H4 Cells Research Articles

Compression force promotes glioblastoma progression through the Piezo1‑GDF15‑CTLA4 axis.

Glioma, a type of brain tumor, is influenced by mechanical forces in its microenvironment that affect cancer progression. However, our understanding of the contribution of compression and its associated mechanisms remains limited. The objective of the present study was to create an environment in which human brain glioma H4 cells experience pressure and thereby investigate the compressive mechanosensors and signaling pathways. Subsequent time‑lapse imaging and wound healing assays confirmed that 12 h of compression significantly increased cell migration, thereby linking compression with enhanced cell motility. Compression upregulated the expression of Piezo1, a mechanosensitive ion channel, and growth differentiation factor 15 (GDF15), a TGF‑β superfamily member. Knockdown experiments targeting PIEZO1 or GDF15 using small interfering RNA resulted in reduced cell motility, with Piezo1 regulating GDF15 expression. Compression also upregulated CTLA4, a critical immune checkpoint protein. The findings of the present study therefore suggest that compression enhances glioma progression by stimulating Piezo1, promoting GDF15 expression and increasing CTLA4 expression. Thus, these findings provide important insights into the influence of mechanical compression on glioma progression and highlight the involvement of the Piezo1‑GDF15 signaling pathway. Understanding tumor responses to mechanical forces in the brain microenvironment may guide the development of targeted therapeutic strategies to mitigate tumor progression and improve patient outcomes.

Stress granules formation in HEI-OC1 auditory cells and in H4 human neuroglioma cells secondary to cisplatin exposure

Abstract Stress granules (SGs) are highly dynamic micromolecular membraneless condensates that generate in cells subjected to stress. Formed from pools of untranslating messenger ribonucleoproteins (RNP), SGs dynamics constitute vital processes essential for cell survival. Here, we investigate whether established cytotoxic agents, such as the platinum-based chemotherapeutic agent cisplatin and the aminoglycoside antibiotic gentamicin, elicit SG formation in the House Ear Institute-Organ of Corti-1 (HEI-OC1) auditory cell line, H4 human neuroglioma cells and HEK-293T human embryonic kidney cells. Cells were treated with cisplatin or gentamicin for specific durations at designated concentrations. SG formation was assessed using immunocytochemistry and live cell imaging. Levels of essential proteins involved in SG assembly were evaluated using immunoblotting. We observed cisplatin-associated SG assembly in HEI-OC1 and H4 cells via confocal microscopy through antibody colabeling of G3BP1 with PABP or Caprin1. While maintaining an unchanged pattern of expression of main constituent SG proteins, cisplatin-related SGs in H4 cells persisted for at least 12 h after drug removal. Cells subjected to gentamicin exposure did not exhibit SGs. Our findings offer insights into subcellular mechanisms related to cisplatin-associated cytotoxicity, highlighting the need for future studies to further investigate this stress-response mechanism.

High-throughput screening for small-molecule stabilizers of misfolded glucocerebrosidase in Gaucher disease and Parkinson’s disease

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.

5-Aminolevulinic acid-mediated photodynamic therapy in combination with kinase inhibitor lapatinib enhances glioblastoma cell death

5-Aminolevulinic acid (ALA) is an intraoperative imaging agent approved for protoporphyrin IX (PpIX) fluorescence-guided resection of glioblastoma (GBM). It is currently under clinical evaluation for photodynamic therapy (PDT) after the completion of GBM surgery. We previously showed that lapatinib, a clinical kinase inhibitor of epidermal growth factor receptor 1 & 2 (EGFR and HER2), enhanced PpIX fluorescence in a panel of GBM cell lines by blocking ABCG2 (ATP-binding cassette super-family G member 2)-mediated PpIX efflux, which suggests its potential for improving ALA for GBM surgery and PDT. Here we show that lapatinib enhanced PDT-induced cytotoxicity by promoting GBM cell death with the induction of apoptosis followed by necrosis. While the induction of tumor cell apoptosis was massive and rapid in the H4 cell line with no detectable Bcl-2 and a low level of Bcl-xL, it was delayed and much less in extent in A172, U-87 and U-118 cell lines with higher levels of pro-survival Bcl-2 family proteins. Lapatinib treatment alone neither reduced GBM cell viability nor had any significant effect on EGFR downstream signaling. Its enhancement of ALA–PDT was largely due to the increase of intracellular PpIX particularly in the mitochondria, resulting in the activation of mitochondria-mediated apoptosis in H4 cells. Our present study demonstrates that lapatinib inhibits ABCG2-mediated PpIX efflux and sensitizes GBM cells to ALA–PDT by inducing tumor cell death.

