Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

Poly(rC)-binding protein 1 alleviates neurotoxicity in 6-OHDA-induced SH-SY5Y cells and modulates glial cells in neuroinflammation.

BACKGROUND: Parkinson's disease (PD) is a debilitating neurodegenerative condition characterized by the loss of dopaminergic neurons and neuroinflammation. Previous research has identified the involvement of Poly (rC)-binding protein 1 (PCBP1) in certain degenerative diseases; however, its specific mechanisms in PD remain incompletely understood. METHODS: In this study, 6-OHDA-induced neurotoxicity in the cell lines SH-SY5Y, BV-2 and HA, was used to evaluate the protective effects of PCBP1. We assessed alterations in BDNF levels in SY5Y cells, changes in GDNF expression in glial cells, as well as variations in HSP70 and NF-κB activation. Additionally, glial cells were used as the in vitro model for neuroinflammation mechanisms. RESULTS: The results indicate that the overexpression of PCBP1 significantly enhances cell growth compared to the control plasmid pEGFP/N1 group. Overexpression of PCBP1 leads to a substantial reduction in early apoptosis rates in SH-SY5Y, HA, and BV-2 cells, with statistically significant differences (p < 0.05). Furthermore, the overexpression of PCBP1 in cells results in a marked increase in the expression of HSP70, GDNF, and BDNF, while reducing NF-κB expression. Additionally, in SH-SY5Y, HA, and BV-2 cells overexpressing PCBP1, there is a decrease in the inflammatory factor IL-6 compared to the control plasmid pEGFP/N1 group, while BV-2 cells exhibit a significant increase in the anti-inflammatory factor IL-10. CONCLUSION: Our findings suggest that PCBP1 plays a substantial role in promoting cell growth and modulating the balance of neuroprotective and inflammatory factors. These results offer valuable insights into the potential therapeutic utility of PCBP1 in mitigating neuroinflammation and enhancing neuronal survival in PD.

Retinoic acid attenuates ischemic injury-induced activation of glial cells and inflammatory factors in a rat stroke model.

Stroke is a leading cause of death and long-term disability which can cause oxidative damage and inflammation of the neuronal cells. Retinoic acid is an active metabolite of vitamin A that has various beneficial effects including antioxidant and anti-inflammatory effects. In this study, we investigated whether retinoic acid modulates oxidative stress and inflammatory factors in a stroke animal model. A middle cerebral artery occlusion (MCAO) was performed on adult male rats to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the peritoneal cavity for four days before MCAO surgery. The neurobehavioral tests were carried out 24 h after MCAO and cerebral cortex tissues were collected. The cortical damage was assessed by hematoxylin-eosin staining and reactive oxygen species assay. In addition, Western blot and immunohistochemical staining were performed to investigate the activation of glial cells and inflammatory cytokines in MCAO animals. Ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used as markers of microglial and astrocyte activation, respectively. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were used as representative pro-inflammatory cytokines. Results showed that MCAO damage caused neurobehavioral defects and histopathological changes in the ischemic region and increased oxidative stress. Retinoic acid treatment reduced these changes caused by MCAO damage. We detected increases in Iba-1 and GFAP in MCAO animals treated with vehicle. However, retinoic acid alleviated increases in Iba-1 and GFAP caused by MCAO damage. Moreover, MCAO increased levels of nuclear factor-κB and pro-inflammatory cytokines, including TNF-α and IL-1ß. Retinoic acid alleviated the expression of these inflammatory proteins. These findings elucidate that retinoic acid regulates microglia and astrocyte activation and modulates pro-inflammatory cytokines. Therefore, this study suggests that retinoic acid exhibits strong antioxidant and anti-inflammatory properties by reducing oxidative stress, inhibiting neuroglia cell activation, and preventing the increase of pro-inflammatory cytokines in a cerebral ischemia.

Cerebellar granule cell migration and folia development require Mllt11/Af1q/Tcf7c.

