BADGE·2H₂O), which is the most detected BADGE derivative not only in canned foods but also in human specimens, during gestation and lactation, could accelerate neuronal differentiation in the cortex of fetuses and induce anxiety-like behavior in juvenile mice. In this study, we investigated the effects of low-dose BADGE·2H₂O (1–100 pM) treatment on neurites and the mechanism of neurite outgrowth in cortical neurons. BADGE·2H₂O exposure significantly increased the number of dendrites and neurite length in cortical neurons; these accelerating effects were inhibited by estrogen receptor (ER) antagonist ICI 182,780 and G-protein-coupled estrogen receptor (GPER) antagonist G15. BADGE·2H₂O down-regulated Hes1 expression, which is a transcriptional repressor, and increased levels of neuritogenic factor neurogenin-3 (Ngn3) in the cortical neurons; the changes were significantly blocked by G15. These data suggest that direct BADGE·2H₂O exposure can accelerate neuritogenesis and outgrowth in cortical neurons through down-regulation of Hes1 and by increasing Ngn3 levels through ERs, particularly GPER. Full article

The neuronal progenitor NG108-15 neuroblastoma x glioma cell line proliferates indefinitely in vitro and is capable of directed differentiation into cholinergic neurons. The cell line is a robust model for investigating neuronal differentiation and function in vitro. The lineage-specific transcription factor-mediated differentiation of pluripotent stem cell lines (PSCs) leads to more rapid, efficient, and functional neurons. In this study, we tested the hypothesis that transcription factors could also drive the fate of an immortalised cell line. We first established a stable NG108-15 cell line, by piggyBac (pBac) transposition, that conditionally expresses neurogenin-2 (Ngn2), a common transcription factor for specifying neuronal fate. Following doxycycline-induction of Ngn2, we observed more rapid and efficient differentiation, and improved neurite outgrowth and viability compared with the WT cell line. Moreover, when co-cultured with C2C12 mouse myotubes, the modified NG108-15 cells resulted in significantly larger acetylcholine receptor (AChR) aggregates, suggesting enhanced neuromuscular junction (NMJ) formation. These findings describe a novel methodology for differentiating NG108-15 cells more efficiently, to enhance the usefulness of the cell line as a motor neuron model. Full article

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion. Full article

Maternal obesity and/or high-fat diet (HF) consumption can disrupt appetite regulation in their offspring, contributing to transgenerational obesity and metabolic diseases. As fatty acids (FAs) play a role in appetite regulation, we investigated the maternal and fetal levels of FAs as potential contributors to programmed hyperphagia observed in the offspring of obese dams. Female mice were fed either a control diet (CT) or HF prior to mating, and fetal and maternal blood and tissues were collected at 19 days of gestation. Elevated levels of linoleic acid were observed in the serum of HF dams as well as in the serum of their fetuses. An increased concentration of eicosadienoic acid was also detected in the hypothalamus of female HF-O fetuses. HF-O male fetuses showed increased hypothalamic neuropeptide Y (Npy) gene expression, while HF-O female fetuses showed decreased hypothalamic pro-opiomelanocortin (POMC) protein content. Both male and female fetuses exhibited reduced hypothalamic neurogenin 3 (NGN-3) gene expression. In vitro experiments confirmed that LA contributed to the decreased gene expression of Pomc and Ngn-3 in neuronal cells. During lactation, HF female offspring consumed more milk and had a higher body weight compared to CT. In summary, this study demonstrated that exposure to HF prior to and during gestation alters the FA composition in maternal serum and fetal serum and hypothalamus, particularly increasing n-6, which may play a role in the switch from POMC to NPY neurons, leading to increased weight gain in the offspring during lactation. Full article