Metabolomic and Proteomic Analysis of ApoE4-Carrying H4 Neuroglioma Cells in Alzheimer's Disease Using OrbiSIMS and LC-MS/MS.

Growing clinical evidence reveals that systematic molecular alterations in the brain occur 20 years before the onset of AD pathological features. Apolipoprotein E4 (ApoE4) is one of the most significant genetic risk factors for Alzheimer's disease (AD), which is not only associated with the AD pathological features such as amyloid-β deposition, phosphorylation of tau proteins, and neuroinflammation but is also involved in metabolism, neuron growth, and synaptic plasticity. Multiomics, such as metabolomics and proteomics, are applied widely in identifying key disease-related molecular alterations and disease-progression-related changes. Despite recent advances in the development of analytical technologies, screening the entire profile of metabolites remains challenging due to the numerous classes of compounds with diverse chemical properties that require different extraction processes for mass spectrometry. In this study, we utilized Orbitrap Secondary Ion Mass Spectrometry (OrbiSIMS) as a chemical filtering screening tool to examine molecular alterations in ApoE4-carried neuroglioma cells compared to wild-type H4 cells. The findings were compared using liquid chromatography (LC)-MS/MS targeted metabolomics analysis for the confirmation of specific metabolite classes. Detected alterations in peptide fragments by OrbiSIMS provided preliminary indications of protein changes. These were extensively analyzed through proteomics to explore ApoE4's impact on proteins. Our metabolomics approach, combining OrbiSIMS and LC-MS/MS, revealed disruptions in lipid metabolism, including glycerophospholipids and sphingolipids, as well as amino acid metabolism, encompassing alanine, aspartate, and glutamate metabolism; aminoacyl-tRNA biosynthesis; glutamine metabolism; and taurine and hypotaurine metabolism. Further LC-MS/MS proteomics studies confirmed the dysfunction in amino acid and tRNA aminoacylation metabolic processes, and highlighted RNA splicing alterations influenced by ApoE4.

SORL1 regulates tau trafficking

AbstractBackgroundWhile LRP1 is a cellular receptor that mediates tau uptake and potentiates tau seeding, we observed LRP1‐independent uptake of tau in LRP1‐genetically deficient cells and hypothesized that the sortilin‐related receptor (SORL1) could also be involved in tau processing.MethodSurface plasmon resonance was used to assess tau binding to SORL1. 125I‐labelled tau uptake assays were used to investigate the role of SORL1 in endocytosis. The P301S FRET biosensor was used to assess SORL1’s role in cytosolic tau seeding, using both WT and AD‐associated mutations in the SORL1 gene (G511R and N1358S). Confocal microscopy was used to investigate colocalization and intracellular trafficking of tau and SORL1. Finally, we investigated the impact of SORL1 on tau processing in vivo by injecting human pathogenic tau isolated from AD patients into the hippocampus of SORL1 knockout vs wildtype mice and analyzing via immunohistochemistry.ResultWe confirmed high‐affinity binding of tau to SORL1 and found that fluorescently labeled tau colocalizes with overexpressed SORL1 in H4 neuroglioma cells. When we knocked down SORL1 expression using siRNA in H4 cells, we observed a significant increase in the LRP1‐mediated degradation of 125I‐labeled tau, revealing that SORL1 directs tau away from lysosomal degradative pathways. To determine if this occurs in vivo, we injected pathogenic forms of human tau into the hippocampal region of WT and SORL1‐deficient mice. Two days following injection, preliminary results suggest less human tau retention in the brains of SORL1‐deficient mice when compared to WT controls. Together, these data suggest that SORL1 may have a dual function in tau metabolism: impacting both uptake of tau, and trafficking of pathogenic tau to lysosomes. We also found that SORL1 promotes cytosolic tau seeding induced by pathogenic forms of tau. The AD‐associated N1358S mutation SORL1 gene demonstrated a significant increase in tau seeding when compared with WT SORL1.ConclusionThese studies identify SORL1 as a sorting receptor for tau that contributes to seeding of pathogenic tau. Furthermore, they indicated that SORL1 directs tau away from lysosomal degradation pathways, and identify promotion of tau seeding as a potential mechanism by which the SORL1 N1358S mutation contributes to AD.

