Murine Astrocytes Research Articles

3D Bioprinted Neural‐Like Tissue as a Platform to Study Neurotropism of Mouse‐Adapted SARS‐CoV‐2 (Adv. Biology 8/2022)

Bioprinted Neural-Like Tissue In article 2200002, Maricato, Porcionatto, and co-workers use 3D bioprinting technology for the biofabrication of neural-like tissues composed of natural polymers and murine astrocytes and neurons. The mimetic neural tissue is infected with mouse-adapted SARS-CoV-2, showing permissiveness and specificity with the adapted virus, representing a proof-of-a-concept platform for modeling neuroinfection, contributing to the speed of antiviral drug screening.

Nuclear morphology is a deep learning biomarker of cellular senescence

Cellular senescence is an important factor in aging and many age-related diseases, but understanding its role in health is challenging due to the lack of exclusive or universal markers. Using neural networks, we predict senescence from the nuclear morphology of human fibroblasts with up to 95% accuracy, and investigate murine astrocytes, murine neurons, and fibroblasts with premature aging in culture. After generalizing our approach, the predictor recognizes higher rates of senescence in p21-positive and ethynyl-2’-deoxyuridine (EdU)-negative nuclei in tissues and shows an increasing rate of senescent cells with age in H&E-stained murine liver tissue and human dermal biopsies. Evaluating medical records reveals that higher rates of senescent cells correspond to decreased rates of malignant neoplasms and increased rates of osteoporosis, osteoarthritis, hypertension and cerebral infarction. In sum, we show that morphological alterations of the nucleus can serve as a deep learning predictor of senescence that is applicable across tissues and species and is associated with health outcomes in humans.

Dimethyl itaconate reprograms neurotoxic to neuroprotective primary astrocytes through the regulation of NLRP3 inflammasome and NRF2/HO-1 pathways

The activation of neurotoxic reactive astrocytes contributes to the pathogenesis of many neurodegenerative diseases. Itaconate, a product of cellular metabolism, is released from activated macrophage/microglia and has been shown to regulate inflammatory responses in several mammalian cells. This study was designed to investigate the impact of cell-permeable dimethyl itaconate (DI) on reactive astrocyte-dependent neurotoxicity. Primary murine astrocyte cells were isolated and stimulated with lipopolysaccharide (LPS) to generate reactive astrocytes. Treating these activated cells with DI was able to diminish the neurotoxic phenotype of reactive astrocytes, as we found reduced LPS-induced Nod-like receptor protein 3 (NLRP3) inflammasome activation and interleukin-1β (IL-1β) secretion. DI reduced the level of inflammasome components, attenuated inflammasome assembly and subsequently reduced caspase-1 cleavage and IL-1β levels. Additionally, DI attenuated nuclear factor-kappa B (NF-κB) phosphorylation in LPS-activated astrocytes and also protected astrocytes from LPS-induced cytotoxicity, including a lowering of Bax and caspase3. DI-treated reactive astrocytes showed an elevated GSH/GSSG ratio and improved antioxidant defense factors including catalase and superoxide dismutase, while lipid peroxidation was reduced. We found that DI activated the nuclear factor 2 (NRF2) and heme oxygenase-1 (HO-1) pathway in astrocytes and thereby potentially control redox-regulation and the inflammatory state of astrocytes. Collectively, these results indicate the neuroprotective role of DI by reprogramming astrocytes from neurotoxic A1 to neuroprotective A2 states and thereby reveal a novel potential strategy for the treatment of neurodegenerative diseases.

Z-DNA binding protein 1 mediates necroptotic and apoptotic cell death pathways in murine astrocytes following herpes simplex virus-1 infection

BackgroundThe mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined.MethodsWe have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each.ResultsWe show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia.ConclusionsOur findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.

CCN2/CTGF-A Modulator of the Optic Nerve Head Astrocyte.

