Neural Stem Cell Model Research Articles

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUNDOne of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders.AIMTo investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation.METHODSBrain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells.RESULTSIn agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.CONCLUSIONSmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

CSIG-06. ELUCIDATING THE ROLE OF CO-OCCURRING MUTATIONS IN FGFR1-DRIVEN PEDIATRIC LOW-GRADE GLIOMA

Abstract Pediatric low-grade gliomas (pLGGs) have excellent survival, however, with current standard of care, most patients suffer lifelong severe sequalae. pLGGs are almost exclusively driven by single activating mutations in the mitogen-activated protein kinase (MAPK) pathway. As targeted molecular therapy clinical trials focusing on BRAF-altered pLGGs are showing promising results in early clinical trials, similar efforts are underway for FGFR1-altered tumors. In our cohort of FGFR1-4 altered gliomas, we observe frequent occurrence of a second alteration associated with activation of the MAPK or mammalian target of rapamycin (mTOR) pathways in pLGGs. Most commonly in the phosphatase NF1, PIK3CA, PIK3R1 PTPN11 or within the FGFR1 gene itself. However, the impact of second co-occurring mutations on therapeutic response and resistance has not been explored. To address this, we established mouse neural stem cell models over-expressing hot-spot mutated FGFR1 alone or in combination with a second alteration. Immunoblotting revealed that the addition of a second alteration attenuated phosphorylation of ERK, AKT and S6 and influenced cell proliferation both in normal growth conditions and in absence of growth factor. Treatment with an FGFR inhibitor (Infigratinib) showed reduced drug response in double mutant cells compared to hot-spot mutated FGFR1 alone. This was associated with less reduction of phosphorylation of ERK and S6 in the double mutant cells upon treatment. In conclusion, the presence of a second alteration influences proliferation and drug response in models of FGFR1-mutated pLGG, potentially by modulating MAPK and mTOR signaling.

Early Life Irradiation-Induced Hypoplasia and Impairment of Neurogenesis in the Dentate Gyrus and Adult Depression Are Mediated by MicroRNA- 34a-5p/T-Cell Intracytoplasmic Antigen-1 Pathway.

Early life radiation exposure causes abnormal brain development, leading to adult depression. However, few studies have been conducted to explore pre- or post-natal irradiation-induced depression-related neuropathological changes. Relevant molecular mechanisms are also poorly understood. We induced adult depression by irradiation of mice at postnatal day 3 (P3) to reveal hippocampal neuropathological changes and investigate their molecular mechanism, focusing on MicroRNA (miR) and its target mRNA and protein. P3 mice were irradiated by γ-rays with 5Gy, and euthanized at 1, 7 and 120 days after irradiation. A behavioral test was conducted before the animals were euthanized at 120 days after irradiation. The animal brains were used for different studies including immunohistochemistry, CAP-miRSeq, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and western blotting. The interaction of miR-34a-5p and its target T-cell intracytoplasmic antigen-1 (Tia1) was confirmed by luciferase reporter assay. Overexpression of Tia1 in a neural stem cell (NSC) model was used to further validate findings from the mouse model. Irradiation with 5 Gy at P3 induced depression in adult mice. Animal hippocampal pathological changes included hypoplasia of the infrapyramidal blade of the stratum granulosum, aberrant and impaired cell division, and neurogenesis in the dentate gyrus. At the molecular level, upregulation of miR-34a-5p and downregulation of Tia1 mRNA were observed in both animal and neural stem cell models. The luciferase reporter assay and gene transfection studies further confirmed a direct interaction between miR-34a-5p and Tia1. Our results indicate that the early life γ-radiation-activated miR-34a-5p/Tia1 pathway is involved in the pathogenesis of adult depression. This novel finding may provide a new therapeutic target by inhibiting the miR-34a-5p/Tia1 pathway to prevent radiation-induced pathogenesis of depression.

The Beneficial Potential of Genetically Modified Stem Cells in the Treatment of Stroke: a Review.