Investigating ApoE4‐mediated metabolic alteration of H4 neuroglioma cells of Alzheimer’s disease

AbstractBackgroundMetabolites and proteins in ApoE4‐carried H4 neuroglioma cells were analyzed to investigate the potential mechanism of the risk gene ApoE4 involved in Alzheimer’s disease (AD).MethodMetabolomics of ApoE4‐carried and wild‐type H4 cells was performed by applying a novel workflow composed of Orbital trapping secondary ion mass spectrometry (OrbiSIMS) as a screening tool, followed by LC‐MS/MS targeted metabolomics for further confirming polar molecules. Proteins affected by ApoE4 were identified by performing LC‐MS‐based proteomics, followed by investigating the protein network and functional enrichment analysis using the STRING database.ResultThe initial OrbiSIMS approach has shown the advantages of detecting large numbers of metabolites with minimal sample preparation, small sample size and a relatively rapid analysis time, allowing 192 putatively annotated metabolites detected in our study. We found glycerophospholipid metabolism, aspartate, glutamate and alanine are the most affected pathways by ApoE4. Polar‐targeted metabolomics of LC‐MS/MS further revealed that the metabolism of taurine and hypotaurine is also affected by ApoE4. Besides, proteomics results suggest an overall metabolic process, RNA splicing process, and translation associated with ApoE4‐related AD development.ConclusionOur results have elucidated the metabolic alteration of H4 neuroglioma cells in the presence of ApoE4, supporting the hypothesis of the low ability of ApoE4 in transporting lipids and discovering two new potential pathways that may be involved in AD. In addition, proteomics analysis by LC‐MS further proved the ApoE4‐mediated dysfunction of nitrogen compound metabolism and RNA splicing process.

A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

BackgroundMesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.MethodsWe used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.ResultsOur results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.ConclusionsThe MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

Long term administration of loquat leaves and their major component, ursolic acid, attenuated endogenous amyloid-β burden and memory impairment

Loquat (Eriobotrya japonica) leaves contain many bioactive components such as ursolic acid (UA) and amygdalin. We investigated the effects of loquat leaf powder and methanol extract in human neuroglioma H4 cells stably expressing the Swedish-type APP695 (APPNL-H4 cells) and C57BL/6 J mice. Surprisingly, the extract greatly enhanced cellular amyloid-beta peptide (Aβ) 42 productions in APPNL-H4 cells. Administration of leaf powder increased Aβ42 levels after 3 months and decreased levels after 12 months compared to control mice. Leaf powder had no effect on working memory after 3 months, but improved working memory after 12 months. Administration of UA decreased Aβ42 and P-tau levels and improved working memory after 12 months, similar to the administration of leave powder for 12 months. Amygdalin enhanced cellular Aβ42 production in APPNL-H4 cells, which was the same as the extract. Three-month administration of amygdalin increased Aβ42 levels slightly but did not significantly increase them, which is similar to the trend observed with the administration of leaf powder for 3 months. UA was likely the main compound contained in loquat leaves responsible for the decrease in intracerebral Aβ42 and P-tau levels. Also, amygdalin might be one of the compounds responsible for the transiently increased intracerebral Aβ42 levels.

