Murine Neuro-2a Cells Research Articles

Abstract 153: Noncoding elements of MYCN mRNA are powerful drivers on oncogenicity in neuroblastoma

Abstract Precision in genetic modeling of oncogene-driven cancer is crucial for comprehending disease pathology. Traditionally, models focused on protein expression through open-reading-frame (ORF) transgenes. This approach overlooks post-transcriptional regulation in endogenous mRNAs, which include noncoding elements like 5’ and 3’ UTRs and introns. All patient oncogene mRNAs encompass these elements, and grasping the collective impact of noncoding and protein coding components may revolutionize our comprehension of cancer origins.Neuroblastoma (NB) is the most common extracranial solid tumor in children. Genetic amplification of MYCN (MA) in NB results in very high mRNA levels and drives poor prognosis. MYCN mRNA is targeted by multiple microRNAs, including let-7. We previously demonstrated that in MA disease the 3’ UTR of MYCN can sequester let-7, inhibiting its function. MYCN mRNA thus also influences genetic patterning in NB by relieving the selective pressure to lose chromosome arms harboring let-7 loci, an event frequent in MYCN non-amplified NB but exceedingly rare in MA disease. This work established for the first time that noncoding mRNA elements within an oncogenic mRNA can itself contribute to disease pathology. To further understand the roles of noncoding elements within MYCN mRNA, we generated BALB/c 3T3 fibroblasts and murine Neuro-2a cells that express the following transgenes: 1.MYCN-ORF: expresses only MYCN open reading frame sequence.2.MYCN-GL: intact genetic locus of MYCN, expresses full length MYCN mRNA.3.M/GFP-GL: variant of #2 where MYCN protein coding sequences are replaced by EGFP. All three constructs demonstrated significantly enhanced in vitro proliferation compared to GFP control cells. Notably, cells expressing MYCN-GL exhibited best growth in both cell types, despite possessing an intact 3’UTR. Unexpectedly, M/GFP-GL cells demonstrated comparable growth to MYCN-ORF in 3T3s and significantly outperformed MYCN-ORF in Neuro-2a cells. This observation not only validates our prior research on the oncogenic role of the MYCN 3’ UTR but also extends it by revealing direct contributions to growth advantages, occasionally surpassing even MYCN protein. Syngeneic animal studies mirrored these trends, with MYCN-GL and M/GFP-GL inducing nearly 100% tumor incidence, surpassing the 57% incidence associated with MYCN-ORF. Tumors induced by MYCN-GL and M/GFP-GL were more than twice the size of those induced by MYCN-ORF. These observations underscore that full-length MYCN mRNA, encompassing all noncoding elements, serves as a more potent driver of cellular growth and oncogenicity than only MYCN protein expression. These findings have exciting implications for understating neuroblastoma pathology and genetic patterns, with broader implications for other oncogene-driven cancers. We are currently generating transgenic mouse models based on the above three constructs. Citation Format: Vishwa Patel, Lorraine Rana Benhamou, Dr. John T. Powers. Noncoding elements of MYCN mRNA are powerful drivers on oncogenicity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 153.

Soloxolone Methyl Reduces the Stimulatory Effect of Leptin on the Aggressive Phenotype of Murine Neuro2a Neuroblastoma Cells via the MAPK/ERK1/2 Pathway.

Despite the proven tumorigenic effect of leptin on epithelial-derived cancers, its impact on the aggressiveness of neural crest-derived cancers, notably neuroblastoma, remains largely unexplored. In our study, for the first time, transcriptome analysis of neuroblastoma tissue demonstrated that the level of leptin is elevated in neuroblastoma patients along with the severity of the disease and is inversely correlated with patient survival. The treatment of murine Neuro2a neuroblastoma cells with leptin significantly stimulated their proliferation and motility and reduced cell adhesion, thus rendering the phenotype of neuroblastoma cells more aggressive. Given the proven efficacy of cyanoenone-bearing semisynthetic triterpenoids in inhibiting the growth of neuroblastoma and preventing obesity in vivo, the effect of soloxolone methyl (SM) on leptin-stimulated Neuro2a cells was further investigated. We found that SM effectively abolished leptin-induced proliferation of Neuro2a cells by inducing G1/S cell cycle arrest and restored their adhesiveness to extracellular matrix (ECM) proteins to near control levels through the upregulation of vimentin, zonula occludens protein 1 (ZO-1), cell adhesion molecule L1 (L1cam), and neural cell adhesion molecule 1 (Ncam1). Moreover, SM significantly suppressed the leptin-associated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and ribosomal protein S6 kinase A1 (p90RSK), which are key kinases that ensure the survival and proliferation of cancer cells. Further molecular modeling studies demonstrated that the inhibitory effect of SM on the mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathway can be mediated by its direct interaction with ERK2 and its upstream regulators, son of sevenless homolog 1 (SOS) and mitogen-activated protein kinase kinase 1 (MEK1). Taken together, our findings in murine Neuro2a cells provide novel evidence of the stimulatory effect of leptin on the aggressiveness of neuroblastoma, which requires further detailed studies in human neuroblastoma cells and relevant animal models. The obtained results indicate that SM can be considered a promising drug candidate capable of reducing the impact of adipokines on tumor progression.

