Astrocyte Cell Research Articles

Expression patterns of astrocyte mRNA under numerous in vitro disease conditions and its applicability in drug development

AbstractBackgroundReactive astrocytes have been observed surrounding amyloid beta (Aβ) plaques in Alzheimer’s disease (AD) patients and animal models. Researchers have pointed out the shortcomings of A1 and A2 classification. Our study aims to discuss the potentials of applying A1 and A2 genes in indicating astrocyte states and explore the relationship between inflammation‐related protein levels and A1 and A2 expression map under the treatment of human Aβ1‐42 oligomer (hAβ1‐42O) and compound TBN.MethodThis study was conducted using murine astrocyte cell line (C8‐D1A). We compared the mRNAs levels (A1 and A2) under numerous in vitro disease conditions, including lipopolysaccharide (LPS) (800ng/ml), LPS‐induced microglial secretion, oxygen‐glucose deprivation (OGD), hAβ1‐42O (2.5μM), low glucose (11mM), high glucose (44mM) treatment. We also compared mRNA levels (cerebellum, cortex, and hippocampi) in high dose STZ (ip.) (150mg/kg, 4 weeks)‐induced C5BL/6 mouse brain, which is usually applied to as AD model. Furthermore, we explored the relationship between the A1, A2 expression map and inflammation‐related protein expression levels in C8‐D1A treated with hAβ1‐42O and TBN.ResultsA1 and A2 genes were altered in different patterns under numerous stimuli. Microglial secretion (LPS), LPS, OGD, hAβ1‐42O mostly induced mRNA upregulation, while abnormal glucose induced hypofunction of most genes. In STZ brain, mRNA expression exhibited district‐dependent differences. hippocampi mRNA were mostly activated compared to cerebellum and cortex, which indicated that Aβ plaque closely relates to astrocyte activation. In vitro, hAβ1‐42O upregulates inflammation related signal proteins. TBN inhibited hAβ1‐42O induced mRNA upregulation (A1, A2, C3, Il6, Tnfα, Tgfβ) and protein secretion (Il6, Tnfα). TBN could also inhibit LPS‐induced inflammation in astrocytes and increase mRNA levels of Aβ degrading enzymes (Ide, Ece‐1) .ConclusionsIt seems that there is not a strict paradigm of A1 and A2 mRNA expression to indicate astrocyte state and mRNA upregulation is not strictly related to detrimental effects. TBN exerted anti‐inflammatory effects may increase Aβ degrading activity. It also ameliorated inflammation induced by hAβ1‐42O, which may indicate the therapeutical potentials of targeting the activation of mRNA (S1pr3, Timp1, Hsbp1, Cxcl10, Osmr, Cp, Ligp1, Fkbp5, S100a10, slc10a6, B3gnt5) to ameliorate hAβ1‐42O‐induced astrocytic inflammation.Cp, Ligp1, Fkbp5, S100a10, slc10a6, B3gnt5).

How does amyloid beta alter levels of ınflammation parameters in human astrocyte culture?

AbstractBackgroundIn neurodegenerative diseases, specific neuron types and glial cells undergo degeneration leading to the appearance of specific disease symptoms (1). Neurodegenerative diseases in the central nervous system (CNS) are also highly effective on cells that support neuron life. Astrocytes are dynamic support cells that maintain brain balance, regulate the neurotransmitter system, process synaptic information, energy metabolism, antioxidant system, and inflammatory response (2). The blood‐brain barrier (BBB) may lose its permeability in pathological conditions. Both endothelial cells and astrocytes undergo phenotypic changes and become reactive (3). In Alzheimer’s disease (AD), amyloid beta (Aβ) is suggested to induce a pathological pathway that leads to neurodegeneration and dementia (4). A limited number of studies in the literature reveal a general profile of the neuroinflammatory mediators induced by Aβ, one of the main pathological elements of AD in human astrocytes.MethodBased on this information, AD‐like pathology was created in our study with Aβ1‐42 treatment in human astrocyte cultures. The inflammatory response of these pathologies in human astrocytes was compared with the profile that emerges differently from a healthy astrocyte population. Alterations in the levels of released inflammatory proteins and their receptors were detected with the chemiluminescence‐based Western/dot blot method by using the human inflammation antibody array (Ray Biotech C‐Series Human Antibody Inflammation Array C4).ResultOur data show that Aβ1‐42 treatment predominantly releases IL‐6, MCP‐1, and TIMP‐2 proteins into the culture supernatant in human astrocyte cells. On the other hand, whether reactive astrogliosis was triggered in the presence of Aβ in human astrocytes was followed by immunofluorescent labeling (IF).ConclusionThis is the first study that investigated the general profile of released inflammatory factors in human astrocytes and how this profile is altered by Aβ1‐42 treatment.(The present work was supported by the Research Fund of Istanbul University, Project no: 30666)

