N2a Cells Research Articles

Downregulation of miR-337-3p in hypoxia/reoxygenation neuroblastoma cells increases KCTD11 expression.

Neurodegeneration is linked to the progressive loss of neural function and is associated with several diseases. Hypoxia is a hallmark in many of these diseases, and several therapies have been developed to treat this disease, including gene expression therapies that should be tightly controlled to avoid side effects. Cells experiencing hypoxia undergo a series of physiological responses that are induced by the activation of various transcription factors. Modulation of microRNA (miRNA) expression to alter transcriptional regulation has been demonstrated to be beneficial in treating multiple diseases, and in this study, we therefore explored potential miRNA candidates that could influence hypoxia-induced nerve cell death. Our data suggest that in mouse neuroblasts Neuro-2a cells with hypoxia/reoxygenation (H/R), miR-337-3p is downregulated to increase the expression of Potassium channel tetramerization domain containing 11 (KCTD11) and subsequently promote apoptosis. Here, we demonstrate for the first time that KCTD11 plays a role in the cellular response to hypoxia, and we also provide a possible regulatory mechanism by identifying the axis of miR-337-3p/KCTD11 as a promising candidate modulator of nerve cell survival after H/R exposure.

Knockdown of INPP5K compromises the differentiation of N2A cells.

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Preventive effect of propolis on cognitive decline in Alzheimer’s disease model mice

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer’s disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-β. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300–1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

Assessing Radiation Effects on Chemo-Treated BT20 and 4T1 Breast Cancer, and Neuroblastoma Cell Lines: A Study of Single and Multiple-Cell Ionization via Infrared Laser Trapping

Background: Our study aimed to assess the radiation sensitivity of BT20, a human breast tumor cell line, using the laser-trapping technique and compare it with N2a and 4T1 cells. Additionally, we investigated the impact of the antitumor compound 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) on radiation sensitivity. Methods and Materials: We employed laser trapping to calculate both the threshold ionization energy (TIE) and threshold radiation dose (TRD) for BT20, N2a, and 4T1 cells. We assessed the effect of DMDD on BT20 cells’ radiosensitivity and conducted comparisons across these cell lines. Results: Our findings reveal that DMDD significantly enhances the radiosensitivity of BT20 breast carcinoma cells. Moreover, we observed distinct trends in TIE and TRD across the three cell lines, with differences attributed to variations in cell size and composition. When multiple cell ionizations were considered, a notable reduction in TRD was observed, implicating factors such as the chain effect of ionizing radiation and the influence of DMDD. The study found that TIE increased with the number of cells in the trap while TRD consistently decreased across all three cell lines, suggesting comparable radiation sensitivity, and oligostilbene treatment further reduced TRD, presenting the potential for enhancing therapeutic ratios in cancer treatment. Conclusion: The antitumor compound DMDD enhances the radiosensitivity of BT20 breast carcinoma cells, highlighting its potential in cancer treatment. Furthermore, our study underscores the impact of cell size and multiple-cell ionizations on TRD. Leveraging laser trapping techniques, biocompatible nanoparticles, and advanced optical tweezers opens promising avenues for personalized and effective cancer therapy approaches.

Inhibition of Kv2.1 potassium channels by the antidepressant drug sertraline

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In this study, we have used the patch-clamp technique to assess the effects of sertraline on Kv2.1 channels heterologously expressed in HEK-293 cells and on the voltage-gated potassium currents (IKv) of Neuro 2a cells, which are predominantly mediated by Kv2.1 channels. Our results reveal that sertraline inhibits Kv2.1 channels in a concentration-dependent manner. The sertraline-induced inhibition was not voltage-dependent and did not require the channels to be open. The kinetics of activation and deactivation were accelerated and decelerated, respectively, by sertraline. Moreover, the inhibition by this drug was use-dependent. Notably, sertraline significantly modified the inactivation mechanism of Kv2.1 channels; the steady-state inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was enhanced and accelerated, and the recovery from inactivation was slowed, suggesting that this is the main mechanism by which sertraline inhibits Kv2.1 channels. Overall, this study provides novel insights into the pharmacological actions of sertraline on Kv2.1 channels, shedding light on the intricate interaction between SSRIs and ion channel function.

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.