The last two decades have witnessed a surge in investigations proposing stem cells as a promising strategy to treat stroke. Since growth factor release is considered as one of the most important aspects of cell-based therapy, stem cells over-expressing growth factors are hypothesized to yield higher levels of therapeutic efficiency. In pre-clinical studies of the last 15 years that were investigating the efficiency of stem cell therapy for stroke, a variety of stem cell types were genetically modified to over-express various factors. In this review we summarize the current knowledge on the therapeutic efficiency of stem cell-derived growth factors, encompassing techniques employed and time points to evaluate. In addition, we discuss several types of stem cells, including the recently developed model of epidermal neural crest stem cells, and genetically modified stem cells over-expressing specific factors, which could elevate the restorative potential of naive stem cells. The restorative potential is based on enhanced survival/differentiation potential of transplanted cells, apoptosis inhibition, infarct volume reduction, neovascularization or functional improvement. Since the majority of studies have focused on the short-term curative effects of genetically engineered stem cells, we emphasize the need to address their long-term impact.Graphical

Three-Dimensional Human Neural Stem Cell Models to Mimic Heparan Sulfate Proteoglycans and the Neural Niche.

Heparan sulfate proteoglycans (HSPGs) are a diverse family of polysaccharides, consisting of a core protein with glycosaminoglycan (GAG) side chains attached. The heterogeneous GAG side-chain carbohydrates consist of repeating disaccharides, with each side chain possessing a specific sulfation pattern. It is the variable sulfation pattern that allows HSPGs to interact with numerous ligands including growth factors, cytokines, chemokines, morphogens, extracellular matrix (ECM) glycoproteins, collagens, enzymes, and lipases. HSPGs are classified according to their localization within an individual cell, and include the membrane bound syndecans (SDCs) and glypicans (GPCs), with perlecan, agrin, and type-XVIII collagen secreted into the ECM. The stem cell niche is defined as the environment that circumscribes stem cells when they are in their naïve state, and includes the ECM, which provides a complex contribution to various biological processes during development and throughout life. These contributions include facilitating cell adhesion, proliferation, migration, differentiation, specification, and cell survival. In contrast, HSPGs play an anticoagulant role in thrombosis through being present on the luminal surface of cells, while also playing roles in the stimulation and inhibition of angiogenesis, highlighting their varied and systemic roles in cellular control. To fully understand the complexities of cell-cell and cell-matrix interactions, three-dimensional (3D) models such as hydrogels offer researchers exciting opportunities, such as controllable 3D in vitro environments, that more readily mimic the in vivo/in situ microenvironment. This review examines our current knowledge of HSPGs in the stem cell niche, human stem cell models, and their role in the development of 3D models that mimic the in vivo neural ECM.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

The effect of electrospun scaffolds on the glycosaminoglycan profile of differentiating neural stem cells

The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.

LGG-35. FUNCTIONAL GENOMIC APPROACHES TO IDENTIFY THERAPEUTIC TARGETS IN MYB AND MYBL1 EXPRESSING PEDIATRIC LOW-GRADE GLIOMAS

AIMRecurrent structural variants involving MYB and MYBL1 transcription factors were recently identified in pediatric low-grade gliomas (pLGGs), such as the MYB-QKI rearrangement in Angiocentric Gliomas and truncations of MYBL1 (MYBL1tr) in Diffuse Astrocytomas. However, therapeutic dependencies induced by these alterations remain unexplored.METHODOLOGYWe have generated in vitro pLGG mouse neural stem cell (NSCs) models engineered to harbor distinct MYB/MYBL1 genomic alterations. We used single cell RNA sequencing approaches to determine the transcriptional profile and dissect the central regulatory networks of our in vitro pLGG models over time. To identify specific genetic dependencies associated with MYB/MYBL1 mutations, we employed the Brie genome-wide mouse CRISPR lentiviral knockout pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes.RESULTS MYB/MYBL1 expression in neural stem cells induced activation of cell-cycle related, glioma-related and senescence-related pathways that are involved in normal development, including activation of MAPK and mTOR signaling which are also activated in human pLGG samples. Genome-scale CRISPR-cas9 screens in isogenic NSCs expressing MYB-QKI or MYBL1tr identified differential genetic dependencies relative to GFP controls. These included regulators of cell-cycle progression and several modulators of the ubiquitin-proteasome degradation pathway. Analysis of RNA-sequencing data from human tumors revealed several of these dependencies identified in the cell line model to be differentially expressed in MYB-altered pLGG tumors relative to normal brain.CONCLUSIONExpression of MYB family alterations induces expression of key developmental and oncogenic pathways and genetic dependencies that represent potential therapeutic targets for MYB or MYBL1 rearranged pLGGs.