GFAP palmitoylcation mediated by ZDHHC23 in spinal astrocytes contributes to the development of neuropathic pain

BackgroundCancer pain has a significant impact on patient’s quality of life. Astrocytes play an important role in cancer pain signaling. The direct targeting of astrocytes can effectively suppress cancer pain,...

Extracellular Vesicles and Their Renin-Angiotensin Cargo as a Link between Metabolic Syndrome and Parkinson's Disease.

Several studies showed an association between metabolic syndrome (MetS) and Parkinson's disease (PD). The linking mechanisms remain unclear. MetS promotes low-grade peripheral oxidative stress and inflammation and dysregulation of the adipose renin-angiotensin system (RAS). Interestingly, brain RAS dysregulation is involved in the progression of dopaminergic degeneration and PD. Circulating extracellular vesicles (EVs) from MetS fat tissue can cross the brain-blood barrier and may act as linking signals. We isolated and characterized EVs from MetS and control rats and analyzed their mRNA and protein cargo using RT-PCR and the ExoView R200 platform, respectively. Furthermore, cultures of the N27 dopaminergic cell line and the C6 astrocytic cell line were treated with EVs from MetS rats. EVs were highly increased in MetS rat serum, which was inhibited by treatment of the rats with the angiotensin type-1-receptor blocker candesartan. Furthermore, EVs from MetS rats showed increased pro-oxidative/pro-inflammatory and decreased anti-oxidative/anti-inflammatory RAS components, which were inhibited in candesartan-treated MetS rats. In cultures, EVs from MetS rats increased N27 cell death and modulated C6 cell function, upregulating markers of neuroinflammation and oxidative stress, which were inhibited by the pre-treatment of cultures with candesartan. The results from rat models suggest EVs and their RAS cargo as a mechanism linking Mets and PD.

Restoring periodontal tissue homoeostasis prevents cognitive decline by reducing the number of Serpina3nhigh astrocytes in the hippocampus

Cognitive decline has been linked to periodontitis through an undetermined pathophysiological mechanism. This study aimed to explore the mechanism underlying periodontitis-related cognitive decline and identify therapeutic strategies for this condition. Using single-nucleus RNA sequencing we found that changes in astrocyte number, gene expression, and cell‒cell communication were associated with cognitive decline in mice with periodontitis. In addition, activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was observed to decrease the phagocytic capability of macrophages and reprogram macrophages to a more proinflammatory state in the gingiva, thus aggravating periodontitis. To further investigate this finding, lipid-based nanoparticles carrying NLRP3 siRNA (NPsiNLRP3) were used to inhibit overactivation of the NLRP3 inflammasome in gingival macrophages, restoring the oral microbiome and reducing periodontal inflammation. Furthermore, gingival injection of NPsiNLRP3 reduced the number of Serpina3nhigh astrocytes in the hippocampus and prevented cognitive decline. This study provides a functional basis for the mechanism by which the destruction of periodontal tissues can worsen cognitive decline and identifies nanoparticle-mediated restoration of gingival macrophage function as a novel treatment for periodontitis-related cognitive decline.

Competing endogenous RNAs in human astrocytes: crosstalk and interacting networks in response to lipotoxicity.

