Astrocyte Cell Research Articles

Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. We identified ADAMTS3 as a potential therapeutic target for GBM.

Protective Actions of α-Tocopherol on Cell Membrane Lipids of Paraquat-Stressed Human Astrocytes Using Microarray Technology, MALDI-MS and Lipidomic Analysis.

Cellular senescence is one of the main contributors to some neurodegenerative disorders. The early detection of senescent cells or their related effects is a key aspect in treating disease progression. In this functional deterioration, oxidative stress and lipid peroxidation play an important role. Endogenous antioxidant compounds, such as α-tocopherol (vitamin E), can mitigate these undesirable effects, particularly lipid peroxidation, by blocking the reaction between free radicals and unsaturated fatty acid. While the antioxidant actions of α-tocopherol have been studied in various systems, monitoring the specific effects on cell membrane lipids at scales compatible with large screenings has not yet been accomplished. Understanding the changes responsible for this protection against one of the consequences of senescence is therefore necessary. Thus, the goal of this study was to determinate the changes in the lipid environment of a Paraquat-treated human astrocytic cell line, as a cellular oxidative stress model, and the specific actions of the antioxidant, α-tocopherol, using cell membrane microarray technology, MALDI-MS and lipidomic analysis. The stress induced by Paraquat exposure significantly decreased cell viability and triggered membrane lipid changes, such as an increase in certain species of ceramides that are lipid mediators of apoptotic pathways. The pre-treatment of cells with α-tocopherol mitigated these effects, enhancing cell viability and modulating the lipid profile in Paraquat-treated astrocytes. These results demonstrate the lipid modulation effects of α-tocopherol against Paraquat-promoted oxidative stress and validate a novel analytical high-throughput method combining cell cultures, microarray technology, MALDI-MS and multivariate analysis to study antioxidant compounds against cellular senescence.

Cell-Type-Dependent Role for nsP3 Macrodomain ADP-Ribose Binding and Hydrolase Activity during Chikungunya Virus Infection.

Chikungunya virus (CHIKV) causes outbreaks of rash, arthritis, and fever associated with neurologic complications, where astrocytes are preferentially infected. A determinant of virulence is the macrodomain (MD) of nonstructural protein 3 (nsP3), which binds and removes ADP-ribose (ADPr) from ADP-ribosylated substrates and regulates stress-granule disruption. We compared the replication of CHIKV 181/25 (WT) and MD mutants with decreased ADPr binding and hydrolase (G32S) or increased ADPr binding and decreased hydrolase (Y114A) activities in C8-D1A astrocytic cells and NSC-34 neuronal cells. WT CHIKV replication was initiated more rapidly with earlier nsP synthesis in C8-D1A than in NSC-34 cells. G32S established infection, amplified replication complexes, and induced host-protein synthesis shut-off less efficiently than WT and produced less infectious virus, while Y114A replication was close to WT. However, G32S mutation effects on structural protein synthesis were cell-type-dependent. In NSC-34 cells, E2 synthesis was decreased compared to WT, while in C8-D1A cells synthesis was increased. Excess E2 produced by G32S-infected C8-D1A cells was assembled into virus particles that were less infectious than those from WT or Y114A-infected cells. Because nsP3 recruits ADP-ribosylated RNA-binding proteins in stress granules away from translation-initiation factors into nsP3 granules where the MD hydrolase can remove ADPr, we postulate that suboptimal translation-factor release decreased structural protein synthesis in NSC-34 cells while failure to de-ADP-ribosylate regulatory RNA-binding proteins increased synthesis in C8-D1A cells.

Single-nuclei transcriptome analysis of Huntington disease iPSC and mouse astrocytes implicates maturation and functional deficits

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene that alters cellular homeostasis, particularly in the striatum and cortex. Astrocyte signaling that establishes and maintains neuronal functions are often altered under pathological conditions. We performed single-nuclei RNA-sequencing on human HD patient-induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from R6/2 HD mice to investigate high-resolution HD astrocyte cell state transitions. We observed altered maturation and glutamate signaling in HD human and mouse astrocytes. Human HD astrocytes also showed upregulated actin-mediated signaling, suggesting that some states may be cell-autonomous and human specific. In both species, astrogliogenesis transcription factors may drive HD astrocyte maturation deficits, which are supported by rescued climbing deficits in HD drosophila with NFIA knockdown. Thus, dysregulated HD astrocyte states may induce dysfunctional astrocytic properties, in part due to maturation deficits influenced by astrogliogenesis transcription factor dysregulation.

