Search Results (133)

α-Latrotoxin stimulates neurotransmitter release by binding to a presynaptic receptor and then forming ion-permeable membrane pores and/or stimulating the receptor, latrophilin-1, or Adhesion G-protein-coupled receptor type L1 (ADGRL1). To avoid pore formation, we use the mutant α-latrotoxin (LTX^N4C), which does not form pores and only acts through ADGRL1. ADGRL1 is cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), which behave as independent cell-surface proteins, reassociating upon binding LTX^N4C. We investigated the role of the NTF-CTF association in LTX^N4C action, using perfluorooctanoic acid (PFOA). We demonstrate that at low concentrations (≤100 μM) PFOA does not adversely affect ADGRL1-expressing neuroblastoma cells or inhibit LTX^N4C binding. However, it causes the dissociation of the NTF-CTF complexes, independent redistribution of the fragments on the cell surface, and their separate internalization. PFOA also promotes the dissociation of NTF-CTF complexes induced by LTX^N4C binding. When applied to mouse neuromuscular junctions, PFOA inhibits LTX^N4C-induced neurotransmitter release in a concentration-dependent manner. Our results indicate that ADGRL1 can mediate LTX^N4C signaling only while its fragments remain associated. These findings explain some aspects of receptor-dependent toxin action and contribute to a mechanistic understanding of ADGRL1 functions in neurons. Full article

SH-SY5Y neuroblastoma cells can be effectively differentiated into a neuronal phenotype using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), making them a valuable in vitro model for studying neuronal differentiation. This study aimed to investigate the electrophysiological properties of SH-SY5Y cells following prolonged differentiation, with a focus on membrane characteristics, evoked action potentials, and the functionality of cellular components such as N-methyl-D-aspartate (NMDA) receptor. Whole-cell patch-clamp recordings were employed to evaluate ionic currents and action potentials in embryonic mouse cortical neurons (mCNs) and in both differentiated and undifferentiated SH-SY5Y neuroblastoma cells. Differentiated SH-SY5Y cells exhibited neurite outgrowth, evoked action potential firing, and functional NMDA receptor-mediated currents. Notably, atorvastatin significantly modulated the duration and firing of action potentials as well as NMDA receptor-mediated currents in differentiated SH-SY5Y cells. These findings highlight that neuronally differentiated SH-SY5Y cells expressing functional NMDA receptor-mediated currents serve as a robust and convenient model for investigating the molecular mechanisms of NMDA receptor function and for screening pharmacological agents targeting these receptors. Full article

Basic and translational cancer biology research requires model systems that recapitulate the features of human tumors. While two-dimensional (2D) cell cultures have been foundational and allowed critical advances, they lack the organizational complexity, cellular interactions, and extracellular matrix present in vivo. Mouse models have thus remained the gold standard for studying cancer. In addition to high cost and low throughput, mouse models can also suffer from reduced tumor heterogeneity and species-specific differences. Three-dimensional (3D) culture models have emerged as a key intermediary between 2D cell lines and mouse models, with lower cost and greater flexibility than mouse models and a more accurate representation of the tumor microenvironment than 2D cell lines. In neuroblastoma, an aggressive childhood cancer, 3D models have been applied to study drug responses, cell motility, and tumor–matrix interactions. Recent advances include the integration of immune cells for immunotherapy studies, mesenchymal stromal cells for tumor–stroma interactions, and bioprinted systems to manipulate matrix properties. This review examines the use of 3D culture systems in neuroblastoma, highlighting their advantages and limitations while emphasizing their potential to bridge gaps between in vitro, preclinical, and clinical applications. By improving our understanding of neuroblastoma biology, 3D models hold promise for advancing therapeutic strategies and outcomes in this childhood cancer. Full article

The neuronal progenitor NG108-15 neuroblastoma x glioma cell line proliferates indefinitely in vitro and is capable of directed differentiation into cholinergic neurons. The cell line is a robust model for investigating neuronal differentiation and function in vitro. The lineage-specific transcription factor-mediated differentiation of pluripotent stem cell lines (PSCs) leads to more rapid, efficient, and functional neurons. In this study, we tested the hypothesis that transcription factors could also drive the fate of an immortalised cell line. We first established a stable NG108-15 cell line, by piggyBac (pBac) transposition, that conditionally expresses neurogenin-2 (Ngn2), a common transcription factor for specifying neuronal fate. Following doxycycline-induction of Ngn2, we observed more rapid and efficient differentiation, and improved neurite outgrowth and viability compared with the WT cell line. Moreover, when co-cultured with C2C12 mouse myotubes, the modified NG108-15 cells resulted in significantly larger acetylcholine receptor (AChR) aggregates, suggesting enhanced neuromuscular junction (NMJ) formation. These findings describe a novel methodology for differentiating NG108-15 cells more efficiently, to enhance the usefulness of the cell line as a motor neuron model. Full article