DIPG-22. DISSECTING THE ONCOGENIC ROLE OF FOXR2 IN DIFFUSE INTRINSIC PONTINE GLIOMA

BACKGROUNDDiffuse intrinsic pontine gliomas (DIPGs) pose particular challenges for treatment. We recently completed a genomic analysis of close to 200 DIPGs and high-grade gliomas. We identified that nearly 10% of all DIPGs have increased expression of the fork head domain transcription factor FOXR2. We hypothesize that FOXR2 accelerates gliomagenesis in histone mutant DIPGs and represents a previously unexplored therapeutic target.METHODSTo determine whether FOXR2 is sufficient to mediate gliomagenesis, we applied an integrative genomics approach using both in vitro and in vivo DIPG models: mouse neural stem cell models expressing FOXR2, in vivo mouse models using in utero brainstem electroporation, patient-derived DIPG cell lines, and RNA sequencing analysis of human and mouse tumors expressing FOXR2.RESULTSOur data shows that FOXR2 indeed is an oncogene that rapidly accelerates gliomagenesis using an in vivo brainstem in utero electroporation model of DIPG. In human tumors, increased FOXR2 expression is mutually exclusive with MYC amplification suggesting functional redundancy. In vivo, FOXR2 results in large brainstem gliomas and rapid neurologic decline of animals. Transcriptional profiling of these tumors demonstrates activation of MYC signaling pathways. In vitro, we have further identified patient-derived cell lines with increased expression of FOXR2.CONCLUSION FOXR2 is sufficient to enhance gliomagenesis and represents a previously understudied therapeutic target for patients with the devastating disease DIPG.

The Nootropic Drug Α-Glyceryl-Phosphoryl-Ethanolamine Exerts Neuroprotective Effects in Human Hippocampal Cells

Brain aging involves changes in the lipid membrane composition that lead to a decrease in membrane excitability and neurotransmitter release. These membrane modifications have been identified as contributing factors in age-related memory decline. In this sense, precursors of phospholipids (PLs) can restore the physiological composition of cellular membranes and produce valuable therapeutic effects in brain aging. Among promising drugs, alpha-glycerylphosphorylethanolamine (GPE) has demonstrated protective effects in amyloid-injured astrocytes and in an aging model of human neural stem cells. However, the compound properties on mature neuronal cells remain unexplored. Herein, GPE was tested in human hippocampal neurons, which are involved in learning and memory, and characterized by a functional cholinergic transmission, thus representing a valuable cellular model to explore the beneficial properties of GPE. GPE induced the release of the main membrane phospholipids and of the acetylcholine neurotransmitter. Moreover, the compound reduced lipid peroxidation and enhanced membrane fluidity of human brain cells. GPE counteracted the DNA damage and viability decrease observed in in vitro aged neurons. Among GPE treatment effects, the autophagy was found positively upregulated. Overall, these results confirm the beneficial effects of GPE treatment and suggest the compound as a promising drug to preserve hippocampal neurons and virtually memory performances.