Vitamin D3 actions on astrocyte cells: A target for therapeutic strategy in Parkinson’s disease?

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic cells in the substantia nigra pars compacta. PD patients’ brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The present study aims to evaluate the neuroprotective activity of VD3 on astrocytes after their exposure to rotenone (ROT) a natural pesticide known to exhibit neurotoxic potential via the inhibition of mitochondrial complex I. Cell viability parameters were evaluated by the MTT test and staining with 7-AAD in cultures of astrocytes treated and untreated with VD3 (0.1, 0.5, and 1.0 ng/mL) and/or ROT (10 µg/mL or 5 µg/mL), and the cytoplasmic production of ROS and the cell death profile were measured by flow cytometry. Glutathione accumulation and ultrastructural changes were evaluated and immunocytochemistry assays for NF-kB and Nrf2 were also carried out. The results showed that VD3 improved the viability of cells previously treated with VD3 and then exposed to ROT, reducing the occurrence of necrotic and apoptotic events. Furthermore, cells exposed to ROT showed increased production of ROS, which decreased significantly with previous treatment with VD3. Importantly, the decrease by ROT in the mitochondrial transmembrane potential was significantly prevented after treating cells with VD3, especially at a concentration of 1 ng/mL. Therefore, treatment with VD3 protected astrocytes from damage caused by ROT, decreasing oxidative stress, decreasing NF-kB and Nrf2 expressions, and improving mitochondrial function. However, further investigation is needed regarding the participation and mechanism of action of VD3 in this cellular model of PD focusing on the crosstalk between Nrf2 and NF-kB.

GAS5 attenuates the malignant progression of glioma stem-like cells by promoting E-cadherin.

It has been widely reported that glioma stem-like cells (GSCs) serve a crucial role in the malignant progression of glioma. In particular, recent studies have reported that long non-coding RNAs (lncRNAs) are closely associated with glioma development. However, the underlying molecular regulatory mechanistic role of GSCs remains poorly understood. The present study established two highly malignant glioma stem-like cell lines from clinical surgical specimens. In these, it was found that the lncRNA growth arrest-specific 5 (GAS5) expression was downregulated in GSCs and high-grade glioma tissues, compared with normal human astrocyte cells (NHAs) and normal brain tissues, respectively, which also showed a positive correlation with patient survival. Functional assays revealed that knocking down GAS5 expression promoted the proliferation, invasion, migration, stemness, and tumorigenicity of GSGs, while suppressing their apoptosis. Mechanistically, GAS5 directly sponged miR-23a, which in turn functioned as an oncogene by inhibiting E-cadherin, through the assays of reverse transcription-quantitative PCR (RT-qPCR) and luciferase reports. In addition, rescue experiments demonstrated that GAS5 could promote the expression and function of E-cadherin in a miR-23a-dependent manner. Collectively, these data suggest that GAS5 functions as a suppressor in GSCs by targeting the miR-23a/E-cadherin axis, which may be a promising therapeutic target against glioma.

Role of ABCA7 in Neural Lipid Metabolism and Its Association with Alzheimer's Disease Neuropathology