In primary open-angle glaucoma (POAG), a neurodegenerative disease of the optic nerve (ON) and leading cause of blindness, the optic nerve head (ONH) undergoes marked structural extracellular matrix (ECM) changes, which contribute to its permanent deformation and to degeneration of ON axons. The remodeling process of the ECM causes changes in the biomechanical properties of the ONH and the peripapillary sclera, which is accompanied by an increased reactivity of the resident astrocytes. The molecular factors involved in the remodeling process belong to the Transforming growth factor (TGF)-β superfamily, especially TGF-β2. In previous publications we showed that TGF-β2 induced ECM alterations are mediated by Cellular Communication Network Factor (CCN)2/Connective Tissue Growth Factor (CTGF) and recently we showed that CCN2/CTGF is expressed by astrocytes of the ON under normal conditions. In this study we wanted to get a better understanding of the function of CCN2/CTGF under normal and pathologic conditions. To this end, we analyzed the glial lamina and peripapillary sclera of CCN2/CTGF overexpressing mice and studied the effect of CCN2/CTGF and increasing substratum stiffness on murine ON astrocytes in vitro. We observed enhanced astrocyte reactivity in the ONH, increased ECM protein synthesis in the peripapillary sclera and increased Ccn2/Ctgf expression in the ONH during the pathologic development in situ. CCN2/CTGF treatment of primary murine ON astrocytes induced a higher migration rate, and increase of ECM proteins including fibronectin, elastin and collagen type III. Furthermore, the astrocytes responded to stiffer substratum with increased glial fibrillary acidic protein, vimentin, actin and CCN2/CTGF synthesis. Finally, we observed the reinforced appearance of CCN2/CTGF in the lamina cribrosa of glaucomatous patients. We conclude that reactive changes in ONH astrocytes, induced by the altered biomechanical characteristics of the region, give rise to a self-amplifying process that includes increased TGF-β2/CCN2/CTGF signaling and leads to the synthesis of ECM molecules and cytoskeleton proteins, a process that in turn augments the stiffness at the ONH. Such a scenario may finally result in a vicious circle in the pathogenesis of POAG. The transgenic CTGF-overexpressing mouse model might be an optimal model to study the chronic pathological POAG changes in the ONH.

A Neuron, Microglia, and Astrocyte Triple Co-culture Model to Study Alzheimer's Disease.

Glial cells are essential to understand Alzheimer’s disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.

Ultrasound-targeted microbubble cavitation increases paracellular gaps in an in vitro blood brain barrier model

Background: Ultrasound-targeted microbubble cavitation (UTMC) can transiently open the blood brain barrier (BBB). We sought to determine the timeline of paracellular gap formation after UTMC in an in vitro model of the BBB. Methods: We utilized a transwell model with murine brain endothelial cells (EC) and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 10 μs duration, 10 ms pulse interval) at 250 kPa was applied to lipid microbubbles in contact with ECs for 20 s. Z-stacks of transwells acquired by confocal microscopy were converted to maximum intensity projections to quantify number and size of paracellular gaps (NIS-Elements). Endothelial barrier function was assessed using transendothelial electrical resistance (TEER). One-way ANOVA with post-hoc t-testing was performed. Results: Compared to no UTMC, there was an increase in the number and size of paraceullar gaps two minutes after UTMC that lasted at least 60 min (p < 0.05). The increase in gaps was associated with a decrease in TEER (p < 0.05). The number of gaps started to decrease 15 min after UTMC, and total gap area started to decrease 30 min after UTMC (p < 0.05). Conclusions: In our model of the BBB, UTMC induces endothelial hyperpermeability, which appears mediated by dynamic paracellular gap formation.