MicroRNAs are tissue-specific expressed short RNAs that serve post-transcriptional gene regulation. A specific microRNA can bind to mRNAs of different genes and thereby suppress their protein production. In the context of the complex phenotype of fibromyalgia, we used the Axiom miRNA Target Site Genotyping Array to search genome-wide for DNA variations in microRNA genes, their regulatory regions, and in the 3’UTR of protein-coding genes. To identify disease-relevant DNA polymorphisms, a cohort of 176 female fibromyalgia patients was studied in comparison to a cohort of 162 healthy women. The association between 48,329 markers and fibromyalgia was investigated using logistic regression adjusted for population stratification. Results show that 29 markers had p-values < 1 × 10⁻³, and the strongest association was observed for rs758459 (p-value of 0.0001), located in the Neurogenin 1 gene which is targeted by hsa-miR-130a-3p. Furthermore, variant rs2295963 is predicted to affect binding of hsa-miR-1-3p. Both microRNAs were previously reported to be differentially expressed in fibromyalgia patients. Despite its limited statistical power, this study reports two microRNA-related polymorphisms which may play a functional role in the pathogenesis of fibromyalgia. For a better understanding of the disease pattern, further functional analyses on the biological significance of microRNAs and microRNA-related polymorphisms are required. Full article

The trigeminal ganglion contains the cell bodies of sensory neurons comprising cranial nerve V, which relays information related to pain, touch, and temperature from the face and head to the brain. Like other cranial ganglia, the trigeminal ganglion is composed of neuronal derivatives of two critical embryonic cell types, neural crest and placode cells. Neurogenesis within the cranial ganglia is promoted by Neurogenin 2 (Neurog2), which is expressed in trigeminal placode cells and their neuronal derivatives, and transcriptionally activates neuronal differentiation genes such as Neuronal Differentiation 1 (NeuroD1). Little is known, however, about the role of Neurog2 and NeuroD1 during chick trigeminal gangliogenesis. To address this, we depleted Neurog2 and NeuroD1 from trigeminal placode cells with morpholinos and demonstrated that Neurog2 and NeuroD1 influence trigeminal ganglion development. While knockdown of both Neurog2 and NeuroD1 affected innervation of the eye, Neurog2 and NeuroD1 had opposite effects on ophthalmic nerve branch organization. Taken together, our results highlight, for the first time, functional roles for Neurog2 and NeuroD1 during chick trigeminal gangliogenesis. These studies shed new light on the molecular mechanisms underlying trigeminal ganglion formation and may also provide insight into general cranial gangliogenesis and diseases of the peripheral nervous system. Full article

In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy. Full article

Neurogenin 1 (Ngn1) belongs to the basic helix–loop–helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption. Full article

Human peripheral neuropathies are poorly understood, and the availability of experimental models limits further research. The PeriTox test uses immature dorsal root ganglia (DRG)-like neurons, derived from induced pluripotent stem cells (iPSC), to assess cell death and neurite damage. Here, we explored the suitability of matured peripheral neuron cultures for the detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A two-step differentiation protocol, involving the transient expression of ectopic neurogenin-1 (NGN1) allowed for the generation of homogeneous cultures of sensory neurons. After >38 days of differentiation, they showed a robust response (Ca²⁺-signaling) to the P2X3 ligand α,β-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at ≥5 nM, while 50–200 nM was required in the PeriTox test to identify neurite damage and cell death. A 24 h treatment with low nM concentrations of bortezomib led to moderate increases in resting cell intracellular Ca²⁺ concentration but signaling through transient receptor potential V1 (TRPV1) receptors or depolarization-triggered Ca²⁺ influx remained unaffected. We interpreted the specific attenuation of purinergic signaling as a functional cell stress response. A reorganization of tubulin to form dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomicin, and MG-132 showed similar stress responses. Thus, the model presented here may be used for the profiling of new proteasome inhibitors in regard to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signaling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons. Full article

The production of pancreatic β cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use. Full article