AbstractBackgroundStrong genetic evidence supports involvement of adenosine triphosphate binding‐cassette transporter subfamily A member 7 (ABCA7) in Alzheimer’s disease (AD) pathogenesis. ABCA7 closely resembles ABCA1, a cholesterol transporter, in structure. However, molecular specificity of ABCA7‐mediated lipid transport and the role of this protein in AD pathophysiology are understood incompletely. We advance the hypothesis that ABCA7 protects against AD development by removing neurodegenerative lipid from the brain.MethodA validated monoclonal antibody was used to measure ABCA7 protein in a cohort of 104 individuals represented by the hippocampus and a different cohort of 119 individuals represented by the parietal cortex. Both cohorts included individuals with Braak stages 0‐V. Changes in ABCA7 level in response to treatment with lipid metabolism modulators were investigated in human microglial C20 and HMC3 and human astrocytic A172 cell lines.ResultTo match for age, the cohorts were subdivided into 63‐78 and 79‐94 age‐at‐death brackets. There was no significant correlation between ABCA7 protein levels and Braak stages. However, individuals with Braak stages II‐V as a group had significantly lower ABCA7 levels than individuals with Braak stages 0/I as a group in the 63‐78 age bracket of both cohorts (Mann‐Whitney p<0.0001 and p=0.003, respectively) and in the 79‐94 bracket of the parietal cortex cohort (p=0.037) but not in the hippocampus cohort (p=0.33). One of the reasons for this difference between the hippocampus and parietal cortex was that ABCA7 levels decreased significantly with age in the Braak stage 0/I group in the hippocampus but not the parietal cortex (p=0.02 vs p=0.77 for ABCA7 levels in the 63‐78 vs 79‐94 age bracket, respectively). An analysis of lipid metabolism using pharmacological modulators revealed that ABCA7 expression in microglial and astrocytic cells does not respond to the intracellular cholesterol level or regulators of intracellular cholesterol metabolism.ConclusionThese results are in line with the hypothesis that ABCA7 prevents AD neuropathogenesis, i.e., loss of ABCA7 protein occurs sharply and early between Braak stages I and II. There also appear to be brain region‐specific changes in ABCA7 expression with age. The neurodegenerative lipid that ABCA7 removes from the brain remains to be identified.

Investigating Distinct Alpha Synuclein Oligomer‐Mediated Cellular Toxicity

AbstractBackgroundAlpha Synuclein (ASyn), an amyloidogenic protein observed in Parkinson’s (PD) and Alzheimer’s (AD) disease, oligomers are toxic and polymorphic protein aggregates that with further investigation could be essential to improving potential neurodegenerative therapeutics.MethodUsing monoclonal antibodies (SynTCs) that bind toxic conformations of ASyn, we utilize cellular models such as primary cortical neurons and primary astrocytes isolated from C57/BL6 mice to investigate differences in the neurotoxic effects of ASyn oligomeric strains. These differences were evaluated by both immunocytochemistry and cell‐based assays. In addition, we analyzed the thermodynamic binding occurring between SynTCs and ASyn oligomeric strains.ResultUtilizing isothermal titration calorimetry (ITC), we confirm the thermodynamic binding between SynTCs and ASyn oligomers in contrast to ASyn monomers and fibrils. Asyn oligomeric strains exhibit differential toxicity in both primary neuron and astrocyte cells. When immunodepleted by SynTCs, cytotoxicity and endogenous ASyn aggregation decreased.ConclusionInvestigating mechanisms of neurotoxicity mediated by Asyn oligomeric strains is valuable in determining biological effects and the therapeutic potential of targeting and reducing toxic ASyn oligomers that contribute to the neurotoxicity observed in multiple neurodegenerative diseases.

Withdrawn: Glycine transporter 1 (GlyT1) is a novel therapeutic target for Alzheimer’s disease

Withdrawal: Miran Yoo, Seong Muk Kim, Juho Lee, Hye‐Ju Kim, Seong Jeong Park “Glycine transporter 1 (GlyT1) is a novel therapeutic target for Alzheimer's disease” Alzheimer's & Dementia, Volume 18, Supplemental Issue 10.The above abstract, published online on 20th December 2022, on Wiley Online Library (https://alz‐journals.onlinelibrary.wiley.com/doi/abs/10.1002/alz.064468) has been withdrawn by agreement between the authors, the Senior Director of Scientific Programs and Outreach, Claire Sexton, the Director of Scientific Programs at the Alzheimer's Association, Ozama Ismail, and John Wiley & Sons Inc. The abstract has been withdrawn at the authors’ request because the authors did not have Amyloid Solution's authorization to publish the information contained in it.

A human-derived neurovascular unit in vitro model to study the effects of cellular cross-talk and soluble factors on barrier integrity.

The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the "neurovascular unit" (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson's disease and Alzheimer's disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.