426 Long non-coding RNA WNT5A-AS1 shows sex-dimorphic differences in survival for males with glioblastoma

OBJECTIVES/GOALS: The goal of this study is to evaluate the role of WNT5A and WNT5a-AS1 in sex-differences of GBM progression. In our preliminary studies, we found that a long non-coding RNA WNT5A-AS1 is overexpressed in male GBM patients. We also found that WNT5A-AS1s expression shows a negative correlation with overall survival within male patients. METHODS/STUDY POPULATION: We will define the mechanism by which WNT5A-AS1 regulates WNT5a-mediated glioma stem cell (GSC) maintenance by assessing the effects of inhibiting WNT5A-AS1 expression on transcriptional activity and stemness in GSCs. We will determine if there are distinct Wnt-signaling patterns in male and female isogenic murine astrocytes by examining the expression of downstream proteins in the Wnt signaling pathway and how inhibition of WNT5A-AS1 alters this expression. We will then examine the impact of WNT5A-AS1 on temozolomide (TMZ) resistance in-vitro and in-vivo. We will assess the cell viability and survival of GBM PDX cells upon treatment with TMZ in vitro. Next, we will assess the capacity of knockdown of WNT5A-AS1 to increase sensitivity to TMZ-induced cell death and prolong survival in vivo in intracranial models. RESULTS/ANTICIPATED RESULTS: We hypothesize that WNT5A-AS1 targets Wnt5a and regulates its expression. We anticipate that knockdown of WNT5A-AS1 will upregulate WNT5A expression. We also expect that inhibiting WNT5A-AS1 will alter GSC stem maintenance and functional effects. We expect to see an increase in downstream Wnt5a signaling proteins in males vs females when treated with exogenous Wnt5a. We hypothesize that knockdown of both, WNT5A-AS1 and WNT5A will alter the expression of downstream proteins. We hypothesize that knockdown of WNT5A-AS1 will decrease tumor growth and therapeutic resistance to TMZ while increasing survival in patient derived xenographs in vivo and in vitro. DISCUSSION/SIGNIFICANCE: This study will provide insight into the mechanisms underlying the difference in GBM onset and progression between male and female patients, which is clinically important. We will also characterize the biological role WNT5A-AS1 which is currently unknown to date and elucidate differential role of GSCs in GBM progression between male and female.

Extracellular Cardiolipin Modulates Select Immune Functions of Astrocytes in Toll-Like Receptor (TLR) 4-Dependent Manner.

Alzheimer's disease (AD) is characterized by chronic neuroinflammation, which is partially mediated by dysregulated functions of glial cells. Cardiolipin (CL) is a phospholipid normally confined to the inner mitochondrial membrane; however, it has been detected in human sera, indicating that it can exist in the extracellular space where it may interact with nearby cells. Although CL has been shown to modulate several functions of microglia in a toll-like receptor (TLR) 4-dependent manner, the effects of extracellular CL on astrocytes are unknown. In addition to their homeostatic functions, astrocytes participate in neuroimmune responses of the brain and express TLR 4. Therefore, we hypothesized that extracellular CL (1) modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity, and (2) acts by interacting with astrocyte TLR 4. We demonstrate that CL inhibits the lipopolysaccharide- (LPS-) induced secretion of cytotoxins and expression of glial fibrillary acidic protein (GFAP) by human U118 MG astrocytic cells. CL alone upregulates the phagocytic activity of human astrocytic cells and primary murine astrocytes. CL in combination with LPS upregulates secretion of interleukin (IL)-1β by astrocytic cells. Furthermore, CL alone increases the secretion of monocyte chemoattractant protein (MCP)-1 by astrocytic cells, which is blocked by the TLR 4-specific antagonist TAK-242. We demonstrate that CL upregulates MCP-1 secretion in the absence of its natural carrier protein, β2-glycoprotein 1, indicating that CL may be bioactive in the brain where this protein is not present. Lastly, we show that CL downregulates the expression of astrocytic TLR 4, implying that CL engages this receptor, as its activation has been shown to lead to its degradation. Overall, our study extends the list of cell type functions of which CL modulates and provides evidence that CL, or liposomes containing this phospholipid can be used to modulate specific neuroimmune functions of astrocytes.

Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNF\u03b1-induced inflammatory responses

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 μM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.