Abstract 3920: Differential effects of Jumonji family demethylase and EZH2 H3K27 methyltransferase inhibition on radiation sensitivity of H3K27M mutant DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with a 5-year survival of <1% (1). Up to 80% of DIPG tumors contain a specific K27M mutation in one of two genes encoding histone H3 (H3K27M). 60-75% of H3K27M mutations occur in gene encoding histone variant H3.3, which has been linked to reduced overall survival and poor response to first line radiation therapy (1). Recent evidence indicates that H3K27M inhibits the enzymatic activity of Polycomb Repressive Complex 2 through interaction with the EZH2 histone methyl transferase (2). A specific inhibitor of Jumonji family histone demethylase GSK-J4 was recently reported to restore H3K27 tri-methylation patterns in human DIPG cells and improve survival of H3K27M mutant orthotopic xenograft brainstem tumor models (3). Furthermore, a genetic deletion of EZH2 had also been shown to improve survival of H3K27M mouse model of DIPG, suggesting that EZH2 may be a potential therapeutic target (4). Given these data, we investigated the effects of Jumonji demethylase inhibitor GSK-J4 and EZH2 inhibitor tazemetostat on radiosensitization of human H3K27M DIPG cells. We found that GSK-J4 inhibitor had a growth inhibitory effect on H3K27M mutant DIPG cells as a single agent (96% growth inhibition at 1.5 micro-molar concentration, P<0.0001). Strikingly, GSK-J4 inhibitor also displayed radiosensitizing effects when combined with ionizing radiation at sub-micromolar concentrations of the drug (34.4% growth inhibition at 0.37 micro-molar concentration when combined with 4 Gy XRT vs 23.3% with XRT alone, P<0.0001 for both; no significant growth inhibition as single agent). Contrary to previously reported results with EZH2 inhibition in H3K27M neural stem cell model of DIPG, EZH2 inhibitor tazemetostat had no effect on human H3K27M DIPG cells as a single agent or when combined with ionizing radiation. This result is, however, not unexpected, given the proposed inhibitory effect of H3K27M mutation on EZH2 activity in DIPG disease pathogenesis. In conclusion, Jumonji family methyltransferase inhibitor GSK-4J sensitizes H3K27M DIPG cells to ionizing radiation, while EZH2 methyltransferase inhibitor has no effect. This result appears to be consistent with the recently proposed epigenetic mechanism of DIPG pathogenesis (1, 2). 1. Johung TB, et al. Diffuse intrinsic pontine glioma: new pathophysiological insights and emerging therapeutic targets. Curr Neuropharmacol. 2017;15:88-97. 2. Lewis PW, et al. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science. 2013;340:857-61. 3. Hashizume R, et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat Med. 2014;20:1394-6. 4. Mohammad F, et al. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med. 2017;23:483-492. Citation Format: Anatoly Y. Nikolaev, John B. Fiveash, Eddy S. Yang. Differential effects of Jumonji family demethylase and EZH2 H3K27 methyltransferase inhibition on radiation sensitivity of H3K27M mutant DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3920.

Downregulation of an Evolutionary Young miR-1290 in an iPSC-Derived Neural Stem Cell Model of Autism Spectrum Disorder.

The identification of several evolutionary young miRNAs, which arose in primates, raised several possibilities for the role of such miRNAs in human-specific disease processes. We previously have identified an evolutionary young miRNA, miR-1290, to be essential in neural stem cell proliferation and neuronal differentiation. Here, we show that miR-1290 is significantly downregulated during neuronal differentiation in reprogrammed induced pluripotent stem cell- (iPSC-) derived neurons obtained from idiopathic autism spectrum disorder (ASD) patients. Further, we identified that miR-1290 is actively released into extracellular vesicles. Supplementing ASD patient-derived neural stem cells (NSCs) with conditioned media from differentiated control-NSCs spiked with “artificial EVs” containing synthetic miR-1290 oligonucleotides significantly rescued differentiation deficits in ASD cell lines. Based on our earlier published study and the observations from the data presented here, we conclude that miR-1290 regulation could play a critical role during neuronal differentiation in early brain development.

Mechanical Characterization of Human Brain Tissue and Soft Dynamic Gels Exhibiting Electromechanical Neuro-Mimicry.

Synthetic hydrogels are an important class of materials in tissue engineering, drug delivery, and other biomedical fields. Their mechanical and electrical properties can be tuned to match those of biological tissues. In this work, hydrogels that exhibit both mechanical and electrical biomimicry are reported. The presented dual networks consist of supramolecular networks formed from 2:1 homoternary complexes of imidazolium-based guest molecules in cucubit[8]uril and covalent networks of oligoethylene glycol-(di)methacrylate. The viscoelastic properties of human brain tissues are also investigated. The mechanical properties of the dual network gels are benchmarked against the human tissue, and it is found that they both are neuro-mimetic and exhibit cytocompatibility in a neural stem cell model.