Anti-TNF-α restricts dengue virus-induced neuropathy.

Proinflammatory TNF-α facilitates dengue virus (DENV) infection in endovascular dysfunction and neurotoxicity. The introduction of TNF-α blocking therapy with Abs is performed to test its therapeutic effect in this study. In DENV-infected mice, TNF-α production in the brain accompanied the progression of neurotoxicity and encephalitis. DENV infection caused the loss of hippocampal neurons with TNF-α expression around damaged regions, and immunostaining showed the induction of apoptosis in hippocampal neurons. TNF-α was expressed in active microglia and astrocytes in DENV-infected mice. TNF-α facilitated DENV-induced neurotoxicity in vitro in murine Neuro-2a cells. Using a currently established encephalitic mouse model in which DENV infection causes progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days postinfection, we showed that TNF-α transgenic mice represented the progressive disease development and administration of neutralizing TNF-α Ab reduced dengue encephalitis and mortality. These results demonstrate an immunopathogenesis of TNF-α for mediating DENV-induced encephalitis-associated neurotoxicity and that targeting TNF-α can be used as a strategy against dengue encephalitis.

Targeting TNF-α against dengue virus-induced neurotoxicity and acute viral encephalitis-like symptoms

Abstract Pro-inflammatory tumor necrosis factor α (TNF-α) facilitates dengue virus (DENV) infection in endovascular dysfunction and neurotoxicity. The introduction of TNF-α blocking therapy with antibodies and antagonists is performed for testing its therapeutic effects in this study. In DENV-infected mice, TNF-α was produced in the brains accompanied by the progression of neurotoxicity and encephalitis. DENV infection caused loss in hippocampal neurons with TNF-α expression around the damaged regions. Additionally, immunostaining showed the induction of apoptosis in hippocampal neurons. TNF-α was expressed in active microglia as well as astrocytes of DENV-infected mice. TNF-α facilitated DENV-induced neurotoxicity in vitro in murine Neuro-2a cells. By using a currently established encephalitic mice model that DENV infection caused progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days post-infection, TNF-α transgenic mice represented the progressive disease development while administration of neutralizing TNF-α antibody reduced dengue encephalitis and mortality. These results demonstrate an immunopathogenesis of TNF-α for mediating DENV-induced encephalitis-associated neurotoxicity and targeting TNF-α can be used as a strategy against dengue encephalitis.

Pharmaco-toxicological effects of the novel third-generation fluorinate synthetic cannabinoids, 5F-ADBINACA, AB-FUBINACA, and STS-135 in mice. In vitro and in vivo studies.

5F-ADBINACA, AB-FUBINACA, and STS-135 are 3 novel third-generation fluorinate synthetic cannabinoids that are illegally marketed as incense, herbal preparations, or research chemicals for their psychoactive cannabis-like effects. The present study aims at investigating the in vitro and in vivo pharmacological activity of 5F-ADBINACA, AB-FUBINACA, and STS-135 in male CD-1 mice, comparing their in vivo effects with those caused by the administration of Δ9 -THC and JWH-018. In vitro competition binding experiments revealed a nanomolar affinity and potency of the 5F-ADBINACA, AB-FUBINACA, and STS-135 on mouse and human CB1 and CB2 receptors. Moreover, these synthetic cannabinoids induced neurotoxicity in murine neuro-2a cells. In vivo studies showed that 5F-ADBINACA, AB-FUBINACA, and STS-135 induced hypothermia; increased pain threshold to both noxious mechanical and thermal stimuli; caused catalepsy; reduced motor activity; impaired sensorimotor responses (visual, acoustic, and tactile); caused seizures, myoclonia, and hyperreflexia; and promoted aggressiveness in mice. Behavioral and neurological effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM 251. Differently, the visual sensory response induced by STS-135 was only partly prevented by the AM 251, suggesting a CB1 -independent mechanism. For the first time, the present study demonstrates the pharmaco-toxicological effects induced by the administration of 5F-ADBINACA, AB-FUBINACA, and STS-135 in mice and suggests their possible detrimental effects on human health.

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells.

The established primary trigger of Alzheimer's disease's is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane ( Koo et al., 1996 ; Ubelmann et al., 2017 ). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line ( Ubelmann et al., 2017 ).

Apolipoprotein B is a new target of the GDNF/RET and ET‐3/EDNRB signalling pathways

GDNF/RET and Endothelin-3 (ET-3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest-derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1. Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest-derived cell development, along with a mouse knock-in for the Hirschsprung-associated mutation Ret(C620R). Silencing for Apob and Ret has been performed via shRNA. GDNF/RET and ET-3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the Ret(C620R) mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53-dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression. Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.

Biosynthetically intriguing chlorinated lipophilic metabolites from geographically distant tropical marine cyanobacteria.