The Involvement of RAGE and Its Ligands during Progression of ALS in SOD1 G93A Transgenic Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive degeneration of upper and lower motor neurons that causes paralysis and muscle atrophy. The pathogenesis of the disease is still not elucidated. Receptor for Advanced Glycation End Product (RAGE) is a major component of the innate immune system and has implications in ALS pathogenesis. Multiple studies suggest the role of RAGE and its ligands in ALS. RAGE and its ligands are overexpressed in human and murine ALS motor neurons, astrocytes, and microglia. Here, we demonstrated the expression of RAGE and its ligands during the progression of the disease in the transgenic SOD1 G93A mouse lumbar spinal cord. We observed the highest expression of HMGB1 and S100b proteins at ALS onset. Our results highlight the potential role of RAGE and its ligands in ALS pathogenesis and suggest that some of the RAGE ligands might be used as biomarkers in early ALS diagnosis and potentially be useful in targeted therapeutic interventions at the early stage of this devastating disease.

Astrocyte in prion disease: a double-edged sword.

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein (PrPC) into the pathologic isoform PrPSc. Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc, neuronal loss, spongiform degeneration and astrogliosis in the brain. Prion diseases affect human and animals, there is no effective therapy, and they invariably remain fatal. For a long time, neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis, and the contribution of non-neuronal cells like microglia and astrocytes was considered less important. Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain, but the exact role of these non-neuronal cells during prion pathology is still elusive. Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions. However, astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains. Additionally, sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified, differentially expressed in the brain during prion infection. Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes. Recently, we established a murine astrocyte cell line persistently infected with mouse-adapted prions, and showed how such astrocytes differentially process various prion strains. Considering the complexity of the role of astrocytes in prion pathogenesis, we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes, their differential regulation of signaling cascades, and the interaction with neurons and microglia during prion pathogenesis. This will help to establish novel in vivo models and define new therapeutic targets against prion diseases. In this review, we will discuss the complex role of astrocytes in prion disease, the existing experimental resources, the challenges to analyze the contribution of astrocytes in prion disease pathogenesis, and future strategies to improve the understanding of their role in prion disease.

Synthetic extracellular matrices and astrocytes provide a supportive microenvironment for the cultivation and investigation of primary pediatric gliomas.

Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patient-derived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Extracellular cardiolipin regulates select immune functions of astrocytes in a toll-like receptor (TLR) 4-dependent manner.

Cardiolipin (CL) is a mitochondrial phospholipid that may be released extracellularly from damaged or dying cells where it can act as a signaling molecule by interacting with the surrounding cells. Previous research demonstrates that extracellular CL regulates select immune functions of microglia in a toll-like receptor (TLR) 4-dependent manner. However, the effects of CL on astrocyte functions are unknown. Astrocytes are essential participants in diverse physiological brain functions; however, they become overactivated in chronic neuroinflammatory states, including Alzheimer's disease, contributing to damage and death of surrounding tissues and cells. Since astrocytes express TLR 4, we hypothesized that, by interacting with this receptor, extracellular CL modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity. We studied the effects of CL added to the culture media of primary murine astrocytes and human U118 MG astrocytic cells, which were used as astrocyte models. Fluorescent latex microspheres and synaptosomes were used to investigate the phagocytic activity of cells. The secretion of inflammatory cytokines was measured by the enzyme-linked immunosorbent assay or immunoblotting. U118 MG astrocyte supernatants were transferred to human SH-SY5Y neuronal cells to assess the secretion of cytotoxins. The expression of TLR 4 and glial fibrillary acidic protein (GFAP) by U118 MG astrocytic cells was quantified by immunostaining and immunoblotting, respectively. Extracellular CL alone upregulated the phagocytic activity of human U118 MG astrocytic cells and their secretion of monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-β. Extracellular CL upregulated the phagocytic activity of primary murine astrocytes. TLR 4-specific antagonist TAK-242 inhibited CL-induced secretion of MCP-1 by human U118 MG cells. CL alone upregulated the cell-surface expression of TLR 4 by the same cell type. Extracellular CL inhibited the lipopolysaccharide (LPS)-induced increase in GFAP expression as well as the secretion of cytotoxins by U118 MG astrocytic cells. Extracellular CL may be used to modulate select functions of astrocytes, which become dysregulated in chronic neuroinflammatory states. Therefore, CL, or liposomes containing this phospholipid, could be used to ameliorate the chronic neuroinflammation and adverse astrocyte activation associated with Alzheimer's disease.