The combination of chemotherapeutic agents with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment. However, its success is often limited by insufficient immune priming in certain tumors, including pediatric malignancies. In this report, we explore clinical trials currently investigating the use of immunogenic cell death (ICD)-inducing chemotherapies in combination with ICIs for both adult and pediatric cancers. Given the limited clinical data available for pediatric tumors, we focused on recent preclinical studies evaluating the efficacy of these combinations in neuroblastoma (NB). Finally, to address this gap, we propose an innovative strategy to assess the impact of ICD-inducing chemotherapies on antitumor immune responses in NB. Using tumor spheroids derived from a transgenic NB mouse model, we validated our previous in vivo findings concerning how anthracyclines, specifically mitoxantrone and doxorubicin, significantly enhance MHC class I surface expression, stimulate IFNγ and granzyme B production by CD8⁺ T cells and NK cells, and promote immune cell recruitment. Importantly, these anthracyclines also upregulated PD-L1 expression on NB spheroids. This screening platform yielded results similar to in vivo findings, demonstrating that mitoxantrone and doxorubicin are the most potent immunomodulatory agents for NB. These data suggest that the creation of libraries of ICD inducers to be tested on tumor spheroids could reduce the number of combinations to be tested in vivo, in line with the principles of the 3Rs. Furthermore, these results highlight the potential of chemo-immunotherapy regimens to counteract the immunosuppressive tumor microenvironment in NB, paving the way for improved therapeutic strategies in pediatric cancers. They provide compelling evidence to support further clinical investigations of these combinations to enhance outcomes for children with malignancies. Full article

α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca²⁺ (Ca²⁺_e). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca²⁺-chelator demonstrates that Ca²⁺_e-independent effects of αLTX require an increase in cytosolic Ca²⁺ (Ca²⁺_cyt). We also show that thapsigargin, which depletes Ca²⁺ stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX’s effects on Ca²⁺_cyt using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca²⁺_e, αLTX forms membrane pores and allows the influx of Na⁺; this reverses the Na⁺-Ca²⁺ exchanger, leading to the release of stored Ca²⁺ and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca²⁺ store and induces transient opening of Ca²⁺ channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca²⁺ entry. These results indicate that Ca²⁺ release from intracellular stores and that Ca²⁺ influx through latrophilin-activated store-operated Ca²⁺ channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals. Full article

Background/Objectives: Neuroblastoma is a genetically diverse, highly metastatic pediatric cancer accounting for 15% of childhood cancer deaths despite only having ~8% of childhood cancer incidence. The current standard of care for high-risk diseases is highly genotoxic. This, combined with less than 50% survival in high-risk diseases and an abysmal 5% survival in relapsed cases, makes discovering novel, effective, and less toxic treatments essential. Methods: A prophylactic syngeneic mouse model was used to test high-dose lipid-mediator highly unsaturated fatty acids on tumorigenesis. Wildtype mice were gavaged with 12.3–14.6 g/d (adult human equivalent) omega-3 EPA, DHA, or oxidation-resistant bis allylic deuterated DHA (D-DHA) and 4.6–6.0 g/d arachidonic acid (ARA). At seven days, MYCN-expressing murine neuro-2a cells syngeneic to the gavaged mice were injected subcutaneously. Oral gavage continued for 10–20 d post-injection when tumors and tissues were harvested. Results: Fifty percent of control (not gavaged) animals form tumors (4/8) at about 10 d. High-dose DHA, D-DHA, and EPA block tumor formation completely in n = 8 or 10 animals. In contrast, ω6 arachidonic acid (4.6–6.0 g/d) enhances tumor formation (6/10 tumors) and reduces latency (5.5 to 10 days) compared to the control. The co-delivery of ARA and EPA results in a reduced tumor burden analogous to the control group, suggesting that EPA directly opposes the mechanism of ARA-mediated tumor formation. DHA acts through a non-oxidative mechanism. Conclusions: Sustained high-dose ω3 (weeks/months) is safe and well-tolerated in humans. These results suggest that ω3 DHA and EPA delivery at ultra-high doses may represent a viable low-toxicity therapy for neuroblastoma. Full article