Human microRNA‐298 plays a vital role in maintaining APP and tau homeostasis and IL‐induced inflammation: Implication in Alzheimer’s disease.

AbstractBackgroundAlzheimer’s disease (AD) is marked by neurofibrillary tangles mainly composed of hyperphosphorylated microtubule‐associated protein tau; senile plaques comprising amyloid β (Aβ) peptides; and neuroinflammation. How these pathological changes are regulated remains unclear. Genetic, epigenetic, and environmental factors play important roles in disease progression. MicroRNA (miRNA), a group of small non‐coding RNAs, regulates multiple biological and pathological processes by binding to target mRNAs and altering translation levels. Disrupting this process could contribute to AD pathogenesis. We hypothesize that miR‐298 plays a vital role in maintaining APP and tau homeostasis and IL‐induced inflammation, particularly gliosis.MethodWe performed miR‐298 treatments and blockade by antisense‐miR298 (antagomiRs) in 3’UTR‐reporter clone‐transfected cultures and protein levels from target native mRNAs (Chopra et al‐Mol. Psychiatry‐2021). We transfected human astrocyte cells with miR‐298 mimics or antagomiRs. We analyzed culture media and lysates for protein and mRNA. We performed ELISA and real‐time‐qPCR assays to determine cytokines, APP and tau levels. We analyzed autopsy brain tissues from non‐cognitively impaired (NCI) and AD subjects by measuring levels of miR‐298, target mRNAs and proteins in cortical samples, as we described (Wang et al‐Mol. Psychiatry‐2022).ResultWe identified putative sites for miR‐298 in mRNAs encoding APP, BACE1, MAPT, IL1α, and IL6. Treatment by miR‐298 significantly increased production and secretion of multiple pro‐inflammatory cytokines, specifically IL‐1, IL‐6 and TNF‐α in human astrocyte cultures. Furthermore, treatment by miR‐298 reduced tau, APP and BACE1 protein and mRNA levels in astrocyte cultures. Levels of miR‐298 varied in postmortem human temporal lobe between AD patients and age‐matched NCI controls.ConclusionMiR‐298 induced a variety of pro‐inflammatory cytokines without administration of other challenges. Induction of multiple cytokines may be due to combinations of direct miRNA activity and regulation of upstream signaling pathways. MiR‐298 may play an important biological role in AD by reducing APP, BACE1 and tau expression while increasing the physiologic production of IL cytokines. This apparent paradox is resolved by noting that early‐stage (prodromal) gliosis may contribute to the clearance of toxic aggregates. We plan to test miR‐298 in AD animal models, including human tau‐overexpressing transgenic mice.We appreciate NIH‐NIA grants: (R01AG051086, R21AG056007, R56AG072810, P01AG014449, P30AG072976).

Cell sorting after tracer injection to elucidate allocation of cellular FDG uptake in different cell types and different microglial activation states

AbstractBackground2‐deoxy‐2‐[18F]fluoro‐d‐glucose positron‐emission‐tomography (FDG‐PET) is widely used to study cerebral glucose metabolism. Radiosorting revealed that the FDG‐PET signal is directly influenced by microglial glucose uptake in mouse models and we found similar associations in patients with neurodegenerative diseases. Now, we questioned the allocation of cellular FDG uptake in the rodent brain.MethodSingle cell [18F]FDG uptake as assessed by magnetic activated cell sorting (MACS) was multiplied with published total cell numbers in the mouse brain, i.e. 71e+6 neurons or 7% (≈ 7.6e+6) microglia cells to estimate the overall uptake per cell fraction. Estimated cellular microglial [18F]FDG uptake as assessed by MACS was cross‐validated with observed %‐FDG‐PET reductions after microglia depletion. Interstitial and vessel [18F]FDG was estimated by interstitial compartment volume and assumed equilibrium of non‐phosphorylated extra‐ and intracellular [14C]Deoxyglucose. Synaptic [18F]FDG uptake was estimated via an enriched synaptosome fraction relative to [18F]FDG uptake of whole‐brain homogenate. A microglia proliferation index of 1.3 was assumed for the whole brain in the Aβ mouse model.ResultMicroglia cells accounted for 8.6% of the overall brain [18F]FDG uptake in wild‐type mice and for 29.9% of the overall brain [18F]FDG uptake in PS2APP mice, confirmed by similar alterations after microglia depletion in PET. Synapses contributed to 13.2‐17.1% of the overall brain [18F]FDG uptake whereas interstitial (5.9‐7.7%) and vessel (2.6‐3.4%) [18F]FDG uptake was of minor magnitude. Neuronal and astrocyte cell bodies accounted for <10% of the overall brain [18F]FDG uptake, but a proportion of 42‐55% was not recovered and likely allocated in lost cell processes, oligodendrocytes and other cell types.ConclusionMicroglial FDG uptake alterations are the source of FDG‐PET changes in models of neurodegenerative diseases. Our data should increase the awareness to consider glia cells as key contributors of FDG uptake and the detailed proportions of these cellular contributions in healthy brains and under disease conditions deserve deeper investigation.