Five new vinylchlorine-containing metabolites, the lipoamides janthielamide A and kimbeamides A-C and the ketide-extended pyranone kimbelactone A, have been isolated from collections of marine cyanobacteria made in Curaçao and Papua New Guinea. Both janthielamide A and kimbeamide A exhibited moderate sodium channel blocking activity in murine Neuro-2a cells. Consistent with this activity, janthielamide A was also found to antagonize veratridine-induced sodium influx in murine cerebrocortical neurons. These lipoamides represent the newest additions to a relatively rare family of marine cyanobacterial-derived lipoamides and a new structural class of compounds exhibiting neuromodulatory activities from marine cyanobacteria.

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.

Immunomodulation with dendritic cells and donor lymphocyte infusion converge to induce graft vs neuroblastoma reactions without GVHD after allogeneic bone marrow transplantation.

Background:Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT.Methods:Mice bearing congenic (H2Ka) Neuro-2a tumours were grafted with allogeneic (H2Kb) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DCNeuro2a) were inoculated (on day +7) in conjunction with donor (H2Kb) and haploidentical (H2Ka/b) lymphocytes.Results:Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DCNeuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2Ka/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT.Conclsions:The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours.

Doppel-induced apoptosis and counteraction by cellular prion protein in neuroblastoma and astrocytes

Expression of a prion-like protein, doppel, induces apoptosis-like changes in cerebellar neuronal granule and Purkinje cells of prion-knockout mice and this effect can be rescued by re-introduction of cellular prion. Since most of those studies were done in transgenic mice, in the present study, we have established a murine neuro-2a cell line and the primary rat adult reactive astrocyte model for studying doppel-induced apoptosis and possible prion counteraction. We demonstrate that expression of doppel in neuro-2a cells causes apoptosis, during which DNA fragmentation occurs as visualized by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining and other intracellular changes characteristic of apoptosis are observed in the electron microscope. Using immunoblot analyses, we further demonstrate that doppel expression activates caspase-10 as well as caspase-3, but does not activate caspase-9. Addition of purified doppel to cultures of neuro-2a cells and the primary astrocytes causes similar apoptotic changes. Significantly, apoptosis induced by doppel is enhanced when cellular prion protein is depleted by RNA interference, suggesting a protective effect of cellular prion against doppel-induced apoptosis. The antagonistic interaction between cellular prion and doppel appears to involve direct protein–protein interaction possibly on cell membrane as cellular prion and doppel physically interact with each other and co-localize on cell membranes. Together, our data show that doppel induces apoptosis in neuroblastoma neuro-2a and rat primary astrocytes via a caspase-10 mediated pathway and that this effect is counteracted by cellular prion through direct interaction with doppel possibly on cell membrane.

Neurotoxic meroditerpenoids from the tropical marine brown alga Stypopodium flabelliforme.

Brine shrimp toxicity and TLC analysis guided the isolation of five new and biologically active meroditerpenoids [2beta,3alpha-epitaondiol (1), flabellinol (2), flabellinone (3), stypotriolaldehyde (4), and stypohydroperoxide (5)] along with five known compounds from the marine brown alga Stypopodium flabelliforme collected in Papua New Guinea. The planar structures of compounds 1-5 were determined by extensive spectroscopic analysis (1D and 2D NMR, LRMS, HRMS, IR, and UV), while relative configuration was determined by 1D and 2D NOE experiments. X-ray crystallography confirmed the relative configuration of 2beta,3alpha-epitaondiol (1), and the modified Mosher's ester method was used to establish its absolute configuration. All of the new metabolites were moderately toxic to murine neuro-2a cells (LC50 2-25 microM), and three [2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3)] possessed potent sodium channel blocking activity. Stypotriolaldehyde (4) had a biphasic effect on the concentration of intracellular Ca2+ in rat cerebellar granule neurons (CGN). The previously known compound, stypoldione (6), also modulated intracellular calcium concentration and was cytotoxic in CGN. Metabolites 2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3) displayed moderate cytotoxicity to the NCI-H460 human lung cancer cell line.

The transcription factor early growth response factor-1 (EGR-1) promotes apoptosis of neuroblastoma cells.

Early growth response factor-1 (EGR-1) is an immediate early gene, which is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. EGR-1 has been involved in diverse biological functions such as cell growth, differentiation and apoptosis. Here we report that enforced expression of the EGR-1 gene induces apoptosis, as determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) analysis, in murine Neuro2A cells. In accordance with this role of EGR-1 in cell death, antisense oligonucleotides increase cell viability in cells cultured in the absence of serum. This apoptotic activity of the EGR-1 appears to be mediated by p73, a member of the p53 family of proteins, since an increase in the amount of p73 is observed in clones stably expressing the EGR-1 protein. We also observed an increase in the transcriptional activity of the mdm2 promoter in cells overexpressing EGR-1, which is paralleled by a marked decrease in the levels of p53 protein, therefore excluding a role of this protein in mediating EGR-1-induced apoptosis. Our results suggest that EGR-1 is an important factor involved in neuronal apoptosis.