Neurodegenerative diseases (NDs) are characterized by a progressive deterioration of neuronal function, leading to motor and cognitive damage in patients. Astrocytes are essential for maintaining brain homeostasis, and their functional impairment is increasingly recognized as central to the etiology of various NDs. Such impairment can be induced by toxic insults with palmitic acid (PA), a common fatty acid, that disrupts autophagy, increases reactive oxygen species, and triggers inflammation. Although the effects of PA on astrocytes have been addressed, most aspects of the dynamics of this fatty acid remain unknown. Additionally, there is still no model that satisfactorily explains how astroglia goes from being neuroprotective to neurotoxic. Current incomplete knowledge needs to be improved by the growing field of non-coding RNAs (ncRNAs), which is proven to be related to NDs, where the complexity of the interactions among these molecules and how they control other RNA expressions need to be addressed. In the present study, we present an extensive competing endogenous RNA (ceRNA) network using transcriptomic data from normal human astrocyte (NHA) cells exposed to PA lipotoxic conditions and experimentally validated data on ncRNA interaction. The obtained network contains 7 lncRNA transcripts, 38 miRNAs, and 239 mRNAs that showed enrichment in ND-related processes, such as fatty acid metabolism and biosynthesis, FoxO and TGF-β signaling pathways, prion diseases, apoptosis, and immune-related pathways. In addition, the transcriptomic profile was used to propose 22 potential key controllers lncRNA/miRNA/mRNA axes in ND mechanisms. The relevance of five of these axes was corroborated by the miRNA expression data obtained in other studies. MEG3 (ENST00000398461)/hsa-let-7d-5p/ATF6B axis showed importance in Parkinson's and late Alzheimer's diseases, while AC092687.3/hsa-let-7e-5p/[SREBF2, FNIP1, PMAIP1] and SDCBP2-AS1 (ENST00000446423)/hsa-miR-101-3p/MAPK6 axes are probably related to Alzheimer's disease development and pathology. The presented network and axes will help to understand the PA-induced mechanisms in astrocytes, leading to protection or injury in the CNS under lipotoxic conditions as part of the intricated cellular regulation influencing the pathology of different NDs. Furthermore, the five corroborated axes could be considered study targets for new pharmacologic treatments or as possible diagnostic molecules, contributing to improving the quality of life of millions worldwide.

Hydrodynamic Assembly of Astrocyte Cells in Conductive Hollow Microfibers.

The manufacturing of 3D cell scaffoldings provides advantages for modeling diseases and injuries as it enables the creation of physiologically relevant platforms. A triple-flow microfluidic device is developed to rapidly fabricate alginate/graphene hollow microfibers based on the gelation of alginate induced with CaCl2 . This five-channel microdevice actualizes continuous mild fabrication of hollow fibers under an optimized flow rate ratio of 300:200:100µLmin-1 . The polymer solution is 2.5% alginate in 0.1% graphene and a 30% polyethylene glycol solution is used as the sheath and core solutions. The biocompatibility of these conductive microfibers by encapsulating mouse astrocyte cells (C8D1A) within the scaffolds is investigated. The cells can successfully survive both the manufacturing process and prolonged encapsulation for up to 8 days, where there is between 18-53% of live cells on both the alginate microfibers and alginate/graphene microfibers. These unique 3D hollow scaffolds can significantly enhance the available surface area for nutrient transport to the cells. In addition, these conductive hollow scaffolds illustrate unique advantages such as 0.728cm3 gr-1 porosity and two times more electrical conductivity in comparison to alginate scaffolds. The results confirm the potential of these scaffolds as a microenvironment that supports cell growth.