BDE209-promoted Dio2 degradation in H4 glioma cells through the autophagy pathway, resulting in hypothyroidism and leading to neurotoxicity

Decabromodiphenyl ether (BDE209), the homologue with the highest number of brominates in polybrominated diphenyl ethers (PBDEs), is one of the most widespread environmental persistent organic pollutants (POPs) due to its mass production and extensive application in recent decades. BDE209 is neurotoxic, possibly related to its interference with the thyroid hormone (TH) system. However, the underlying molecular mechanisms of BDE209-induced TH interference and neurobehavioral disorders remains unknown. Here, we explored how BDE209 manipulated the major enzyme, human type II iodothyronine deiodinase (Dio2), that is most important in regulating local cerebral TH equilibrium by neuroglial cells, using an in vitro model of human glioma H4 cells. Clonogenic cell survival assay and LC/MS/MS analysis showed that BDE209 could induce chronic neurotoxicity by inducing TH interference. Co-IP assay, RT-qPCR and confocal assay identified that BDE209 destroyed the stability of Dio2 without affecting its expression, and promoted its binding to p62, thereby enhancing its autophagic degradation, thus causing TH metabolism disorder and neurotoxicity. Furthermore, molecular docking studies predicted that BDE209 could effectively suppress Dio2 activity by competing with tetraiodothyronine (T4). Collectively, our study demonstrates that BDE209-induced Dio2 degradation and loss of its enzymatic activity in neuroglial cells are the fundamental pathogenic basis for BDE209-mediated cerebral TH disequilibrium and neurotoxicity, providing a target of interest for further investigation using glial/neuronal cell co-culture system and in vivo models.

Haploinsufficiency of progranulin causes cell type specific impairments in PINK1/Parkin mitophagy

AbstractBackgroundGRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia.MethodsWe investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC).ResultsLower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons.ConclusionsThe results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.

Human Cancer Cell Radiation Response Investigated through Topological Analysis of 2D Cell Networks

Clonogenic assays are routinely used to evaluate the response of cancer cells to external radiation fields, assess their radioresistance and radiosensitivity, estimate the performance of radiotherapy. However, classic clonogenic tests focus on the number of colonies forming on a substrate upon exposure to ionizing radiation, and disregard other important characteristics of cells such their ability to generate structures with a certain shape. The radioresistance and radiosensitivity of cancer cells may depend less on the number of cells in a colony and more on the way cells interact to form complex networks. In this study, we have examined whether the topology of 2D cancer-cell graphs is influenced by ionizing radiation. We subjected different cancer cell lines, i.e. H4 epithelial neuroglioma cells, H460 lung cancer cells, PC3 bone metastasis of grade IV of prostate cancer and T24 urinary bladder cancer cells, cultured on planar surfaces, to increasing photon radiation levels up to 6 Gy. Fluorescence images of samples were then processed to determine the topological parameters of the cell-graphs developing over time. We found that the larger the dose, the less uniform the distribution of cells on the substrate—evidenced by high values of small-world coefficient (cc), high values of clustering coefficient (cc), and small values of characteristic path length (cpl). For all considered cell lines, sw>1 for doses higher or equal to 4 Gy, while the sensitivity to the dose varied for different cell lines: T24 cells seem more distinctly affected by the radiation, followed by the H4, H460 and PC3 cells. Results of the work reinforce the view that the characteristics of cancer cells and their response to radiotherapy can be determined by examining their collective behavior—encoded in a few topological parameters—as an alternative to classical clonogenic assays.

“Anti-cancer” effect of ketamine in comparison with MK801 on neuroglioma and lung cancer cells

Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, is commonly used to induce anaesthesia during cancer surgery and relieve neuropathic and cancer pain. This study was conducted to assess whether ketamine has any inhibiting effects on neuroglioma (H4) and lung cancer cells (A549) in vitro.The cultured H4 and A549 cells were treated with ketamine and MK801 (0.1, 1, 10, 100, or 1000 μM) for 24 h. The expressions of glutamate receptors on both types of cancer cells were assessed with qRT-PCR. In addition, cell proliferation and migration were assessed with cell counting Kit-8 and wound healing assays. Cyclin D1, matrix metalloproteinase 9 (MMP9), phosphorylation of extracellular signal-regulated kinase (pERK), and cleaved-caspase-3 expression together with reactive oxygen species (ROS) were also assessed with Western blot, immunostaining, and/or flowcytometry.NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors were expressed on both H4 and A549 cells. Ketamine inhibited cancer cell proliferation and migration in a dose-dependent manner by suppressing the cell cycle and inducing apoptosis. Ketamine decreased cyclin D1, pERK, and MMP9 expression. In addition, ketamine increased ROS and cleaved caspase-3 expression and induced apoptosis. The anti-cancer effect of ketamine was more pronounced in A549 cells when compared with H4 cells. MK801 showed similar effects to those of ketamine.Ketamine suppressed cell proliferation and migration in both neuroglioma and lung cancer cells, likely through the antagonization of NMDA receptors.