Stroke affects over 12 million people annually, leading to high mortality, long-term disability, and substantial healthcare costs. Although East Asian herbal medicines are widely used for stroke treatment, the pathways of operation they use remain poorly understood. Our study investigates the neuroprotective properties of Astragalus mongholicus (AM) in acute ischemic stroke using photothrombotic (PTB) and transient middle cerebral artery occlusion (tMCAO) mouse models, as well as an in vitro oxygen-glucose deprivation (OGD) model. Post-OGD treatment with AM improved cell viability in mouse neuroblastoma cells, likely by reducing reactive oxygen species (ROS). Mice received short-term (0–2 days) or long-term (0–27 days) AM treatment post-stroke. Infarct size was assessed using a 2,3,5-triphenyl tetrazolium chloride (TTC) staining procedure alongside magnetic resonance imaging (MRI). Neuroprotective metabolites including inositol (Ins), glycerophosphocholine+phosphocholine (GPc+ PCh), N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG), creatine + phosphocreatine (Cr+PCr), and glutamine+glutamate (Glx) were analyzed via magnetic resonance spectroscopy (MRS). Gliosis was assessed using GFAP and Iba-1 immunohistochemical markers, while neurological deficits were quantified with modified neurological severity scores (mNSS). Motor and cognitive functions were assessed using cylinder, rotarod, and novel object recognition (NOR) tests. AM treatment significantly reduced ischemic damage and improved neurological outcomes in both acute and chronic stages of PTB and tMCAO models. Additionally, AM increased neuroprotective metabolites levels, reduced gliosis, and decreased oxidative stress, as evidenced by reduced inducible nitric oxide synthase (iNOS). These findings highlight the antioxidant properties of AM and its strong therapeutic potential for promoting recovery after ischemic stroke by alleviating neurological deficits, reducing gliosis, and mitigating oxidative stress. Full article

Background: The Neuro-2a cell line, derived from a murine neuroblastoma (NB), was established as early as 1969 and originates from a transplantable tumor that arose spontaneously in an A/Jax male mouse in 1940. Since then, it has been applied in over 10,000 studies and is used by the World Organization for Animal Health for the routine diagnosis of rabies. Surprisingly, however, Neuro-2a has never been genetically characterized in detail; this study fills that gap. Methods: The Neuro-2a cell line and two of its derivatives, Neuro-2a TR-alpha and Neuro-2a TR-beta, were analyzed for their chromosomal constitution using molecular cytogenetic approaches. Array comparative genomic hybridization was performed to characterize copy number alterations. Results: Neuro-2A has a hyper-tetraploid karyotype with 70 to 97 chromosomes per cell, and the karyotypes of its two examined derivatives were quite similar. Neither of them had a Y-chromosome. The complex karyotype of Neuro-2a includes mitotically stable dicentres, neocentrics, and complex rearrangements resembling chromothripsis events. Although no amplification of euchromatin or oncogenes was detected, there are five derivative chromosomes with the amplification of centromere-near heterochromatic material and 1–5 additional derivatives consisting only of such material. Conclusions: Since satellite DNA amplification has recently been found in advanced human tumors, this finding may be the corresponding equivalent in mice. An in silico translation of the obtained results into the human genome indicated that Neuro-2A is suitable as a model for advanced human NB. Full article

Ferroptosis is an iron-dependent form of programmed cell death that is influenced by biological processes such as iron metabolism and senescence. As brain iron levels increase with aging, ferroptosis is also implicated in the development of age-related pathologic conditions such as Alzheimer’s disease (AD) and related dementias (ADRD). Indeed, inhibitors of ferroptosis have been shown to be protective in models of degenerative brain disorders like AD/ADRD. Given the inaccessibility of the living human brain for metabolic studies, the goal of this work was to characterize an in vitro model for understanding how aging and iron availability influence neuronal iron metabolism and ferroptosis. First, the human (SH-SY5Y) and mouse (Neuro-2a) neuroblastoma lines were terminally differentiated into mature neurons by culturing in all-trans-retinoic acid for at least 72 h. Despite demonstrating all signs of neuronal differentiation and maturation, including increased expression of the iron storage protein ferritin, we discovered that differentiation conferred ferroptosis resistance in both cell lines. Gene expression data indicates differentiated neurons increase their capacity to protect against iron-mediated oxidative damage by augmenting cystine import, and subsequently increasing intracellular cysteine levels, to promote glutathione production and glutathione peroxidase activity (GPX). In support of this hypothesis, we found that culturing differentiated neurons in cysteine-depleted media sensitized them to GPX4 inhibition, and that these effects are mitigated by cystine supplementation. Such findings are important as they provide guidance for the use of in vitro experimental models to investigate the role of ferroptosis in neurodegeneration in pathologies such as ADRD. Full article