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes

BackgroundThe crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. New methodWe modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. ResultsWe generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. Comparison with existing method(s)Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99–100%). ConclusionsWe have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

Transcriptome Profile During Rabies Virus Infection: Identification of Human CXCL16 as a Potential New Viral Target.

Rabies virus (RABV), the causative agent for rabies disease is still presenting a major public health concern causing approximately 60,000 deaths annually. This neurotropic virus (genus Lyssavirus, family Rhabdoviridae) induces an acute and almost always fatal form of encephalomyelitis in humans. Despite the lethal consequences associated with clinical symptoms of rabies, RABV limits neuro-inflammation without causing major histopathological lesions in humans. Nevertheless, information about the mechanisms of infection and cellular response in the central nervous system (CNS) remain scarce. Here, we investigated the expression of inflammatory genes involved in immune response to RABV (dog-adapted strain Tha) in mice, the most common animal model used to study rabies. To better elucidate the pathophysiological mechanisms during natural RABV infection, we compared the inflammatory transcriptome profile observed at the late stage of infection in the mouse brain (cortex and brain stem/cerebellum) with the ortholog gene expression in post-mortem brain biopsies of rabid patients. Our data indicate that the inflammatory response associated with rabies is more pronounced in the murine brain compared to the human brain. In contrast to murine transcription profiles, we identified CXC motif chemokine ligand 16 (CXCL16) as the only significant differentially expressed gene in post-mortem brains of rabid patients. This result was confirmed in vitro, in which Tha suppressed interferon alpha (IFN-α)-induced CXCL16 expression in human CNS cell lines but induced CXCL16 expression in IFN-α-stimulated murine astrocytes. We hypothesize that RABV-induced modulation of the CXCL16 pathway in the brain possibly affects neurotransmission, natural killer (NK) and T cell recruitment and activation. Overall, we show species-specific differences in the inflammatory response of the brain, highlighted the importance of understanding the potential limitations of extrapolating data from animal models to humans.

Self-extracellular RNA promotes pro-inflammatory response of astrocytes to exogenous and endogenous danger signals

ObjectiveAstrocytes participate in the local innate immune response of the central nervous system. In response to stress such as ischemia, activated cells release endogenous factors known as damage-associated molecular patterns (DAMPs). Self-extracellular RNA (eRNA) is such a ubiquitous alarm signal. However, it is unclear whether eRNA is involved in the early acute phase of cerebral ischemia and is sufficient to sensitize astrocytes towards a DAMP or PAMP (pathogen-associated molecular pattern) reaction.MethodsPro-inflammatory activation upon eRNA stimulation was characterized in primary murine astrocyte cultures. In vivo, an experimental stroke model was used to localize and quantify eRNA in murine brain sections. Using primary cortical neurons and the mouse hippocampal neuronal cell line HT-22, neuronal RNA release upon stress conditions related to cerebral hypoxia/ischemia was analyzed.ResultsWhile low-dose eRNA alone did not promote pro-inflammatory activation of astrocytes in culture, it strongly enhanced the expression of pro-inflammatory cytokines in the presence of either Pam2CSK4, a synthetic PAMP molecule that mimics bacterial infection, or high mobility group box 1 (HMGB1), a prominent DAMP. Synergism of eRNA/Pam2CSK4 and eRNA/HMGB1 was prevented by blockage of the astroglial toll-like receptor (TLR)-2. Inhibition of NF-κB- and mitogen-activated protein kinase-dependent signaling pathways hampered eRNA/Pam2CSK4-mediated pro-inflammatory activation of astrocytes. In vivo, the amount of non-nuclear, presumably extracellular ribosomal RNA in close proximity to neurons significantly accumulated across the infarct core and peri-infarct areas that was accompanied by transcriptional up-regulation of various pro-inflammatory factors. Accordingly, the exposure of neurons to hypoxic/ischemic stress in vitro resulted in the release of eRNA, partly mediated by active cellular processes dependent on the cytosolic calcium level.ConclusionThe DAMP signal eRNA can sensitize astrocytes as active players in cerebral innate immunity towards exogenous and endogenous activators of inflammation (PAMPs and DAMPs) in a synergistic manner via TLR2-NF-κB-dependent signaling mechanisms. These findings provide new insights into the pathogenesis of ischemic stroke and other inflammatory neurological disorders. Further studies will clarify whether administration of RNase in vivo may serve as an effective treatment for inflammatory brain pathologies.

