Mouse Neuroblastoma Neuro2a Cells Research Articles

Asiaticoside but not its aglycone exhibits neuritogenicity through TrkA receptor signaling: a bridge between ERK1/2-CREB and Akt-GSK3β/RhoA.

The neuritogenicity and the neuroregenerative potential of asiaticoside (AS) and its aglycone, asiatic acid (AA), has been generally reported. We recently identified the participation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) and protein kinase B (Akt) in the neuritogenic mechanism of AS and AA. In this study, we further investigated the possible upstream target molecule and the associated downstream signaling of both triterpenoids in mouse neuroblastoma Neuro-2a cells. Our immunoblotting and immunofluorescence assays revealed that either AS or AA exerted neurite extension activity through inhibitory effect on glycogen synthase kinase 3β (GSK3β) and Ras homolog gene family member A (RhoA). AS appeared significantly more potent in promoting neurite elongation than AA, and concurrently expressed a higher degree of inhibition on GSK3β and RhoA activations. The mediation of GSK3β and RhoA activities in AS-treated cells involved Akt signaling. Moreover, when using GW441756, a specific tropomyosin receptor kinase A (TrkA) receptor signaling inhibitor, the ERK1/2 and Akt phosphorylation, the inhibitory effects on GSK3β and RhoA and the neurite outgrowth induced by AS, but not AA, were totally suppressed. In conclusion, our findings supported the different upstream regulators of AS and AA in promoting neuritogenicity in Neuro-2a cells. Although both AS and AA could enhance neurite elongation through the suppression of GSK3β and RhoA activities, only AS could modulate the effect through TrkA receptor signaling.

Role of miR-124 in the regulation of retinoic acid-induced Neuro-2A cell differentiation.

Retinoic acid can cause many types of cells, including mouse neuroblastoma Neuro-2A cells, to differentiate into neurons. However, it is still unknown whether microRNAs (miRNAs) play a role in this neuronal differentiation. To address this issue, real-time polymerase chain reaction assays were used to detect the expression of several differentiation-related miRNAs during the differentiation of retinoic acid-treated Neuro-2A cells. The results revealed that miR-124 and miR-9 were upregulated, while miR-125b was downregulated in retinoic acid-treated Neuro-2A cells. To identify the miRNA that may play a key role, miR-124 expression was regulated by transfection of miRNA mimics or inhibitors. Morphological analysis results showed that inhibition of miR-124 expression reversed the effects of retinoic acid on neurite outgrowth. Moreover, miR-124 overexpression alone caused Neuro-2A cells to differentiate into neurons, and its inhibitor could block this effect. These results suggest that miR-124 plays an important role in retinoic acid-induced differentiation of Neuro-2A cells.

Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Voltage-gated potassium (K+) channel subfamily B member 1 (KCNB1, Kv2.1) and integrin-α5 form macromolecular complexes-named integrin-α5-KCNB1 complexes (IKCs)-in the human brain, but their function was poorly understood. Here we report that membrane depolarization triggered IKC intracellular signals mediated by small GTPases of the Ras subfamily and protein kinase B (Akt) to advance the development of filopodia and lamellipodia in Chinese hamster ovary cells, stimulate their motility, and enhance neurite outgrowth in mouse neuroblastoma Neuro2a cells. Five KCNB1 mutants (L211P, R312H G379R, G381R, and F416L) linked to severe infancy or early-onset epileptic encephalopathy exhibited markedly defective conduction. However, although L211P, G379R, and G381R normally engaged Ras/Akt and stimulated cell migration, R312H and F416L failed to activate Ras/Akt signaling and did not enhance cell migration. Taken together, these data suggest that IKCs modulate cellular plasticity via Ras and Akt signaling. As such, defective IKCs may cause epilepsy through mechanisms other than dysregulated excitability such as, for example, abnormal neuronal development and resulting synaptic connectivity.-Yu, W., Shin, M. R., Sesti, F. Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Melatonin Promotes Nerve Regeneration Following End-to-Side Neurorrhaphy by Accelerating Cytoskeletal Remodeling via the Melatonin Receptor-dependent Pathway