Hexabromocyclododecane (HBCD) is widely used in polystyrene foams, building materials, and electrical equipment as a brominated flame retardant (BFR) and persists in the environment and human body matrix. It has attracted increased attention since its neuroendocrine disorder effects have been observed in humans and animals. However, studies evaluating the neurotoxicity of HBCD diastereoisomers and the potential mechanisms involved are still limited. In this study, we compared the cytotoxicity induced by the three HBCD diastereoisomers (i.e., α-, β-, and γ-HBCD) in N2a cells and further investigated the underlying molecular mechanism. Our results showed that HBCD diastereoisomers decreased cell viability in the order of β-HBCD > α-HBCD > γ-HBCD. Moreover, α-HBCD and β-HBCD exposure led to different degrees of cell cycle disruption and oxidative stress of N2a cells, implying that oxidative stress-mediated differential cytotoxicity of HBCD diastereoisomers. The expressions of caspases and Bcl-2 were differentially regulated by α-HBCD and β-HBCD, suggesting that the mitochondrial apoptosis pathway may be critical in HBCDs-mediated N2a cell toxicity. Therefore, our studies provided novel evidence for the underlying mechanisms of the distinct cytotoxicity of HBCD diastereoisomers. Full article

Sodium tungstate (Na₂WO₄) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na₂WO₄ in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na₂WO₄ promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na₂WO₄ increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na₂WO₄ increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na₂WO₄ on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na₂WO₄ were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na₂WO_4. These findings support the role of Na₂WO₄ in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound. Full article

Gamma-oryzanol (ORY), found in rice (Oryza sativa L.), is a mixture of ferulic acid esters with triterpene alcohols, well-known for its antioxidant and anti-inflammatory properties. Our past research demonstrated its positive impact on cognitive function in adult mice, influencing synaptic plasticity and neuroprotection. In this study, we explored whether ORY can exert neuro-differentiating effects by using different experimental models. For this purpose, chemical characterization identified four components that are most abundant in ORY. In human neuroblastoma cells, we showed ORY’s ability to stimulate neurite outgrowth, upregulating the expression of GAP43, BDNF, and TrkB genes. In addition, ORY was found to guide adult mouse hippocampal neural progenitor cells (NPCs) toward a neuronal commitment. Microinjection of ORY in zebrafish Tg (-3.1 neurog1:GFP) amplified neurog1-GFP signal, islet1, and bdnf mRNA levels. Zebrafish nrf2a and nrf2b morphants (MOs) were utilized to assess ORY effects in the presence or absence of Nrf2. Notably, ORY’s ability to activate bdnf was nullified in nrf2a-MO and nrf2b-MO. Furthermore, computational analysis suggested ORY’s single components have different affinities for the Keap1-Kelch domain. In conclusion, although more in-depth studies are needed, our findings position ORY as a potential source of bioactive molecules with neuro-differentiating potential involving the Nrf2 pathway. Full article

The future of therapy for neurodegenerative diseases (NDs) relies on new strategies targeting multiple pharmacological pathways. Our research led to obtaining the compound AR71 [(E)-3-(3,4,5-trimethoxyphenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one], which has high affinity for human H₃R (K_i = 24 nM) and selectivity towards histamine H₁ and H₄ receptors (K_i > 2500 nM), and showed anti-inflammatory activity in a model of lipopolysaccharide-induced inflammation in BV-2 cells. The presented tests confirmed its antagonist/inverse agonist activity profile and good metabolic stability while docking studies showed the binding mode to histamine H₁, H₃, and H₄ receptors. In in vitro tests, cytotoxicity was evaluated at three cell lines (neuroblastoma, astrocytes, and human peripheral blood mononuclear cells), and a neuroprotective effect was observed in rotenone-induced toxicity. In vivo experiments in a mouse neuropathic pain model demonstrated the highest analgesic effects of AR71 at the dose of 20 mg/kg body weight. Additionally, AR71 showed antiproliferative activity in higher concentrations. These findings suggest the need for further evaluation of AR71’s therapeutic potential in treating ND and CNS cancer using animal experimental models. Full article

Glucagon-like peptide-1 (GLP-1)-based drugs have been approved by the United States Food and Drug Administration (FDA) and are widely used to treat type 2 diabetes mellitus (T2DM) and obesity. More recent developments of unimolecular peptides targeting multiple incretin-related receptors (“multi-agonists”), including the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) and the glucagon (Gcg) receptor (GcgR), have emerged with the aim of enhancing drug benefits. In this study, we utilized human and mouse microglial cell lines, HMC3 and IMG, respectively, together with the human neuroblastoma SH-SY5Y cell line as cellular models of neurodegeneration. Using these cell lines, we studied the neuroprotective and anti-inflammatory capacity of several multi-agonists in comparison with a single GLP-1 receptor (GLP-1R) agonist, exendin-4. Our data demonstrate that the two selected GLP-1R/GIPR dual agonists and a GLP-1R/GIPR/GcgR triple agonist not only have neurotrophic and neuroprotective effects but also have anti-neuroinflammatory properties, as indicated by the decreased microglial cyclooxygenase 2 (COX2) expression, nitrite production, and pro-inflammatory cytokine release. In addition, our results indicate that these multi-agonists have the potential to outperform commercially available single GLP-1R agonists in neurodegenerative disease treatment. Full article