A novel phosphoproteomic landscape evoked in response to type I interferon in the brain and in glial cells

BackgroundType I interferons (IFN-I) are key responders to central nervous system infection and injury and are also increased in common neurodegenerative diseases. Their effects are primarily mediated via transcriptional regulation of several hundred interferon-regulated genes. In addition, IFN-I activate several kinases including members of the MAPK and PI3K families. Yet, how changes to the global protein phosphoproteome contribute to the cellular response to IFN-I is unknown.MethodsThe cerebral phosphoproteome of mice with brain-targeted chronic production of the IFN-I, IFN-α, was obtained. Changes in phosphorylation were analyzed by ontology and pathway analysis and kinase enrichment predictions. These were verified by phenotypic analysis, immunohistochemistry and immunoblots. In addition, primary murine microglia and astrocytes, the brain's primary IFN-I-responding cells, were acutely treated with IFN-α and the global phosphoproteome was similarly analyzed.ResultsWe identified widespread protein phosphorylation as a novel mechanism by which IFN-I mediate their effects. In our mouse model for IFN-I-induced neurodegeneration, protein phosphorylation, rather than the proteome, aligned with the clinical hallmarks and pathological outcome, including impaired development, motor dysfunction and seizures. In vitro experiments revealed extensive and rapid IFN-I-induced protein phosphorylation in microglia and astrocytes. Response to acute IFN-I stimulation was independent of gene expression and mediated by a small number of kinase families. The changes in the phosphoproteome affected a diverse range of cellular processes and functional analysis suggested that this response induced an immediate reactive state and prepared cells for subsequent transcriptional responses.ConclusionsOur studies reveal a hitherto unappreciated role for changes in the protein phosphorylation landscape in cellular responses to IFN-I and thus provide insights for novel diagnostic and therapeutic strategies for neurological diseases caused by IFN-I.

Neuroprotective and anti-inflammatory effect of pectolinarigenin, a flavonoid from Amazonian Aegiphila integrifolia (Jacq.), against lipopolysaccharide-induced inflammation in astrocytes via NFκB and MAPK pathways

Neurodegenerative diseases affect millions of people worldwide. Regardless of the underlying cause, neuroinflammation is the greatest risk factor for developing any of these disorders. Pectolinarigenin (PNG) is an active flavonoid with several biological properties, anti-metastatic and anti-inflammatory activity. This study investigate the biological effects of PNG in macrophage and astrocyte cultures, with focus on elucidating the molecular mechanisms involved in the PNG activity. J774A.1 murine macrophage or cerebral cortex primary astrocytes primary cultures were treated with different concentration of PNG (1–160 μM) and the inflammatory process was stimulated by LPS (1 μg/ml) and the effect of PNG in different inflammatory markers were determined. PNG did not affect astrocyte or macrophage viability. Moreover, this flavonoid reduced NO• release in macrophages, attenuated astrocyte activation by preventing the overexpression of glial fibrillary acidic protein, and decreased the release of inflammatory mediators, IL-1β and IL-6 induced by LPS by the glial cell, as well as enhanced basal levels of IL-10. In addition, PNG suppressed NFκB, p38MAPK and ERK1/2 phosphorylation in astrocytes culture induced by LPS. The results show clear evidence that this novel flavonoid protects astrocytes against LPS-induced inflammatory toxicity. In conclusion, PNG presents neuroprotective and anti-inflammatory property through the inhibition of inflammatory signaling pathways.