The organization of neurons into distinct layers, known as lamination, is a common feature of the nervous system. This process, which arises from the direct coupling of neurogenesis and neuronal migration, plays a crucial role in the development of the cerebellum, a structure exhibiting a distinct folding cytoarchitecture with cells arranged in discrete layers. Disruptions to neuronal migration can lead to various neurodevelopmental disorders, highlighting the significance of understanding the molecular regulation of lamination. We report a role Mllt11/Af1q/Tcf7c (myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 fused gene from chromosome 1q, also known as Mllt11 transcriptional cofactor 7; henceforth referred to Mllt11) in the migration of cerebellar granule cells (GCs). We now show that Mllt11 plays a role in both the tangential and radial migration of GCs. Loss of Mllt11 led to an accumulation of GC precursors in the rhombic lip region and a reduction in the number of GCs successfully populating developing folia. Consequently, this results in smaller folia and an overall reduction in cerebellar size. Furthermore, analysis of the anchoring centers reveals disruptions in the perinatal folia cytoarchitecture, including alterations in the Bergmann glia fiber orientation and reduced infolding of the Purkinje cell plate. Lastly, we demonstrate that Mllt11 interacts with non-muscle myosin IIB (NMIIB) and Mllt11 loss-reduced NMIIB expression. We propose that the dysregulation of NMIIB underlies altered GC migratory behavior. Taken together, the findings reported herein demonstrate a role for Mllt11 in regulating neuronal migration within the developing cerebellum, which is necessary for its proper neuroanatomical organization.

The interaction of lipocalin-2 and astrocytes in neuroinflammation: mechanisms and therapeutic application.

Neuroinflammation is a common pathological process in various neurological disorders, including stroke, Alzheimer's disease, Parkinson's disease, and others. It involves the activation of glial cells, particularly astrocytes, and the release of inflammatory mediators. Lipocalin-2 (Lcn-2) is a secretory protein mainly secreted by activated astrocytes, which can affect neuroinflammation through various pathways. It can also act as a pro-inflammatory factor by modulating astrocyte activation and polarization through different signaling pathways, such as NF-κB, and JAK-STAT, amplifying the inflammatory response and aggravating neural injury. Consequently, Lcn-2 and astrocytes may be potential therapeutic targets for neuroinflammation and related diseases. This review summarizes the current knowledge on the role mechanisms, interactions, and therapeutic implications of Lcn-2 and astrocytes in neuroinflammation.

The protective barrier role of satellite glial cells in sensory ganglia.

Neurons in sensory ganglia are wrapped completely by satellite glial cells (SGCs). One putative function of SGCs is to regulate the neuronal microenvironment, but this role has received only little attention. In this study we investigated whether the SGC envelope serves a barrier function and how SGCs may control the neuronal microenvironment. We studied this question on short-term (<24 h) cell cultures of dorsal root ganglia and trigeminal ganglia from adult mice, which contain neurons surrounded with SGCs, and neurons that are not. Using calcium imaging, we measured neuronal responses to molecules with established actions on sensory neurons. We found that neurons surrounded by SGCs had a smaller response to molecules such as adenosine triphosphate (ATP), glutamate, GABA, and bradykinin than neurons without glial cover. When we inhibited the activity of NTPDases, which hydrolyze the ATP, and also when we inhibited the glutamate and GABA transporters on SGCs, this difference in the neuronal response was no longer observed. We conclude that the SGC envelope does not hinder diffusional passage, but acts as a metabolic barrier that regulates the neuronal microenvironment, and can protect the neurons and modulate their activity.

HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.

Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 109, 8.46 × 109, 1.269 × 1010, 1.692 × 1010, 2.115 × 1010, and 2.538 × 1010 gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.

Glial Cx43 hemichannels and neuronal Panx1 hemichannels and P2X7 receptors orchestrate presynaptic homeostatic plasticity.

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.

Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

BACKGROUND: The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. METHODS: Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. RESULTS: STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. CONCLUSIONS: These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Identifying Hmga2 preserving visual function by promoting a shift of Müller glia cell fate in mice with acute retinal injury.