HMGB1‐Mediated Senescence and Brain Inflammation Contributes to Cognitive Dysfunctions

AbstractBackgroundHigh mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein, which is excessively released by glial cells during aging and Alzheimer’s Disease (AD). The extracellular HMGB1 serves as a key initiator of neuroinflammation, which is linked to exacerbated cognitive impairments in AD. Importantly, aging ‐ the strongest risk factor for AD is characterized by a chronic, low‐grade inflammation known as inflammaging. Evidence suggests that senescent cells (SC) accumulate with aging and are characterized by an apoptosis‐resistant, arrested cell cycle with a distinct “inflammatory” phenotype also known as senescence‐associated secretory phenotype (SASP). However, the precise etiology of inflammaging in tauopathies remain largely unknown.MethodWe used brain tissues from AD patients and hTau mouse model and performed immunoflourescence, microscopy, astrocyte cell cultures, pharmacological inhibition of HMGB1 in vitro and in vivo in a hTau mouse model to examine role of HMGB1 in senescence, brain inflammation and cognitive dysfunctions. We scored cellular senescence, inflammation, tau pathology (phospho‐tau, tau oligomers, neurofibrillary tangles), Synaptic and postsynaptic proteins, and cognitive functions (Y‐maze and novel‐objective recognition tests).ResultIn this study, we demonstrate that (1) HMGB1 release is linked to advanced age in hTau tauopathy mice. (2) HMGB1 release is a hallmark of senescent cells both in mice and humans. (3) Astrocytes display a senescence‐like phenotype in the brains of patients with AD. (4) Pathological tau oligomers (TauOs) exposure triggers astrocyte senescence through HMGB1 release, which mediates paracrine senescence in adjacent cells and potentiates inflammatory SASP. (5) Inhibition of HMGB1 release using ethyl pyruvate (EP) and glycyrrhizic acid (GA) results in decreased astrocyte senescence and neuroinflammation. (6) Importantly, despite the developed tauopathy and cognitive deficits in 12‐month‐old hTau mice, EP+GA treatment significantly decreased SCs loads in the brain, reduces inflammaging, and thus ameliorated cognitive deficits. Collectively, our data suggest that HMGB1 release promotes inflammaging, and cellular senescence, which could represent an important pathomechanism in the progression of aging‐related diseases, including AD. Future studies are warranted to develop safe and effective therapeutic strategies to control inflammaging and enhance healthy lifespan in patients with tauopathy.ConclusionThe data suggests that HMGB1 release is a key pathomechanism in AD.

Development of potent and selective FAAH inhibitors with improved drug-like properties as potential tools to treat neuroinflammatory conditions