CNSC-32. SIMULTANEOUS INVESTIGATION OF TUMOR CELL BEHAVIOR AND CONNECTIVITY WITH CORRELATIVE IN VIVO TWO-PHOTON AND EX VIVO EXPANSION MICROSCOPY

Abstract Glioblastomas are the most aggressive primary brain tumors, characterized by whole-brain colonization, high therapeutic resistance, and inevitable recurrence. It has previously been shown that glioblastoma cells can form functionally gap-junction connected tumor cell networks with other tumor cells and/or astrocytes, which play a crucial role in tumor progression and treatment response. Furthermore, there is increasing evidence that synaptic neuron-glioma networks promote tumor growth. However, methodologies to investigate the direct link between synaptic, astrocytic and tumor cell connectivity together with tumor cell behavior including invasion, proliferation and therapeutic resistance are yet lacking. We aimed to characterize neuron-tumor and tumor-tumor/astrocyte cell interactions on a single cell level by using correlative in vivo two-photon and ex vivo expansion microscopy with near synaptic-resolution. We studied glioblastoma cells with intravital two-photon microscopy using patient-derived glioblastoma mouse models and the fluorescent functional imaging dye Sulforhodamine 101 allowing to investigate intratumoral and astrocytic connectivity over an extended period up to weeks. Subsequently, these functional tumor cell subpopulations could be directly correlated ex vivo to identify neuron-glioma synaptic connectivity. We utilized machine learning based segmentation and a semi-automated analysis pipeline to explore the connection of different glioblastoma morphological and behavioral subtypes to direct and indirect neuronal input in multiple xenograft models. With this new correlative method, we were able to simultaneously evaluate tumor cell behavior, synaptic and tumor-tumor/astrocyte connections on a single cell level. In addition, we demonstrated that this pipeline can be used for patient samples, revealing its clinical-translational potential for cancer neuroscience. In summary, we have established a new correlative technology that interlinks two key aspects of brain cancer neuroscience: cancer cell behavior and brain tumor network connectivity. This methodology promises a more thorough understanding of glioblastoma biology, facilitating the discovery of cancer biology, including the role of brain tumor networks in therapeutic resistance.

MODL-47. A NEW MOUSE MODEL OF IDH MUTATED GLIOMAS IDENTIFIES TUMOR CELLS OF ORIGIN AND DETERMINANTS OF SENSITIVITY TO IDH INHIBITORS

Abstract Driver mutations in IDH1 and IDH2 characterize a substantial proportion of diffuse gliomas. These tumors are typically of lower grade at diagnosis, but many eventually transform to more aggressive disease. Furthermore, there is a lack of effective curative treatments. Mutated IDH molecules acquire neomorphic enzymatic activity, favoring the synthesis of D-2-hydroxyglutarate (D-2-HG). Accumulation of this metabolite perturbs many cellular functions and drives gliomagenesis through incompletely understood mechanisms. IDH mutations are early events in gliomagenesis, but the identity of the initiating cell type has been debated. Here, using genetically engineered mice, we found that combining Idh1R132H and Trp53 loss in oligodendrocyte progenitors leads to fully penetrant development of diffuse gliomas. The tumors recapitulate the cardinal features of the corresponding human disease. While heterogeneous, the mouse Idh1R132H and Idh1WT tumors show distinct patterns of transcriptional alterations and oncogenic copy number variants. Mutant IDH itself is an attractive drug target, as it is clonally expressed and usually retained during tumor evolution, even though its relevance for disease progression has been questioned. Nevertheless, recent clinical trials have demonstrated clear, but variable, anti-tumor effects of mutant IDH inhibitors in patients. To better understand the mechanistic basis for heterogeneous responses to mutant IDH inhibition, we performed CRISPR/Cas9 functional genomic screens in cell lines derived from the mouse Idh1R132H;Trp53MUT tumors. These revealed interactions between the mutant IDH inhibitor vorasidenib and molecules related to astrocytic differentiation, Notch signaling, and cell growth and metabolism. The translational relevance of these findings was supported by molecular data from human tumors, patients treated with IDH inhibitors, and human-derived cell models. Overall, these studies nominate oligodendrocyte progenitors as candidate cells of origin for IDH mutated gliomas, and point to strategies that may enhance the efficacy of IDH inhibitors.