BACKGROUND: Unlike in lower vertebrates, Müller glia (MG) in adult mammalian retinas lack the ability to reprogram into neurons after retinal injury or degeneration and exhibit reactive gliosis instead. Whether a transition in MG cell fate from gliosis to reprogramming would help preserve photoreceptors is still under exploration. METHODS: A mouse model of retinitis pigmentosa (RP) was established using MG cell lineage tracing mice by intraperitoneal injection of sodium iodate (SI). The critical time point for the fate determination of MG gliosis was determined through immunohistochemical staining methods. Then, bulk-RNA and single-cell RNA seq techniques were used to elucidate the changes in RNA transcription of the retina and MG at that time point, and new genes that may determine the fate transition of MG were screened. Finally, the selected gene was specifically overexpressed in MG cells through adeno-associated viruses (AAV) in the mouse RP model. Bulk-RNA seq technique, immunohistochemical staining methods, and visual function testing were used to elucidate and validate the mechanism of new genes function on MG cell fate transition and retinal function. RESULTS: Here, we found the critical time point for MG gliosis fate determination was 3 days post SI injection. Hmga2 was screened out as a candidate regulator for the cell fate transition of MG. After retinal injury caused by SI, the Hmga2 protein is temporarily and lowly expressed in MG cells. Overexpression of Hmga2 in MG down-regulated glial cell related genes and up-regulated photoreceptor related genes. Besides, overexpressing Hmga2 exclusively to MG reduced MG gliosis, made MG obtain cone's marker, and retained visual function in mice with acute retinal injury. CONCLUSION: Our results suggested the unique reprogramming properties of Hmga2 in regulating the fate transition of MG and neuroprotective effects on the retina with acute injury. This work uncovers the reprogramming ability of epigenetic factors in MG.

Resveratrol Alleviates the Early Challenges of Implant-Based Drug Delivery in a Human Glial Cell Model.

Brain diseases are oftentimes life-threatening and difficult to treat. The local administration of drug substances using brain implants can increase on-site concentrations and decrease systemic side effects. However, the biocompatibility of potential brain implant materials needs to be evaluated carefully as implants can trigger foreign body reactions, particularly by increasing the microglia and astrocyte reactivity. To date, these tests have been frequently conducted in very simple in vitro models, in particular not respecting the key players in glial cell reactions and the challenges of surgical implantation characterized by the disruption of oxygen and nutrient supply. Thus, we established an in vitro model in which we treated human glial cell lines with reduced oxygen and glucose levels. The model displayed cytokine and reactive oxygen species release from reactive microglia and an increase in a marker of reactive astrocytes, galectin-3. Moreover, the treatment caused changes in the cell survival and triggered the production of hypoxia-inducible factor 1α. In this comprehensive platform, we demonstrated the protective effect of the natural polyphenol resveratrol as a model substance, which might be included in brain implants to ease the undesired glial cell response. Overall, a glial-cell-based in vitro model of the initial challenges of local brain disease treatment may prove useful for investigating new therapy options.

Effect of Inducers and Inhibitors of the Keap1/Nrf2/ARE System on the Viability and Functional Activity of Model Neuronal-Like and Glial Cells.

On mouse neuroblastoma (Neuro-2a) and human glioblastoma (U-87 MG) cell lines, we studied the effect of inducers and inhibitors of redox-sensitive signaling system of the antioxidant-responsive element Keap1/Nrf2/ARE on the main processes that determine nerve cell viability and vital activity (proliferative activity, apoptosis, autophagy, and activation of the Keap1/Nrf2/ARE system). Inhibitors of the Keap1/Nrf2/ARE system stimulate apoptosis more pronouncedly than inducers, have a weaker effect on autophagy, and do not change the nuclear to cytoplasmic Nrf2 ratio. In general, the revealed effects testify in favor of the potential effectiveness of stimulating the Keap1/Nrf2/ARE system for the prevention and adjuvant therapy of neurodegenerative diseases.

ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials.

BACKGROUND: As the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell-cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation. RESULTS: After the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation. CONCLUSION: Despite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.

Vasoactive Intestinal Polypeptide Plays a Key Role in the Microbial-Neuroimmune Control of Intestinal Motility.