The neuroprotective performance against neuroinflammation of the endocannabinoid system (ECS) can be remarkably improved by indirect stimulation mediated by the pharmacological inhibition of the key ECS catabolic enzyme fatty acid amide hydrolase (FAAH). Based on our previous works and aiming to discover new selective FAAH inhibitors , we herein reported a new series of carbamate-based FAAH inhibitors (4a-t) which showed improved drug disposition properties compared to the previously reported analogues 2a-b. The introduction of ionizable functions allowed us to obtain new FAAH inhibitors of nanomolar potency characterized by good water solubility and chemical stability at physiological pH. Interesting structure-activity relationships (SARs), deeply analyzed by molecular docking and molecular dynamic (MD) simulations, were obtained. All the newly developed inhibitors showed an excellent selectivity profile evaluated against monoacylglycerol lipase and cannabinoid receptors. The reversible mechanism of action was determined by a rapid dilution assay. Absence of toxicity was confirmed in mouse fibroblasts NIH3T3 (for compounds 4e, 4g, 4n-o, and 4s) and in human astrocytes cell line 1321N1 (for compounds 4e, 4n, and 4s). The absence of undesired cardiac effects was also confirmed for compound 4n. Selected analogues (compounds 4e, 4g, 4n, and 4s) were able to reduce oxidative stress in 1321N1 astrocytes and exhibited notable neuroprotective effects when tested in an ex vivo model of neuroinflammation.

Amelioration of cholesterol sulfate for lead-induced CTX cell apoptosis based on BDNF signaling pathway mediated cholesterol metabolism

Lead (Pb), as a deleterious heavy metal, ubiquitously exists in environment and industry, which engenders multi-organ disfunction, especially the brain of infants who are vulnerable to attack from lead-induced neurotoxicity. Although cholesterol sulfate (CS) is crucial constituent of cell membranes and precursor of neurosteroids, which maintains the function and survival of neurons, the role of CS in lead-induced neurological damage still remains incomplete. In this work, Rat Brain Astrocytes cell line (CTX cells) was applied into exploration that protective effects of CS on CTX cell apoptosis induced by lead via the regulation of BDNF/TrkB signaling pathway mediated cholesterol metabolism. We found that CTX cells exposed to lead manifested apparent cytotoxicity, where the viability of CTX cells was significantly suppressed, accompanied with the elevation of apoptosis, in response to a trend towards increases in reactive oxygen species (ROS) production and pro-apoptotic protein Cleaved-caspase3, synchronized with the decline in anti-apoptotic protein Bcl-2. Moreover, accumulation of lead in CTX cells showed a dose-dependent increase, and meanwhile, decrements in cholesterol content occurred along with increase in lead exposure, in which expressions of cholesterol metabolism related proteins and transcriptions of its genes (SREBP2, LDLR, and HMGCR) were diminished. Furthermore, BDNF signaling pathway was obviously blocked after lead exposure, down-regulating expressions of proteins BDNF and TrkB. However, pretreatment with CS detoxified the negative impacts of lead-invoked CTX cell damage, acting as an effective remedy for apoptosis, imbalance of cholesterol metabolism and inhibition of BDNF signaling pathway. In addition, the relationship between BDNF signaling pathway and cholesterol metabolism was further verified, in which cholesterol metabolism related proteins and genes were promoted significantly after the activation of BDNF/TrkB signaling pathway using 7,8-Dihydroxyflavone (7,8-DHF), thereby detoxifying lead-induced CTX cell injury. However, the pretreatment of TrkB inhibitor ANA-12 offset the promotion of 7,8-DHF and ultimately inhibit cholesterol metabolism. Overall, our study demonstrated that CS could initiate the BDNF/TrkB signaling pathway, regulating the cholesterol metabolism against CTX cell apoptosis invoked by lead.

Ultrastructural destruction of neurovascular unit in experimental cervical spondylotic myelopathy.