CSIG-18. WNT SIGNALING MODULATES TUMORIGENIC FEATURES AND CELLULAR IDENTITY IN A MODEL OF LOW-GRADE GLIOMAGENESIS

Abstract RATIONALE: Isocitrate dehydrogenase (IDH)-1/2 mutations are known driver mutations for the development of low-grade gliomas (LGGs). However, secondary drivers are needed for tumor growth, and it remains unclear which co-factors and pathways are necessary to develop the LGG tumor phenotype. To identify additional players in IDH mutant-mediated oncogenesis, we investigated the effects of disrupting glioma-associated pathways in a tractable in vitro system of IDH-mutant malignant transformation. METHODS: A doxycycline-inducible IDHR132H expressing normal human astrocyte cell line was concomitantly treated with either a Wnt-activator/inhibitor (CHIR99021/IWP-2), TGF-β inhibitor (Repsox) or Notch inhibitor (YO-01027). IDH-associated CD171 marker expression changes were assessed with Flow cytometry, followed by transcriptional and proteomic analysis with RNA-Seq and mass spectrometry. Functional characterization entailed cell viability and clonogenic assays as well as cell cycle analyses. RESULTS: We observed significant IDH-related CD171 expression changes upon treatment with CHIR99021, Repsox and YO-01027. Wnt activation with CHIR99021 profoundly affected the IDH mutant-induced transcriptional landscape, leading to differential usage of LGG and stemness programs including altered expression of SOX4 and SOX11. On the protein level, Wnt activation additionally impacted pathways relevant for cellular architecture, specifically Rho-Rac-CDC42 signaling. This was also reflected by an altered cell morphology with small, rounded up cells, augmented clonal proliferation capacity and increased proportions of cell in S-phase. CONCLUSION: Wnt activation in a IDHR132H model system modulated LGG development, leading to differential expression of key LGG and stemness associated profiles, morphologic cell changes and increased replication capacity. While aberrant Wnt signaling is a common feature in glioblastoma, our data point to an effect also on low-grade gliomagenesis, therefore requiring further investigation.

Elucidating the Therapeutic Utility of Olaparib in Sulfatide-Induced Human Astrocyte Toxicity and Neuroinflammation

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5–100 μM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca2+) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC50∼20 μM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC50∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD.Graphical Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca2+ concentration. PARP-1 inhibition with Olaparib reduces Ca2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

Regional investigation of UT-B urea transporters in the rat brain

Recent studies have reported increased levels of urea in the aging brain and various neurological disorders. Additionally, these diseased tissues also have increased expression of the UT-B transporter that regulates urea transport in the brain. However, little is known regarding the actual UT-B protein distribution across the brain in either normal or diseased states. This current study investigated UT-B protein abundance across three regions of the rat brain – anterior, posterior and cerebellum. Endpoint RT-PCR experiments showed that there were no regional differences in UT-B RNA expression (NS, N = 3, ANOVA), whilst Western blotting confirmed no difference in the abundance of a 35 kDa UT-B protein (NS, N = 3–4, ANOVA). In contrast, there was a significant variation in a non-UT-B 100 kDa protein (P < 0.001, N = 3–4, ANOVA), which was also detected by anti-UT-B antibodies. Using the C6 rat astrocyte cell line, Western blot analysis showed that 48-h incubation in either 5 mM or 10 mM significantly increased a 30–45 kDa UT-B protein signal (P < 0.05, N = 3, ANOVA). Furthermore, investigation of compartmentalized C6 protein samples showed the 30–45 kDa signal in the membrane fraction, whilst the 100 kDa non-UT-B signal was predominantly in the cytosolic fraction. Finally, immunolocalization studies gave surprisingly weak detection of rat UT-B, except for strong staining of red blood cells in the cerebellum. In conclusion, this study confirmed that RNA expression and protein abundance of UT-B were equal across all regions of the rat brain, suggesting that urea levels were also similar. However, it also highlighted some of the technical challenges of studying urea transporters at the protein level.