Argininyl-fructosyl-glucose (AFG) antagonizes D-galactose-induced cellular senescence of neuro-2a via inhibiting endoplasmic reticulum stress and apoptosis

Argininyl-fructosyl-glucose (AFG), a distinctive non-saponin compound isolated from red ginseng (Panax ginseng. C.A. Meyer), demonstrates neuroprotective effects. However, the molecular mechanism underlying AFG's anti-aging properties remains unclear. This study established an in vitro D-galactose-induced N2a cells subacute aging model to investigate AFG's anti-aging activity and potential mechanisms. Results indicated that AFG treatment (8 μM) significantly reduces the overexpression of aging-related proteins (p53/p21/p16) induced by D-galactose, highlighting its anti-aging efficacy. AFG concurrently diminishes ROS accumulation, lowering extracellular lactate dehydrogenase and alleviating intracellular oxidative stress. Notably, AFG suppresses the PERK/CHOP/caspase 12 pathway, reducing intracellular Ca2+ concentration and mitigating endoplasmic reticulum (ER) stress. Additionally, AFG decreases Bax and caspase 3 expression, elevates Bcl-2 and mitochondrial membrane potential, mitigating apoptosis. NAC with 4-PBA validated the results. Overall, AFG protects N2a cells from aging by reducing ER stress and apoptosis, suggesting its potential as a key substance for treating aging and neurodegenerative diseases.

Heterozygous loss-of-function variants in DOCK4 cause neurodevelopmental delay and microcephaly

Neurons form the basic anatomical and functional structure of the nervous system, and defects in neuronal differentiation or formation of neurites are associated with various psychiatric and neurodevelopmental disorders. Dynamic changes in the cytoskeleton are essential for this process, which is, inter alia, controlled by the dedicator of cytokinesis 4 (DOCK4) through the activation of RAC1. Here, we clinically describe 7 individuals (6 males and one female) with variants in DOCK4 and overlapping phenotype of mild to severe global developmental delay. Additional symptoms include coordination or gait abnormalities, microcephaly, nonspecific brain malformations, hypotonia and seizures. Four individuals carry missense variants (three of them detected de novo) and three individuals carry null variants (two of them maternally inherited). Molecular modeling of the heterozygous missense variants suggests that the majority of them affect the globular structure of DOCK4. In vitro functional expression studies in transfected Neuro-2A cells showed that all missense variants impaired neurite outgrowth. Furthermore, Dock4 knockout Neuro-2A cells also exhibited defects in promoting neurite outgrowth. Our results, including clinical, molecular and functional data, suggest that loss-of-function variants in DOCK4 probable cause a variable spectrum of a novel neurodevelopmental disorder with microcephaly.

Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

A New Oxoaporphine and Liriodenine's Anti-Neuroblastoma Potential from the Roots of Polyalthia bullata King

Polyalthia bullata King’s root yielded a new compound named 5-methylliridine (1) in addition to six previously identified compounds. These known compounds include liriodenine (2), 11-methoxyliriodenine (3), lysicamine (4), onychine (5), 5-hydroxy-6-methoxyonychine (6), and 8-methoxyeupolauridine (7). The structures of compounds 1-7 were determined through spectroscopic analysis. Liriodenine (2) exhibited a remarkable ability to decrease the cell viability of cancerous N2A cells to 22% within a 24 h timeframe, indicating its potential as an anti-neuroblastoma agent. Molecular docking results additionally suggested that oxoaporphines (1-4) have the potential to act as inhibitors of protein kinases. These findings highlight the therapeutic potential of P. bullata constituents in cancer treatment, particularly neuroblastoma, and contribute to understanding its medicinal properties.

Drosophila TMEM63 and mouse TMEM63A are lysosomal mechanosensory ion channels

Cells sense physical forces and convert them into electrical or chemical signals, a process known as mechanotransduction. Whereas extensive studies focus on mechanotransduction at the plasma membrane, little is known about whether and how intracellular organelles sense mechanical force and the physiological functions of organellar mechanosensing. Here we identify the Drosophila TMEM63 (DmTMEM63) ion channel as an intrinsic mechanosensor of the lysosome, a major degradative organelle. Endogenous DmTMEM63 proteins localize to lysosomes, mediate lysosomal mechanosensitivity and modulate lysosomal morphology and function. Tmem63 mutant flies exhibit impaired lysosomal degradation, synaptic loss, progressive motor deficits and early death, with some of these mutant phenotypes recapitulating symptoms of TMEM63-associated human diseases. Importantly, mouse TMEM63A mediates lysosomal mechanosensitivity in Neuro-2a cells, indicative of functional conservation in mammals. Our findings reveal DmTMEM63 channel function in lysosomes and its physiological roles in vivo and provide a molecular basis to explore the mechanosensitive process in subcellular organelles.