The pathogenesis of cervical spondylotic myelopathy (CSM) remains unclear. This study aimed to explore the ultrastructural pathology of neurovascular unit (NVU) during natural development of CSM. A total of 24 rats were randomly allocated to the control group and the CSM group. Basso-Beattie-Bresnahan (BBB) scoring and somatosensory evoked potentials (SEP) were used as functional assessments. Hematoxylin-eosin (HE), toluidine blue (TB), and Luxol fast blue (LFB) stains were used for general structure observation. Transmission electron microscopy (TEM) was applied for investigating ultrastructural characteristics. The evident compression caused significant neurological dysfunction, which was confirmed by the decrease in BBB score and SEP amplitude, as well as the prolongation of SEP latency (P < 0.05). The histopathological findings verified a significant decrease in the amount of Nissl body and myelin area and an increase in vacuolation compared with the control group (P < 0.05). The TEM results revealed ultrastructural destruction of NVU in several forms, including: neuronal degeneration and apoptosis; disruption of axonal cytoskeleton (neurofilaments) and myelin sheath and dystrophy of axonal terminal with dysfunction mitochondria; degenerative oligodendrocyte, astrocyte, and microglial cell inclusions with degenerating axon and dystrophic dendrite; swollen microvascular endothelium and loss of tight junction integrity; corroded basement membrane and collapsed microvascular wall; and proliferated pericyte and perivascular astrocytic endfeet. In the CSM group, reduction was observed in the amount of mitochondria with normal appearance and the number of cristae per mitochondria (P < 0.05), while no substantial drop of synaptic vesicle number was seen (P > 0.05). Significant narrowing of microvascular lumen size was also observed, accompanied by growth in the vascular wall area, endothelial area, basement membrane thickness, astrocytic endfeet area, and pericyte coverage area (rate) (P < 0.05). Altogether, the findings of this study demonstrated ultrastructural destruction of NVU in an experimental CSM model with dorsal-lateral compression, revealing one of the crucial pathophysiological mechanisms of CSM.

Anencephaly, Bifid Tongue, and Cleft Palate in a Pomeranian Dog: GFAP and NeuN Immunoreactivities.

Anencephaly is a congenital disease manifesting with the absence of the brain due to the failure of the cranial part of the neural tube to close during the embryonic stage. The disease may be accompanied by other anomalies and usually results in premature death. A stillborn puppy of a 2-year-old female Pomeranian dog is examined in this case. The lack of brain tissue and accompanying abnormal skull formation was noted macroscopically. The eyes were protruding out of their normal position (exophthalmos), and a bifid tongue together with a secondary cleft palate was present. On serial sections stained with Haematoxylin-Eosin, only the medulla spinalis among the central nervous system structures could be inspected microscopically. Immunohistochemistry staining revealed GFAP immunoreactivity in the astrocytic glial cells. NeuN immunoreactivity was detected in the neurons in the medulla spinalis and spinal ganglions. Incomplete retinal layers were observed on the eye sections stained with Haematoxylin-Eosin and NeuN. The case was concluded to be coherent with skull and nervous system congenital malformations rarely observed in dogs. To the best of our knowledge, this represents the first description of a dog with anencephaly, bifid tongue and cleft palate.

Carbon Monoxide Stimulates Both Mitophagy And Mitochondrial Biogenesis to Mediate Protection Against Oxidative Stress in Astrocytes.

Astrocytes are key glial cells for the metabolic and functional support of the brain. Mitochondrial quality control (MQC), in particular the balance between mitophagy and mitochondrial biogenesis, is a major event for the maintenance of cellular homeostasis. Carbon monoxide (CO) is an endogenous gasotransmitter that inhibits cell death and inflammation by targeting mitochondria. It is well established that CO promotes cytoprotection by increasing mitochondrial population and metabolism (oxidative phosphorylation). Thus, it is hypothesized that CO-induced cytoprotection may also be mediated by the balance between mitophagy and mitochondrial biogenesis. Herein, the carbon monoxide releasing molecule-A1 (CORM-A1) was used in primary cultures of astrocytes to assess CO role on mitochondrial turnover. PINK1/Parkin-dependent mitophagy was stimulated by CORM-A1 following 1h of treatment. While at 24h after treatment, CORM-A1 increased mitochondrial population, which may indicate mitochondrial biogenesis. In fact, mitochondrial biogenesis was confirmed by the enhancement of PGC-1α expression that upregulates several mitochondrial transcription factors. Furthermore, inhibition of mitophagy by knocking down PINK1 expression reverted CO-induced mitochondrial biogenesis, indicating that mitochondrial turnover is dependent on modulation of mitophagy. Finally, CORM-A1 prevented astrocytic cell death induced by oxidative stress in a mitophagy-dependent manner. In fact, whenever PINK1 was knocked down, CORM-A1-induced cytoprotection was lost. In summary, CORM-A1 stimulates mitochondrial turnover, which in turn prevents astrocytic cell death. CO cytoprotection depends on increasing mitochondrial population and on eliminating dysfunctional mitochondria.