Choice of Alternative Polyadenylation Sites, Mediated by the RNA-Binding Protein Elavl3, Plays a Role in Differentiation of Inhibitory Neuronal Progenitors.

Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3′ untranslated region (3′UTR). Variations in length and sequence of the 3′UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3′UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3′UTRs during differentiation. Enriched expression of the longer 3′UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3′UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3′UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.

Effect of Angelica polysaccharide on brain senescence of Nestin-GFP mice induced by D-galactose

The incidence of neurodegenerative diseases is severely increasing with the aging. It has been proposed that NSCs (neural stem cells) help to control aging, but the mechanisms responsible remain unclear. Angelica polysaccharide is an active ingredient of Angelica sinensis in traditional Chinese medicine, which possesses versatile pharmacological activities including anti-oxidative and anti-aging effects. In this study, D-gal (D-galactose) was used to construct an aging model of Nestin-GFP transgenic mice brain tissues and NSCs. Mouse model was subcutaneously injected with D-gal, as we observed that mice consistently displayed acceleration of aging-like behavior change, energy metabolism decreased, the expression of aging-related genes was up-regulated. Conversely, aging retardation was achieved in Nestin-GFP mice Induced by D-gal that was locally injected with ASP (Angelica polysaccharide). Mechanistically, we isolated and cultured NSCs in vitro. ASP protected NSCs by increasing the cell proliferation; decreasing the number of SA-β-gal stained neurons; increasing the activity of SOD(superoxide dismutase) and T-AOC(total antioxidant capacity), decreasing the content of MDA(malondialdehyde); decreasing the levels of IL-1b,IL-6,TNF-a and ROS; and down-regulated the expression of cellular senescence associated genes p53, p21 in the aging NSCs. In conclusion, ASP can delay aging speed by protecting NSCs and promote neurogenesis by enhancing the antioxidant and anti-inflammatory capacity, up-regulation of p53/p21 signaling pathway. As to provide theoretical basis for treatment for brain aging related diseases, add new scientific connotation for “qi and blood theory” and “supplement blood and delay aging” of Traditional Chinese Medicine.

Abstract 3179: TORC1/2 inhibition sensitizes MYC-driven medulloblastoma cells to carboplatin chemotherapy

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of high medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. We performed an in silico analysis of these cells, which predicted that mTOR inhibitors would be potential therapeutic agents. The dual TORC1/2 inhibitor TAK228 (also known as MLN128 and INK128) is orally bioavailable and penetrates the brain. TAK228 is currently in early clinical trials for adults with glioblastoma. We hypothesized that TAK228 would have activity against MYC-driven medulloblastoma. We tested this hypothesis using the MYC amplified medulloblastoma cell lines D425 and D283 as well as our human neural stem cell models. We determined that TAK228 was effective in inhibiting both mTORC1/2 as measured by western blots showing decreased p-S6 and p-AKT473 expression. P-S6 expression is decreased by 90% and p-AKT expression is decreased by 70% in D425 cell lines treated with 20nM TAK228. We found that treatment with TAK228 decreases cell growth in D425 and D283 high-MYC medulloblatoma cell lines at concentrations of 20nM (D425 p<0.005 vs vehicle, D283 p<0.0005 vs vehicle). We also found that TAK228 induces apoptosis as measured by cleaved caspase 3 staining and an increased expression of cleaved PARP (D425 p<0.05 vs vehicle, D283 p<0.0005 vs vehicle). Because mTOR inhibition can suppress glutathione production, and glutathione is required to detoxify platinum containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin, which is part of the current standard treatment for high-risk medulloblastoma. TAK228 in combination with carboplatin inhibits cell growth as measured by MTS assay (D425 p<0.05 vs vehicle, D283 p<0.05 vs vehicle) and induces apoptosis as measured by cleaved PARP expression. Combination therapy of TAK228 and carboplatin treatment leads to a 7-fold increase in cleaved PARP compared to cells treated with the vehicle. In vivo testing in orthotopic xenografts is currently underway to determine the survival benefit of TAK228 in combination with carboplatin. TAK228 is effective at inhibiting growth and inducing apoptosis in high-MYC medulloblastoma cell lines and shows strong combinatorial efficacy with carboplatin. Citation Format: Rachael E. Maynard. TORC1/2 inhibition sensitizes MYC-driven medulloblastoma cells to carboplatin chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3179.