Type 2 diabetes is caused by impaired insulin secretion and/or insulin resistance. Loss of pancreatic β-cell mass detected in human diabetic patients has been considered to be a major cause of impaired insulin secretion. Additionally, apoptosis is found in pancreatic β-cells; β-cell mass loss is induced when cell death exceeds proliferation. Recently, however, β-cell dedifferentiation to pancreatic endocrine progenitor cells and β-cell transdifferentiation to α-cell was reported in human islets, which led to a new underlying molecular mechanism. Hyperglycemia inhibits nuclear translocation and expression of forkhead box-O1 (FoxO1) and induces the expression of neurogenin-3 (Ngn3), which is required for the development and maintenance of pancreatic endocrine progenitor cells. This new hypothesis (Foxology) is attracting attention because it explains molecular mechanism(s) underlying β-cell plasticity. The lineage tracing technique revealed that the contribution of dedifferentiation is higher than that of β-cell apoptosis retaining to β-cell mass loss. In addition, islet cells transdifferentiate each other, such as transdifferentiation of pancreatic β-cell to α-cell and vice versa. Islet cells can exhibit plasticity, and they may have the ability to redifferentiate into any cell type. This review describes recent findings in the dedifferentiation and transdifferentiation of β-cells. We outline novel treatment(s) for diabetes targeting islet cell plasticity. Full article

We investigated the effect of low molecular weight barley β-glucan (LMW-BG) on cecal fermentation, glucose, and lipid metabolism through comparisons to high molecular weight β-glucan (HMW-BG). C57BL/6J male mice were fed a moderate-fat diet for 61 days. LMW-BG or HMW-BG was added to the diet corresponding to 4% β-glucan. We measured the apparent absorption of fat, serum biomarkers, the expression levels of genes involved in glucose and lipid metabolism in the liver and ileum, and bacterial counts of the major microbiota groups using real time PCR. The concentration of short-chain fatty acids (SCFAs) in the cecum was analyzed by GC/MS. Significant reductions in serum leptin, total- and LDL-cholesterol concentrations, and mRNA expression levels of sterol regulatory element-binding protein-1c (SREBP-1c) were observed in both BG groups. HMW-BG specific effects were observed in inhibiting fat absorption and reducing abdominal deposit fat, whereas LMW-BG specific effects were observed in increasing bacterial counts of Bifidobacterium and Bacteroides and cecal total SCFAs, acetate, and propionate. mRNA expression of neurogenin 3 was increased in the LMW-BG group. We report that LMW-BG affects glucose and lipid metabolism via a prebiotic effect, whereas the high viscosity of HMW-BG in the digestive tract is responsible for its specific effects. Full article

Diabetes mellitus is a chronic metabolic disease, and its progression leads to serious complications. Although various novel therapeutic approaches for diabetes mellitus have developed in the last three decades, its prevalence has been rising more rapidly worldwide. Silk-related materials have been used as anti-diabetic remedies in Oriental medicine and many studies have shown the effects of silk fibroin (SF) in both in vitro and in vivo models. In our previous works, we reported that hydrolyzed SF improved the survival of HIT-T15 cells under high glucose conditions and ameliorated diabetic dyslipidemia in a mouse model. However, we could not provide a precise molecular mechanism. To further evaluate the functions of hydrolyzed SF on the pancreatic β-cell, we investigated the effects of hydrolyzed SF on the pancreatic β-cell proliferation and regeneration in the mouse model. Hydrolyzed SF induced the expression of the proliferating cell nuclear antigen (PCNA) and reduced the apoptotic cell population in the pancreatic islets. Hydrolyzed SF treatment not only induced the expression of transcription factors involved in the pancreatic β-cell regeneration in RT-PCR results but also increased neurogenin3 and Neuro D protein levels in the pancreas of those in the group treated with hydrolyzed SF. In line with this, hydrolyzed SF treatment generated insulin mRNA expressing small cell colonies in the pancreas. Therefore, our results suggest that the administration of hydrolyzed SF increases the pancreatic β-cell proliferation and regeneration in C57BL/KsJ-Leprdb/db mice. Full article

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies. Full article