Characterization and identification of primarily cultured astrocytes and microglia

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

The antidepressant-like and glioprotective effects of the Y2 receptor antagonist SF-11 in the astroglial degeneration model of depression in rats: Involvement of glutamatergic inhibition

In this study, we explored the potential antidepressant-like properties of the brain-penetrant Y2 receptor (Y2R) antagonist SF-11 [N-(4-ethoxyphenyl)− 4-(hydroxydiphenylmethyl)− 1-piperidinecarbothioamide] in the astroglial degeneration model of depression with an emphasis on checking the possible mechanisms implicated in this antidepressant-like effect. The model of depression relies on the loss of astrocytes in the medial prefrontal cortex (mPFC) in Sprague-Dawley rats after administering the gliotoxin L-alpha-aminoadipic acid (L-AAA). SF-11 was administered intraperitoneally (i.p.) once (10 mg/kg) or for three consecutive days (10 mg/kg/day), and the effects of L-AAA and SF-11 injected alone or in combination were investigated using the forced swim test (FST), sucrose intake test (SIT), Western blotting, immunohistochemical staining, and microdialysis. SF-11 produced an antidepressant-like effect after single or three-day administration in rats subjected to astrocyte impairment, as demonstrated by the FST and SIT, respectively. Immunoblotting and immunohistochemical analyses showed that SF-11 reversed the L-AAA-induced astrocyte cell death in the mPFC, suggesting it is glioprotective. Microdialysis studies showed that SF-11 decreased extracellular glutamate (Glu) levels compared to basal value when administered alone and compared to the basal value and control group in LAAA-treated rats. The results from immunoblotting analysis indicated the involvement of Y2Rs in the astrocyte ablation model of depression and the antidepressant-like effect of SF-11. In addition, we observed the participation of the caspase-3 apoptotic pathway in the mechanism of gliotoxin action induced by L-AAA. These findings demonstrate that SF-11, a Y2R antagonist, elicited a rapid antidepressant-like response, possibly linked to its ability to inhibit glutamatergic neurotransmission.

Expression profiles and function prediction of tRNA-derived fragments in glioma

BackgroundGlioblastoma (GBM) is the most aggressive malignant primary brain tumor. The transfer RNA-derived fragments (tRFs) are a new group of small noncoding RNAs, which are dysregulated in many cancers. Until now, the expression and function of tRFs in glioma remain unknown.MethodsThe expression profiles of tRF subtypes were analyzed using the Cancer Genome Atlas (TCGA)-low-grade gliomas (LGG)/GBM dataset. The target genes of tRFs were subjected to Gene Ontology, Kyoto Encyclopedia and Gene set enrichment analysis of Genes and Genomes pathway enrichment analysis. The protein-protein interaction enrichment analysis was performed by STRING. QRT-PCR was performed to detect the expressions of tRFs in human glioma cell lines U87, U373, U251, and human astrocyte cell line SVG p12. Western blot assay was used to detect to the expression of S100A11. The interaction between tRF-19-R118LOJX and S100A11 mRNA 3’UTR was detected by dual-luciferase reporter assay. The effects of tRF-19-R118LOJX, tRF-19-6SM83OJX and S100A11 on the glioma cell proliferation, migration and in vitro vasculogenic mimicry formation ability were examined by CCK-8 proliferation assay, EdU assay, HoloMonitor cell migration assay and tube formation assay, respectively.ResultstRF-19-R118LOJX and tRF-19-6SM83OJX are the most differentially expressed tRFs between LGG and GBM groups. The functional enrichment analysis showed that the target genes of tRF-19-R118LOJX and tRF-19-6SM83OJX are enriched in regulating blood vessel development. The upregulated target genes are linked to adverse survival outcomes in glioma patients. tRF-19-R118LOJX and tRF-19-6SM83OJX were identified to suppress glioma cell proliferation, migration, and in vitro vasculogenic mimicry formation. The mechanism of tRF-19-R118LOJX might be related to its function as an RNA silencer by targeting the S100A11 mRNA 3’UTR.ConclusiontRFs would become novel diagnostic biomarkers and therapeutic targets of glioma, and the mechanism might be related to its post-transcriptionally regulation of gene expression by targeting mRNA 3’UTR.

Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 μm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

Testosterone upregulates glial cell line-derived neurotrophic factor (GDNF) and promotes neuroinflammation to enhance glioma cell survival and proliferation

BackgroundTestosterone contributes to male organism development, such as bone density, muscle development, and fat repartition. Estrogen (derived from testosterone) also contributes to female reproductive system development. Here, we investigated the effect of testosterone on glioma cells and brain neuron inflammation essential for cancer development and progression.MethodsThe human astrocyte and glioma cell lines were treated with 6 ng/ml exogenous testosterone in vitro. We performed cell counting kit-8, transwell, and wound healing assays to determine the effect of testosterone on glioma cell proliferation, migration, and invasion. The glioma cells were injected into the xenograft and treated with 5 µl concentrated testosterone. Transcriptional suppression of glial cell line-derived neurotrophic factor (GDNF) was performed to evaluate brain neuron inflammation and survival. The tumor tissues were assessed by hematoxylin–eosin staining and immunohistochemistry.ResultsTestosterone upregulates GDNF to stimulate proliferation, migration, and invasion of glioma cells. Pathologically, the augmentation of GDNF and cyclophilin A contributed to neuroprotection when treated with testosterone. Our investigation showed that testosterone contributes to brain neuron and astrocyte inflammation through the upregulation of nuclear factor erythroid 2-related factor 2 (NRF2), glial fibrillary acid protein (GFAP), and sirtuin 5 (SIRT5), resulting in pro-inflammatory macrophages recruitments into the neural microenvironment. Mechanically, testosterone treatment regulates GDNF translocation from the glioma cells and astrocyte nuclei to the cytoplasm.ConclusionTestosterone upregulates GDNF in glioma cells and astrocytes essential for microglial proliferation, migration, and invasion. Testosterone contributes to brain tumor growth via GDNF and inflammation.Graphical The contribution of testosterone, macrophages, and astrocytes, in old neuron rescue, survival, and proliferation. During brain neuron inflammation, the organism activates and stimulates the neuron rescue through the enrichment of the old neuron microenvironment with growth factors such as GDNF, BDNF, SOX1/2, and MAPK secreted by the surrounding neurons and glial cells to maintain the damaged neuron by inflammation alive even if the axon is dead. The immune response also contributes to brain cell survival through the secretion of proinflammatory cytokines, resulting in inflammation maintenance. The rescued old neuron interaction with infiltrated macrophages contributes to angiogenesis to supplement the old neuron with more nutrients leading to metabolism activation and surrounding cell uncontrollable cell growth.

Chaotic resonance in an astrocyte-coupled excitable neuron

We study the chaotic resonance phenomenon whereby the response of a neuron to a weak signal is amplified with the help of chaotic current stemming from background activity in the brain. This resonance behavior exhibits a bell-shaped curve in terms of detection quality due to increasing chaotic current intensity. Recent experimental studies have shown that astrocytes, which are the most abundant types of glial cells, may be responsible for the regulation of electrophysiological events in neuronal medium. Hence, we consider here a realistic neuronal system which is constituted by a bipartite network consisting of an excitable neuron and an astrocyte. Our analysis reveals that signal detection quality can be greatly enhanced with the astrocyte contribution obtained by appropriate neuronal and astrocytic cell dynamics. We find that depolarization-induced astrocytic glutamate release is able to improve chaotic resonance performance considerably in the presence of an adequately strong interaction between the astrocyte and the excitable neuron receiving a weak signal with a relatively higher frequency. We also show that a moderate production rate of gliotransmitters is required for the astrocyte to affect resonance performance of the neuron. Except for those conditions where the facilitating effect of astrocyte is observed, it can also reduce signal detection performance in the neuron. Furthermore, we demonstrated that intrinsic neuronal excitability is regulated by the astrocyte, via a comparison of resonance behaviors under effects of bias and astrocytic current separately. Taken together, our findings provide a novel insight into the functioning of astrocyte-neuron circuits, in particular the encoding weak signals via chaotic resonance, and suggest that astrocytes play a key role in intrinsic regulation and selectivity in neuronal information processing.