A Golgi lipid signaling pathway that controls neural stem cell polarity in the developing neocortex

The Golgi apparatus is a central regulator of membrane trafficking and cell polarity in mammalian cells. I will discuss our recent work in a murine embryonic neural stem cell (NSC) model demonstrating that a Golgi phosphatidylinositol 4‐phosphate (PI4P)‐dependent pathway promotes loading of the Golgi system into NSC apical processes. The physiological significance of this mechanism is demonstrated by defects in apical loading of the Golgi system resulting in cell‐autonomous disruption in NSC polarity and, when imposed on a tissue‐wide scale, catastrophic failure in development of the neocortex. This pathway requires the action of a pair of phosphatidylinositol transfer proteins (PITPNA and (PITPNB) to promote PI4P production on Golgi membranes, and the actions of downstream effector proteins that respond to PI4P signaling. These effectors include GOLPH3, the nonconventional myosin Myo18A, and the ceramide transfer protein CERT. Finally, we find the PI4P‐dependent regulation of Golgi apical positioning is transmitted to the differentiated progeny of the affected NSCs and influences later steps in development of the embryonic mouse neocortex. Taken together, we have identified a novel mechanism by which PITPs, PI4P, and Golgi apparatus position regulate cell polarity and stem cell biology in the developing mammalian forebrain.Support or Funding InformationThis research was funded by NIH grant R01‐GM112591 and grant BE‐0017 from the Robert A. Welch Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RIP140/PGC-1α axis involved in vitamin A-induced neural differentiation by increasing mitochondrial function

Vitamin A deficiency and mitochondrial dysfunction are both associated with neural differentiation-related disorders, such as Alzheimer’s disease (AD) and Down syndrome (DS). The mechanism of vitamin A-induced neural differentiation and the notion that vitamin A can regulate the morphology and function of mitochondria in its induction of neural differentiation through the RIP140/PGC-1α axis are unclear. The aim of this study was to investigate the roles and underlying mechanisms of RIP140/PGC-1α axis in vitamin A-induced neural differentiation. Human neuroblastoma cells (SH-SY5Y) were used as a model of neural stem cells, which were incubated with DMSO, 9-cis-retinoic acid (9-cis-RA), 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (at-RA). Neural differentiation of SH-SY5Y was evaluated by Sandquist calculation, combined with immunofluorescence and real-time polymerase chain reaction (PCR) of neural markers. Mitochondrial function was estimated by ultrastructure assay using transmission electron microscopy (TEM) combined with the expression of PGC-1α and NEMGs using real-time PCR. The participation of the RA signaling pathway was demonstrated by adding RA receptor antagonists. Vitamin A derivatives are able to regulate mitochondrial morphology and function, and furthermore to induce neural differentiation through the RA signaling pathway. The RIP140/PGC-1α axis is involved in the regulation of mitochondrial function in vitamin A derivative-induced neural differentiation.

Glypican-2 levels in cerebrospinal fluid predict the status of adult hippocampal neurogenesis

Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis in vivo. Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.

Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein

Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the gene encoding the microtubule-associated protein TAU (MAPT). Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 genome editing in human induced pluripotent stem (iPS) cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs derived from one patient carrying the N279K MAPT mutation, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU isoforms, demonstrated increased vulnerability to oxidative stress and elevated protein ubiquitination and exhibited disease-associated changes in transcriptome profiles when compared to astrocytes derived from one control individual and to the isogenic control. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study highlights the utility of iPS cell-derived NPCs to elucidate the role of astrocytes in the pathogenesis of FTD.