Acceleration of cytoskeletal remodeling in regenerated axons is crucial for a fully functional recovery following peripheral nerve injury (PNI). Melatonin plays important roles in cell differentiation and protection of cytoskeleton stability, thus, the present study aimed to investigate whether melatonin can enhance neurite outgrowth and promote cytoskeletal remodeling in a PNI animal model and in differentiated neurons.End-to-side neurorrhaphy (ESN) rat model was used for assessing cytoskeletal rearrangement in regenerated axon. Subject rats received 1 mg/kg/day melatonin injection for one month. The amplitude of compound muscle action potentials and the number of re-innervated motor end plates on target muscles were assessed to represent the functional recovery after ESN.Melatonin treatment enhanced functional recovery after ESN, compared to the saline treated group. Additionally, in spinal cord and peripheral nerve tissue, animals receiving melatonin displayed enhanced expression of GAP43 and β3-tubulin one month after ESN, and an increased number of re-innervated motor end plates on their target muscle. In vitro analysis revealed that melatonin treatment significantly promoted neurite outgrowth, and increased expression of melatonin receptors as well as β3-tubulin in mouse neuroblastoma Neuro-2a (N2a) cells. Treatment with a melatonin receptor antagonist, luzindole, significantly suppressed melatonin receptors and β3-tubulin expression.Importantly, we found that melatonin treatment suppressed activation of calmodulin-dependent protein kinase II (CaMKII) in vitro and in vivo, suggesting that the β3-tubulin remodeling may occur via CaMKII-mediated Ca2+ signaling. These results suggested that melatonin may promote functional recovery after PNI by accelerating cytoskeletal remodeling through the melatonin receptor-dependent pathway.

Repeated exposure of cocaine alters mitochondrial dynamics in mouse neuroblastoma Neuro2a

Chronic abuse of psychoactive drugs including cocaine causes addiction, dependence, and brain disorders through the alteration of neuronal plasticity. Mitochondrial fission and fusion, collectively called as mitochondrial dynamics, participates in not only the maintenance of neuronal homeostasis but also reorganization of neuronal circuits. The purpose of this study is to examine effects of direct and repeated exposure of cocaine on mitochondrial dynamics in neuronal cells in vitro. Repeated exposure to 600 μM cocaine (2∼3 times per week) of Neuro2a mouse neuroblastoma cells for 3 weeks resulted in decrease of mitochondrial transmembrane potential, activation of autophagy, and upregulation of Parkin, a protein involved in mitochondrial autophagy. Increased expression of mitochondrial fission genes and significant increase in the ser-616 phosphorylated-DRP1, the key regulator of mitochondrial fission, were observed in the cells exposed repeatedly to 600 μM cocaine. Electron microscopy showed significant increase in the number of mitochondria in cocaine-treated cells compared with control cells. Thus, our results show that repeated cocaine exposure not only causes mitochondrial dysfunction but also alters mitochondrial dynamics in Neuro2a cells. Changes in the mitochondrial dynamics might be involved in neural adaptation during repeated cocaine exposure.

Psolusosides C3 and D2-D5, Five Novel Triterpene Hexaosides From the Sea Cucumber Psolus fabricii (Psolidae, Dendrochirotida): Chemical Structures and Bioactivities

Five new triterpene glycosides, psolusosides C3 (1), D2 (2), D3 (3), D4 (4), and D5 (5), have been isolated from the sea cucumber Psolus fabricii. The structures of these glycosides were elucidated by 2-dimensional nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. All the compounds contain hexasaccharide carbohydrate chains, differing from each other in the third monosaccharide residue: xylose was found in psolusosides of group C and glucose in group D. Aglycones of the isolated compounds belong to the holostane type, contain 9(11)-double bond and 16-keto-group and have different side chains. The hemolytic activity against mouse erythrocytes and cytotoxic activity against mouse Ehrlich carcinoma cells (ascite form) and neuroblastoma Neuro 2A cells of 1 to 5 as well as the psolusosides isolated by us earlier, C1, C2, and D1, 26-nor-25-oxo-holotoxin A1, and holotoxin A1, have been studied. Psolusosides C2, D1, and D2 (2) having the aglycones without hydroxy group in the side chain showed the strongest hemolytic action in this series. Psolusoside D3 (3) containing a peroxide group in the side chain of its aglycone was surprisingly highly cytotoxic in all the tests.

The formyl peptide receptor agonist FPRa14 induces differentiation of Neuro2a mouse neuroblastoma cells into multiple distinct morphologies which can be specifically inhibited with FPR antagonists and FPR knockdown using siRNA.