BACKGROUND & AIMS: Although chronic diarrhea and constipation are common, the treatment is symptomatic because their pathophysiology is poorly understood. Accumulating evidence suggests that the microbiota modulates gut function, but the underlying mechanisms are unknown. We therefore investigated the pathways by which microbiota modulates gastrointestinal motility in different sections of the alimentary tract. METHODS: Gastric emptying, intestinal transit, muscle contractility, acetylcholine release, gene expression, and vasoactive intestinal polypeptide (VIP) immunoreactivity were assessed in wild-type and Myd88-/-Trif-/- mice in germ-free, gnotobiotic, and specific pathogen-free conditions. Effects of transient colonization and antimicrobials as well as immune cell blockade were investigated. VIP levels were assessed in human full-thickness biopsies by Western blot. RESULTS: Germ-free mice had similar gastric emptying but slower intestinal transit compared with specific pathogen-free mice or mice monocolonized with Lactobacillus rhamnosus or Escherichia coli, the latter having stronger effects. Although muscle contractility was unaffected, its neural control was modulated by microbiota by up-regulating jejunal VIP, which co-localized with and controlled cholinergic nerve function. This process was responsive to changes in the microbial composition and load and mediated through toll-like receptor signaling, with enteric glia cells playing a key role. Jejunal VIP was lower in patients with chronic intestinal pseudo-obstruction compared with control subjects. CONCLUSIONS: Microbial control of gastrointestinal motility is both region- and bacteria-specific; it reacts to environmental changes and is mediated by innate immunity-neural system interactions. By regulating cholinergic nerves, small intestinal VIP plays a key role in this process, thus providing a new therapeutic target for patients with motility disorders.

Cut homeodomain transcription factor is a novel regulator of growth and morphogenesis of cortex glia niche around neural cells.

Cortex glia in Drosophila central nervous system form a niche around neural cells for necessary signals to establish cross talk with their surroundings. These cells grow and expand their thin processes around neural cell bodies. Although essential for the development and function of the nervous system, how these cells make extensive and intricate connected networks remains largely unknown. In this study, we show that Cut, a homeodomain transcription factor, directly regulates the fate of the cortex glia, impacting neural stem cell (NSC) homeostasis. Focusing on the thoracic ventral nerve cord, we found that Cut is required for the normal growth and development of cortex glia and timely increase in DNA content through endocycle to later divide via acytokinetic mitosis. Knockdown of Cut in cortex glia significantly reduces the growth of cellular processes, the network around NSCs, and their progeny's cell bodies. Conversely, overexpression of Cut induces overall growth of the main processes at the expense of side ones. Whereas the Cut knockdown slows down the timely increase of DNA, the Cut overexpression results in a significant increase in nuclear size and volume and a 3-fold increase in DNA content of cortex glia. Further, we note that constitutively high Cut also interfered with nuclei separation during acytokinetic mitosis. Since the cortex glia form syncytial networks around neural cells, the finding identifies Cut as a novel regulator of glial growth and variant cell cycles to support a functional nervous system.

Flavonoids and ischemic stroke-induced neuroinflammation: Focus on the glial cells.

Ischemic stroke is one of the most cases worldwide, with high rate of morbidity and mortality. In the pathological process of ischemic stroke, neuroinflammation is an essential process that defines the functional prognosis. After stroke onset, microglia, astrocytes and the infiltrating immune cells contribute to a complicated neuroinflammation cascade and play the complicated roles in the pathophysiological variations of ischemic stroke. Both microglia and astrocytes undergo both morphological and functional changes, thereby deeply participate in the neuronal inflammation via releasing pro-inflammatory or anti-inflammatory factors. Flavonoids are plant-specific secondary metabolites and can protect against cerebral ischemia injury via modulating the inflammatory responses. For instances, quercetin can inhibit the expression and release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-6 and IL-1ß, in the cerebral nervous system (CNS). Apigenin and rutin can promote the polarization of microglia to anti-inflammatory genotype and then inhibit neuroinflammation. In this review, we focused on the dual roles of activated microglia and reactive astrocyte in the neuroinflammation following ischemic stroke and discussed the anti-neuroinflammation of some flavonoids. Importantly, we aimed to reveal the new strategies for alleviating the cerebral ischemic stroke.