Chloride intracellular channel protein-1 (CLIC1) antibody in multiple sclerosis patients with predominant optic nerve and spinal cord involvement.

Antibodies to cell surface proteins of astrocytes have been described in chronic inflammatory demyelinating disorders (CIDD) of the central nervous system including multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Our aim was to identify novel anti-astrocyte autoantibodies in relapsing remitting MS (RRMS) patients presenting predominantly with spinal cord and optic nerve attacks (MS-SCON). Sera of 29 MS-SCON patients and 36 healthy controls were screened with indirect immunofluorescence to identify IgG reacting with human astrocyte cultures. Putative target autoantigens were investigated with immunoprecipitation (IP) and liquid chromatography-mass/mass spectrometry (LC-MS/MS) studies using cultured human astrocytes. Validation of LC-MS/MS results was carried out by IP and ELISA. Antibodies to astrocytic cell surface antigens were detected in 5 MS-SCON patients by immunocytochemistry. LC-MS/MS analysis identified chloride intracellular channel protein-1 (CLIC1) as the single common membrane antigen in 2 patients with MS-SCON. IP experiments performed with the commercial CLIC1 antibody confirmed CLIC1-antibody. Home made ELISA using recombinant CLIC1 protein as the target antigen identified CLIC1 antibodies in 9/29 MS-SCON and 3/15 relapsing inflammatory optic neuritis (RION) patients but in none of the 30 NMOSD patients, 36 RRMS patients with only one or no myelitis/optic neuritis attacks and 36 healthy controls. Patients with CLIC1-antibodies showed trends towards exhibiting reduced disability scores. CLIC1-antibody was identified for the first time in MS and RION patients, confirming once again anti-astrocytic autoimmunity in CIDD. CLIC1-antibody may potentially be utilized as a diagnostic biomarker for differentiation of MS from NMOSD.

Newly Diagnosed Anaplastic Astrocytoma: &gt; 23 Year Survival in a 31-Year and 11-Month-Old Female Treated with Antineoplastons

Rationale: Anaplastic astrocytoma (AA), a malignant brain tumor that arises from astrocytic cells, has a poor prognosis. It accounts for 6–7% of gliomas and 1-2% of brain tumors. Objectives: A 31-year and 11-month-old female with an AA is presented to discuss the efficacy of Antineoplastons A10 (Atengenal) and AS2-1 (Astugenal) in the treatment of AA. She presented to the Burzynski Clinic (BC) for treatment and was enrolled in Protocol BT-08, “Phase II Study of Antineoplastons A10 and AS2-1 in Adult Patients with Anaplastic Astrocytoma”, receiving both intravenous (IV) and oral Antineoplastons A10 and AS2-1 (ANP therapy). IV ANP therapy was delivered continuously via subclavian catheter and infusion pump. Tumor response was determined by comparison of baseline brain magnetic resonance imaging (MRI) to sequential brain MRIs during therapy. Findings: The patient presented to the BC with no prior treatment and a one-month history of right arm clumsiness and right leg weakness. Brain MRI and stereotactic biopsy performed elsewhere had demonstrated an AA. Baseline MRI at the BC showed a 2.0 cm2 nonenhancing lesion and two enhancing lesions (0.02 cm2 and 0.15 cm2 ) in the left parietal lobe. IV ANP therapy was given over 56 days, and a complete response (CR) was demonstrated after one month of therapy. Oral ANP therapy was subsequently provided for 17 months. At last follow-up, in June 2023, the patient was healthy and showed no evidence of recurrent disease. She had an overall survival (OS) of &gt; 23 years and one month since diagnosis and an OS of &gt; 23 years since the start of IV ANP therapy. Conclusions: The utilization of ANP therapy in an adult female patient with AA is presented. The patient achieved a CR and prolonged OS, suggesting that ANP therapy may be an effective therapeutic option for adults with AA.