The N-formyl peptide receptors (FPRs) have been identified within neuronal tissues and may serve as yet undetermined functions within the nervous system. The FPRs have been implicated in the progression and invasiveness of neuroblastoma and other cancers. In this study the effects of the synthetic FPR agonist FPRa14, FPR antagonists and FPR knockdown using siRNA on mouse neuroblastoma neuro2a (N2a) cell differentiation plus toxicity were examined. The FPRa14 (1–10μM) was found to induce a significant dose-dependent differentiation response in mouse neuroblastoma N2a cells. Interestingly, three distinct differentiated morphologies were observed, with two non-archetypal forms observed at the higher FPRa14 concentrations. These three forms were also observed in the human neuroblastoma cell-lines IMR-32 and SH-SY5Y when exposed to 100μM FPRa14. In N2a cells combined knockdown of FPR1 and FPR2 using siRNA inhibited the differentiation response to FPRa14, suggesting involvement of both receptor subtypes. Pre-incubating N2a cultures with the FPR1 antagonists Boc-MLF and cyclosporin H significantly reduced FPRa14-induced differentiation to near baseline levels. Meanwhile, the FPR2 antagonist WRW4 had no significant effect on FPRa14-induced N2a differentiation. These results suggest that the N2a differentiation response observed has an FPR1-dependent component. Toxicity of FPRa14 was only observed at higher concentrations. All three antagonists used blocked FPRa14-induced toxicity, whilst only siRNA knockdown of FPR2 reduced toxicity. This suggests that the toxicity and differentiation involve different mechanisms. The demonstration of neuronal differentiation mediated via FPRs in this study represents a significant finding and suggests a role for FPRs in the CNS. This finding could potentially lead to novel therapies for a range of neurological conditions including neuroblastoma, Alzheimer’s disease, Parkinson’s disease and neuropathic pain. Furthermore, this could represent a potential avenue for neuronal regeneration therapies.

New magnetic bimetallic yanus-like Ag-Fe nanoparticles for antitumine therapy

The purpose of the study was to analyze the antitumor activity of magnetic bimetallic Ag-Fe nanoparticles having the structure of Yanus particles produced by the electrical explosion of wires. material and methods. For the synthesis of bimetallic Ag-Fe nanoparticles, a simultaneous electric explosion of silver and iron wires in an argon atmosphere was used. The morphology and size of the nanoparticles and their agglomerates were determined by transmission electron microscopy. To determine the average size of nanoparticles, particle size distribution histograms approximated by log-normal distribution were plotted. The phase composition was determined using x-ray method. The cytotoxic effect of the nanoparticles was evaluated by MTT assay using mouse neuro-2a (N2a) neuroblastoma cell lines and J774 histiocytic sarcoma cell lines. results. In case of simultaneous electric explosion of silver and iron wires in an argon atmosphere, the spherical 72 nm particles were obtained. Silver and iron were shown to be unevenly distributed in particles. There were areas enriched with one of the components with clear boundaries of phase separation or Janus nanoparticles. On the diffractogram of the sample, the main reflections correspond to the phases of metallic Ag and Fe. Aqueous suspensions of monometallic Ag and Fe nanoparticles reduced cell viability to a lesser extent than bimetallic nanoparticles. Ag-Fe nanoparticles showed the highest dose-dependent antitumor activity. The higher corrosion rate of bimetallic Ag-Fe particles compared to monometallic nanoparticles was due to the larger contact area between metallic phases in Janus nanoparticles, which determined a larger number of corrosion centers in bimetallic nanoparticles. conclusion. As a result of an electric explosion of wires from immiscible metals of iron and silver, bimetallic Ag-Fe nanoparticles with Janus particle structure were obtained. Ag-Fe nanoparticles exhibite higher antitumor activity than the individual metals from which they are composed and are a promising material for the fight against cancer cells.

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.

In Vitro Cytotoxicity Induced by the Bufadienolides 1α,2α-Epoxyscillirosidine and Lanceotoxin B on Rat Myocardial and Mouse Neuroblastoma Cell Lines.