Antinociceptive effect and anti-inflammatory activity of 1,4-naphthoquinones in mice

Aim: The ability of synthetic 1,4-naphthoquinones (1,4-NQs) to prevent adenosine triphosphate (ATP)-induced and purinergic P2X7 receptor (P2X7R) mediated inflammation in macrophage and neurodegeneration of neuronal cells in vitro was previously established. The aim of the present study was to investigate analgesic-like and anti-inflammatory activity of 1,4-NQs thioglucoside derivatives, compounds U-286 and U-548, in in vivo experiments. Methods: Spectrofluorimetry approach and YO-PRO-1 fluorescent dye uptake determination were applied to study the effect of 1,4-NQs upon ATP-induced P2X7R mediated macropore formation in mouse neuroblastoma Neuro-2a cells and macrophages RAW 264.7 cells. An acetic acid-induced writhing test, hot plate test, and carrageenan-induced paw edema test were used as an in vivo mouse models to study the ability of 1,4-NQs to inhibit pain and inflammation. In the in vivo experiments, compounds were administered to mice intraperitoneally at dosages of 0.1 mg/kg, 1.0 mg/kg and 10.0 mg/kg. A group of animals that received injections of sterile water was used as a control. Each dosage group and the control group consisted of 6 mice. Results: In the present work the analgesic-like and anti-inflammatory activity of 1,4-NQs, U-286 and U-548, was demonstrated. Compound U-548 showed a significant inhibitory effect in antinociceptive tests reducing the number of mouse writhings and eliminating the latent time of mouse hind paw licking, correspondingly. Selected compounds were able to almost completely reduce the size of carrageenan-induced paw edema 24 h after injection and had a potent anti-inflammatory activity. Observed effects were accompanied with aptitude of studied 1,4-NQs to inhibit the formation of purinergic P2X7R macropore associated with inflammation and nociceptive pain. Conclusions: The results obtained allow to consider compounds U-286 and U-548 and as a pharmacological basis for the development of new analgesic-like and anti-inflammatory drugs.

Neuroprotection by Cerebrolysin and Citicoline Through the Upregulation of Brain-Derived Neurotrophic Factor (BDNF) Expression in the Affected Neural Cells: A Preliminary Clue Obtained Through an In Vitro Study.

Citicoline and cerebrolysin are two unique yet contentious medications because of inconsistencies in efficacy as well as the mystery surrounding their mode of action. The current study aimed to re-validate the neuroprotective benefits of these medications and investigate the possible molecular mechanism. Neuro-2A cells were exposed to tert-butyl hydroperoxide, a consistent in vitro model of neuronal damage caused by oxidative stress. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, acridine orange/ethidium bromide (AO-EtBr) staining, and phase-view examinations were utilized to evaluate cell survival and cytotoxicity. Real-time reverse transcription-polymerase chain reaction (RT-PCR)-based gene expression studies were conducted. Observations revealed that these two medications had modest but considerable neuroprotective effects. While the majority of the genes' expressions remained unchanged, cerebrolysin upregulated Neuregulin 1, and both upregulated brain-derived neurotrophic factor (BDNF) expression. The findings of the current study may be the first to suggest that citicoline and cerebrolysin may increase host cells' defense mechanisms (secretion neurotrophic factors) rather than carrying nutrients for cell survival. Because of its simplicity, the current study can readily be repeated to learn more about these two disputed medications for treating ischemic stroke.

A Curcumin-Decorated Nanozyme with ROS Scavenging and Anti-Inflammatory Properties for Neuroprotection.

Disordered reactive oxygen/nitrogen species are a common occurrence in various diseases, which usually cause cellular oxidative damage and inflammation. Despite the wide range of applications for biomimetic nanoparticles with antioxidant or anti-inflammatory properties, designs that seamlessly integrate these two abilities with a synergistic effect in a simple manner are seldom reported. In this study, we developed a novel PEI-Mn composite nanoparticle (PM NP) using a chelation method, and the curcumin was loaded onto PM NPs via metal-phenol coordination to form PEI-Mn@curcumin nanoparticles (PMC NPs). PMC NPs possessed excellent dispersibility and cytocompatibility, was engineered to serve as an effective nanozyme, and exhibited specific SOD-like and CAT-like activities. In addition, the incorporation of curcumin granted PMC NPs the ability to effectively suppress the expression of inflammatory cytokines in microglia induced by LPS. As curcumin also has antioxidant properties, it further amplified the synergistic efficiency of ROS scavenging. Significantly, PMC NPs effectively scavenged ROS triggered by H2O2 in SIM-A9 microglia cells and Neuro-2a cells. PMC NPs also considerably mitigated DNA and lipid oxidation in Neuro-2a cells and demonstrated an increase in cell viability under various H2O2 concentrations. These properties suggest that PMC NPs have significant potential in addressing excessive ROS and inflammation related to neural diseases.

H3K4 Trimethylation Mediate Hyperhomocysteinemia Induced Neurodegeneration via Suppressing Histone Acetylation by ANP32A.