EPCO-33. DIFFERENTIAL EXPRESSION OF AN ENDOGENOUS RETROVIRAL ELEMENT [HERV-K(HML-6)] IS ASSOCIATED WITH REDUCED SURVIVAL IN GLIOBLASTOMA PATIENTS

Abstract INTRODUCTION Comprising approximately 8% of our genome, Human Endogenous RetroViruses (HERVs) represent a class of germline retroviral infections that are regulated through epigenetic modifications. In cancer cells, which often have epigenetic dysregulation, HERVs have been implicated as potential oncogenic drivers. However, their role in gliomas is not known.Given the link between HERV expression in cancer cell lines and the distinct epigenetic dysregulation in gliomas, we utilized a tailored bioinformatic pipeline to characterize and validate the glioma retrotranscriptome and correlate HERV expression with locus-specific epigenetic modifications.Method:A custom workflow was used to quantify HERV expression in our cell lines of interest. Cell-line methylation was quantified using a custom script. We generated primers specific for the Human endogenous Mouse mammary tumor (MMTV)-Like virus 6 (HML-6). Visualization of RNA transcripts was performed using RNA-scope. Clinical data was obtained using the R package, TCGABiolinks. RESULTS The A172 cell line had significantly higher mean overall HERV expression relative to the M059J and H4 cell lines (p< 0.0001 for both). A172 cells had significantly lower mean number of CpG islands relative to M059J cells and H4 cells (p< 0.0001 for both). There was a significant inverse correlation between mean beta value and FC HERV expression (R=-0.57, p=0.01). qPCR confirmed robust expression of the HML-6 locus in cell culture and neurospheres. Elevated ERVK3-1 expression was associated reduced survival among IDHwt GBM patients (18.3 vs. 15.1 months, p=0.039). This was preserved among IDH mutant (IDHm) GBM as well (17.9 months vs. 14.0 months, p=0.0088). CONCLUSION In gliomas, HERV expression correlates with loss of DNA methylation at HERV loci. HML-6 is overexpressed in highly invasive glioblastoma cell lines and patient-derived neurospheres. We have demonstrated a potential survival detriment associated with elevated expression of the HML-6 product, ERVK3-1.

Identification of Novel Kinases of Tau Using Fluorescence Complementation Mass Spectrometry (FCMS)

Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using invitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.

JM-20, a Benzodiazepine-Dihydropyridine Hybrid Molecule, Inhibits the Formation of Alpha-Synuclein-Aggregated Species.

Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Hsa_circ_0076931 suppresses malignant biological properties, down-regulates miR-6760-3p through direct binding, and up-regulates CCBE1 in glioma.