Consumption of bufadienolide-containing plants are responsible for many livestock mortalities annually. Bufadienolides are divided into two groups; non-cumulative bufadienolides and cumulative bufadienolides. Cumulative bufadienolides are referred to as neurotoxic, as the chronic intoxication with this type of bufadienolide results in a paretic/paralytic syndrome known as ‘krimpsiekte’. The in vitro cytotoxicity of a non-cumulative bufadienolide, 1α,2α-epoxyscillirosidine, and a cumulative bufadienolide, lanceotoxin B, were compared using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction) assay after exposing rat myocardial (H9c2) and mouse neuroblastoma (Neuro-2a) cell lines. The effect of these two bufadienolides on cell ultrastructure was also investigated using transmission electron microscopy (TEM). H9c2 cells exhibited greater cytotoxicity when exposed to 1α,2α-epoxyscillirosidine, compared to lanceotoxin B. In contrast, Neuro-2a cells were more susceptible to lanceotoxin B. The EC50 (half maximal effective concentration) of lanceotoxin B exposure of Neuro-2a cells for 24–72 h ranged from 4.4–5.5 µM compared to EC50s of 35.7–37.6 µM for 1α,2α-epoxyscillirosidine exposure of Neuro-2a cells over the same period. 1α,2α-Epoxyscillirosidine induced extensive vacuolization in both cell types, with swollen RER (rough endoplasmic reticulum) and perinuclear spaces. Lanceotoxin B caused swelling of the mitochondria and sequestration of cytoplasmic material within autophagic vesicles. These results corroborate the notion that cumulative bufadienolides are neurotoxic.

Palmitate-Induced SREBP1 Expression and Activation Underlies the Increased BACE 1 Activity and Amyloid Beta Genesis.

Numerous cross-sectional and longitudinal studies have implicated saturated fat-enriched diets in the etio-pathogenesis of Alzheimer's disease (AD). Emerging evidence shows that saturated fat-enriched diets, such as palmitate-enriched diets, increase amyloid-beta (Aβ) production, the histopathological hallmark of AD. However, the molecular mechanisms that underlie the deleterious effects of palmitate-enriched diets in the augmentation of Aβ genesis are yet to be characterized. Sterol response element binding protein 1 (SREBP1) is a transcription factor that is modulated by saturated fatty acids, such as palmitate, and consequently regulates the expression of genes that code for proteins involved in almost all facets of lipid metabolism. Herein, we determined the role of changes in SREBP1 expression and transcriptional activity in the palmitate-induced effects on Aβ genesis and BACE1 expression, the enzyme that catalyzes the rate-limiting step in Aβ biosynthesis. We demonstrate that palmitate-induced SREBP1 activation directly regulates BACE1 expression at the transcriptional level in the mouse hippocampus and mouse Neuro-2a (N2a) neuroblastoma cells. Chromatin immunoprecipitation (ChIP) studies show that palmitate increases the binding of SREBP1 to the Bace1 promoter region in the mouse hippocampus and mouse N2a neuroblastoma cells. Ectopic expression of the dominant negative SREBP1 mutant and knocking-down SREBP1 expression significantly reduced the palmitate-induced increase in BACE1 expression and subsequent Aβ genesis in mouse N2a neuroblastoma cells. Our study unveils SREBP1 activation as a novel molecular player in the palmitate-induced upregulation of BACE1 expression and subsequent Aβ genesis.

Kalirin-7 plays a neuroprotective role in Neuro-2A cells injured by oxygen-glucose deprivation and reperfusion through Rac1 activation.

Objective(s):The study explored the neuroprotective role of Kalirin-7 (Kal-7) in Neuro-2A cells after oxygen-glucose deprivation and reperfusion (OGD/R) treatment.Materials and Methods:The study used an OGD/R model of mouse Neuro-2A neuroblastoma cells in vitro. Cells were transfected with pCAGGS-Kal-7 to up-regulating kal-7. Then cell proliferation and apoptosis were respectively analyzed by Trypan blue exclusion method and flow cytometry. To examine the involvement of Rac1, cells were treated with Rac1-GTP inhibitor NSC23766 before treatment with OGD/R. Expressions of Bax, Bcl-2, Rac1, and down-stream targets of Rac1 were analyzed by Western blot.Results:Kal-7 significantly decreased OGD/R induced cell apoptosis (P<0.01), but no significant effects were observed on cell proliferation. Kal-7 increased the expressions of apoptosis-related protein of Bcl-2 and Rac1, but decreased the expression of Bax in Neuro-2A cells stimulated to OGD/R. Rac1 was activated by Kal-7 due to the increased levels of its down-stream targets, p-p38 and p-PAK1. NSC23766 reduced the anti-apoptotic effect of Kal-7 as the enhanced apoptotic cell rate and increased Bax/Bcl-2 ratio. Conclusion:These findings suggest that the protective effects of Kal-7 against OGD/R injury in Neuro-2A cells were dependent in a Rac1 activation signaling.