Homocysteine (Hcy) is an independent and serious risk factor for dementia, including Alzheimer's disease (AD), but the precise mechanisms are still poorly understood. In the current study, we observed that the permissive histone mark trimethyl histone H3 lysine 4 (H3K4me3) and its methyltransferase KMT2B were significantly elevated in hyperhomocysteinemia (HHcy) rats, with impairment of synaptic plasticity and cognitive function. Further research found that histone methylation inhibited synapse-associated protein expression, by suppressing histone acetylation. Inhibiting H3K4me3 by downregulating KMT2B could effectively restore Hcy-inhibited H3K14ace in N2a cells. Moreover, chromatin immunoprecipitation revealed that Hcy-induced H3K4me3 resulted in ANP32A mRNA and protein overexpression in the hippocampus, which was regulated by increased transcription Factor c-fos and inhibited histone acetylation and synapse-associated protein expression, and downregulating ANP32A could reverse these changes in Hcy-treated N2a cells. Additionally, the knockdown of KMT2B restored histone acetylation and synapse-associated proteins in Hcy-treated primary hippocampal neurons. These data have revealed a novel crosstalk mechanism between KMT2B-H3K4me3-ANP32A-H3K14ace, shedding light on its role in Hcy-related neurogenerative disorders.

Consequences of GMPPB deficiency for neuromuscular development and maintenance.

Guanosine diphosphate-mannose pyrophosphorylase B (GMPPB) catalyzes the conversion of mannose-1-phosphate and GTP to GDP-mannose, which is required as a mannose donor for the biosynthesis of glycan structures necessary for proper cellular functions. Mutations in GMPPB have been associated with various neuromuscular disorders such as muscular dystrophy and myasthenic syndromes. Here, we report that GMPPB protein abundance increases during brain and skeletal muscle development, which is accompanied by an increase in overall protein mannosylation. To model the human disorder in mice, we generated heterozygous GMPPB KO mice using CIRSPR/Cas9. While we were able to obtain homozygous KO mice from heterozygous matings at the blastocyst stage, homozygous KO embryos were absent beyond embryonic day E8.5, suggesting that the homozygous loss of GMPPB results in early embryonic lethality. Since patients with GMPPB loss-of-function manifest with neuromuscular disorders, we investigated the role of GMPPB in vitro. Thereby, we found that the siRNA-mediated knockdown of Gmppb in either primary myoblasts or the myoblast cell line C2C12 impaired myoblast differentiation and resulted in myotube degeneration. siRNA-mediated knockdown of Gmppb also impaired the neuron-like differentiation of N2A cells. Taken together, our data highlight the essential role of GMPPB during development and differentiation, especially in myogenic and neuronal cell types.

Advancing Peripheral Nerve Regeneration: 3D Bioprinting of GelMA-Based Cell-Laden Electroactive Bioinks for Nerve Conduits.

Peripheral nerve injuries often result in substantial impairment of the neurostimulatory organs. While the autograft is still largely used as the "gold standard" clinical treatment option, nerve guidance conduits (NGCs) are currently considered a promising approach for promoting peripheral nerve regeneration. While several attempts have been made to construct NGCs using various biomaterial combinations, a comprehensive exploration of the process science associated with three-dimensional (3D) extrusion printing of NGCs with clinically relevant sizes (length: 20 mm; diameter: 2-8 mm), while focusing on tunable buildability using electroactive biomaterial inks, remains unexplored. In addressing this gap, we present here the results of the viscoelastic properties of a range of a multifunctional gelatin methacrylate (GelMA)/poly(ethylene glycol) diacrylate (PEGDA)/carbon nanofiber (CNF)/gellan gum (GG) hydrogel bioink formulations and printability assessment using experiments and quantitative models. Our results clearly established the positive impact of the gellan gum on the enhancement of the rheological properties. Interestingly, the strategic incorporation of PEGDA as a secondary cross-linker led to a remarkable enhancement in the strength and modulus by 3 and 8-fold, respectively. Moreover, conductive CNF addition resulted in a 4-fold improvement in measured electrical conductivity. The use of four-component electroactive biomaterial ink allowed us to obtain high neural cell viability in 3D bioprinted constructs. While the conventionally cast scaffolds can support the differentiation of neuro-2a cells, the most important result has been the excellent cell viability of neural cells in 3D encapsulated structures. Taken together, our findings demonstrate the potential of 3D bioprinting and multimodal biophysical cues in developing functional yet critical-sized nerve conduits for peripheral nerve tissue regeneration.

MLKL overexpression leads to Ca2+ and metabolic dyshomeostasis in a neuronal cell model

The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca2+ levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca2+ elevations in response to Ca2+ influx from the extracellular space. On the contrary, Ca2+ signals were elevated after mobilizing Ca2+ from the endoplasmic reticulum. Transient elevations in cytosolic Ca2+, mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24–48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca2+ and metabolic dyshomeostasis, which are cellular hallmarks of brain aging.