The function of circular RNAs (circRNAs) in gliomas is as yet unknown. The present study explored role of hsa_circ_0076931 in glioma. circRNA expression profiles were identified via RNA-seq followed by qRT-PCR validation in three pairs of glioma and normal brain tissues (NBT). The function of hsa_circ_0076931 was investigated in vitro using cell lines as well as in vivo using a xenograft tumor. Hsa_circ_0076931 was up-regulated by overexpression and an mRNA profile compared with wild-type was identified by RNA-seq. The relationship between miR-6760-3p and hsa_circ_0076931 or CCBE1 was confirmed via luciferase reporter or AGO2-RIP assays. A total of 507 circRNAs were identified in glioma tissues that were differentially expressed compared with that in NBT, and the sequencing data were deposited in BioProject (ID: PRJNA746438). Hsa_circ_0007694 and hsa_circ_0008016 were memorably increased whereas hsa_circ_0076931 and hsa_circ_0076948 decreased in glioma compared with those in NBT. Additionally, hsa_circ_0076931 expression was negatively correlated with histological grade. Overexpression of hsa_circ_0076931 inhibited proliferation, migration, and invasion while promoting apoptosis of glioma cells. A total of 4383 and 537 aberrantly expressed genes were identified between the hsa_circ_0076931-overexpressed and control groups in H4 and U118-MG cells, respectively; the sequencing data were deposited in BioProject (ID: PRJNA746438). These differentially expressed genes were mainly enriched in cancer-related pathways. In addition, elevated hsa_circ_0076931 levels induced the expression of CCBE1 while suppressing miR-6760-3p expression. miR-6760-3p can bind to hsa_circ_0076931. The experimental evidence supports using hsa_circ_0076931 as a marker for glioma and to help prevent malignant progression. The mechanism might be relevant to miR-6760-3p and CCBE1.

N6-methyladenosine(m6A) demethylase FTO regulates cellular apoptosis following cobalt-induced oxidative stress

Cobalt is an environmental toxicant that is known to damage human health. However, the molecular mechanisms underlying cobalt-induced neurotoxicity have not been elucidated in detail. In the present research, we used human neuroglioma H4 cells as an in vitro model. Cells were exposed to CoCl2 (0, 100, 200, 400 μM) for 24 h. We performed m6A sequencing techniques and constructed FTO-knockdown/FTO-overexpressing cells to investigate the role of FTO-mediated m6A modification in regulating apoptosis following CoCl2 induced oxidative stress. Our study has shown CoCl2 exposure led to the decrease of demethylase FTO as well as elevated oxidative stress. However, NAC treatment could partly reverse the reduction of FTO expression as well as the degree of ROS via eliminating oxidative stress. Meanwhile, MeRIP-seq and RNA-seq further revealed the potential function m6A modification in regulating apoptosis. More importantly, KEGG pathway and Gene ontology (GO) analyses further elucidated that the differentially m6A-modified genes were aggregated in apoptosis-related pathways. Mechanistic analysis indicated that knockdown of FTO facilitated CoCl2-induced apoptosis via caspase activation and G1/S cell cycle arrest. Nevertheless, overexpression of FTO partly attenuated the increased apoptosis following CoCl2 exposure. More notably, we observed that FTO regulated apoptosis in an m6A-dependent manner. Therefore, our findings reveal that CoCl2 induced ROS affected the m6A modification of apoptosis-related genes by decreasing the expression of FTO, thereby resulting in the activation of apoptosis. These findings provide important insights into CoCl2-induced apoptosis and m6A modification and propose a novel strategy for studying environmental toxicant-related neurodegeneration.

Mass Spectrometry and Computer Simulation Predict the Interactions of AGPS and HNRNPK in Glioma.

Ether lipids are overexpressed in malignant tumor and play an important role in tumor process. Glioma is the most common malignant central nervous system tumor, and the content of ether lipids is higher than that of normal tissues. Alkylglycerone phosphate synthase (AGPS) is a key enzyme in the synthesis of ether esters and plays a vital role in maintaining the morphology and pathogenic properties of tumor cells. The cell proliferation and the content of tumor-related lipid such as monoalkylglycerol ether (MAGe), lysophosphatidic acid ether (LPAe), lysophosphatidylcholine ether (LPCe), lysophosphatidylethanolamine ether (LPEe), phosphatidyl inositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were suppressed after AGPS silencing in U251, H4, and TJ905 cells; however, heterogeneous nuclear ribonucleoprotein K (HNRNPK) could reverse the above phenomenon such as cellar proliferation and ether lipid secretion. We found that HNRNPK was the target protein of AGPS by coimmunoprecipitation and mass spectrometry assay and verified by western blot assay in U251 cells. It confirmed that AGPS and HNRNPK are coexpressed in the cellular nucleus by a confocal laser microscope. The main protein-protein interaction mechanism between AGPS and HNRNPK is hydrogen bond, conjugation bond, hydrophobic bond, and electrostatic force by computer simulation prediction.