EPCO-22. SINGLE CELL ANALYSIS TO UNDERSTAND SEX DIFFERENCES IN GBM OUTCOME

Abstract Males have a higher incidence and mortality in certain cancers compared to females. The same is true in glioblastoma (GBM) regardless of age, suggesting that cell-intrinsic factors rather than extrinsic factors such as circulating sex hormones are causing these sex differences. Our lab has demonstrated cell-intrinsic sex differences with respect to growth, clonogenicity, and in vivo tumorigenesis using an isogenic murine astrocyte cell culture model of GBM (Nf1 -/-; DNp53). Male astrocytes are more likely to adapt and grow when exposed to irradiation-induced stress compared to female astrocytes, which are more likely to undergo growth arrest after the same exposures. These sex differences in response to treatment can be explained by the different chromatin and transcriptional profiles established from the moment of fertilization that can result in different levels of transcriptional heterogeneity, a well-established cause for resistance to induced stress in GBM. However, so far sex has not been considered when exploring transcriptional heterogeneity. As such, we used 10x Genomics technology to extract single nuclei RNA (snRNA) and ATAC (snATAC) sequencing from the same nuclei and identified different levels of transcriptional heterogeneity in male and female astrocytes cells from our model. We also identified different chromatin profiles of crucial genes, such as Sox2, an important TF for the reprogramming of differentiated GBM into stem-like cells, which can then form tumors in vivo. We expect for the male astrocytes to be more effective in escaping growth arrest and adapting to irradiation-induced stress because of these different transcriptional and chromatin profiles. Our work will identify epigenetic mechanisms by which male cells are more able to adapt to treatment-induced stress compared to female cells and will further advance our understanding of sex differences in treatment-response in GBM and cancer overall, while giving avenues for the development of novel treatments.

STEM-12. DISCOVERY THE ORIGIN-CELLS FOR HUMAN GLIOBLASTOMA GENESIS IN SUBVENTRICULAR ZONE

Abstract Human glioblastoma (GBM), originating from the subventricular zone (SVZ), occurs due to molecular disruptions in chromosomes. Most GBM tissues exhibit definitive chromosomal patterns: copy-number-variations (CNV) in chromosome 7 (gain) and 10 (loss), known as the earliest molecular events. Herein, we hypothesised that the origin-cells in SVZ of GBM patients can provide clues regarding these chromosomal alterations. We compared bulk RNA sequencing (RNAseq) data of GBM tumor tissue (n=126), tumour free GBM SVZ (n=40), and tumor-free control SVZ of non-glial tumor (n=9). Paired single-cell-level RNAseq samples of tumor free GBM SVZ (n=7) and GBM tumor tissue (n=10), were done to see cell specific CNVs. Using human SVZ and GBM samples as a background, we generated origin-cell and origin-cell-derived tumour cell using CRISPR/Cas9. In this work, we identified two GBM-origin-cell types with stem-cell signatures during single-cell level analyses of 60 SVZ tissue samples obtained from tumor-free regions of GBM patients. Furthermore, single-cell level analysis revealed that two origin-cell types in SVZ harbor ongoing patterns of CNV alterations. Among the origin-cells found in the SVZ of GBM patients, NO-like cells showed neural progenitor plus oligodendrocyte progenitor (NO) signature, while AN-like cells showed astrocyte plus neural stem cell (AN) signature. For the interconnectedness, we subjected single-cell-level RNA-seq data to ligand-receptor connection analysis. In the stem cell mode, NO-like cells was connected to AN-like cells in SVZ samples and while in the tumor samples, cycling cell was connected to AN-like cells. NO-like cells was common in TERT promoter wildtype GBM and AN-like cells was more common in TERT promoter mutant GBM. CRISPR/Cas9 models revealed accumulation copy-number alterations from non-tumorigenic origin-cells to tumor cells. These two origin-cells (NO-like cells and AN-like cells) derived from the SVZ of the adult human brain will facilitate the understanding of GBM genesis and development of potential novel therapeutic targets.