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients.We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

Polypyrrole increases branching and neurite extension by Neuro2A cells on PBAT ultrathin fibers

We present a methodology for production and application of electrospun hybrid materials containing commercial polyester (poly (butylene adipate-co-terephthalate; PBAT), and a conductive polymer (polypyrrole; PPy) as scaffold for neuronal growth and differentiation. The physical–chemical properties of the scaffolds and optimization of the electrospinning parameters are presented. The electrospun scaffolds are biocompatible and allow proper adhesion and spread of mesenchymal stem cells (MSCs). Fibers produced with PBAT with or without PPy were used as scaffold for Neuro2a mouse neuroblastoma cells adhesion and differentiation. Neuro2a adhered to PBAT and PBAT/PPy2% scaffolds without laminin coating. However, Neuro2a failed to differentiate in PBAT when stimulated by treatment with retinoic acid (RA), but differentiated in PBAT/PPy2% fibers. We hypothesize that PBAT hydrophobicity inhibited proper spreading and further differentiation, and inhibition was overcome by coating the PBAT fibers with laminin. We conclude that fibers produced with the combination of PBAT and PPy can support neuronal differentiation.

4-((5-(Tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2-hydroxybenzoic acid protects against oxygen-glucose deprivation/reperfusion injury

AimsOxidative stress is one of the most important pathological mechanisms which could aggravate ischemic stroke injury. In order to seek for better treatment therapies to alleviate stroke injury, novel chemicals have been synthetized. In the present study, a new compound 4-((5-(tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2- hydroxybenzoic acid, named LX009, was used to determine whether it could reduce the oxidative stress caused by oxygen-glucose deprivation (OGD)/reperfusion (RP) and exert neuroprotective effect both in mouse Neuro 2A (N2A) neuroblastoma cells and mouse primary cortical neurons. Main methodsOGD/RP was performed as an in vitro model to mimic the pathologic process of ischemic stroke. We explored the anti-apoptosis effect of LX009 through CCK8 assay, calcein acetoxymethylester/propidium iodide (calcein-AM/PI) staining, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit, caspase-3 activity assay. Besides, the anti-oxidative stress effect of the drug was determined by intracellular reactive oxygen species (ROS) detection, nitrite analysis, measurement of mitochondrial membrane potential (MMP), intracellular catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activity. Key findingsOur results indicated that LX009 could alleviate OGD/RP-induced cell apoptosis. Furthermore, OGD/RP induced oxidative stress could be reserved by LX009, including measurements of intracellular ROS production, MMP, CAT and Mn-SOD activity. Mechanistically, the phosphorylation level of Akt, as well as the expression of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were elevated after LX009 treatment. SignificanceOur present study indicated that LX009 might have the potential to be an anti-oxidative stress agent in the future.

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Regulation of serum‐ and glucocorticoid‐inducible kinase phosphorylation and catalytic activity in ventral tegmental area by chronic drugs of abuse

Changes in the activity and output of dopamine neurons in the ventral tegmental area (VTA) by chronic drugs of abuse contribute to addiction‐related behaviors. Our lab has previously demonstrated that serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) phosphorylation and catalytic activity are increased by chronic administration of cocaine and morphine. Further, using viral‐mediated overexpression studies, we have observed that decreasing VTA SGK1 catalytic activity decreases drug reward and intake, and that altering VTA SGK1 phosphorylation at serine 78 (S78) is sufficient to modulate cocaine reward and morphine intake, as measured by conditioned place preference and a two‐bottle choice task, respectively. However, little is known about regulation of SGK1 S78 phosphorylation and its role in kinase activity. Therefore, the aim of my study is to identify the kinases responsible for SGK1 S78 phosphorylation by chronic drugs of abuse, since this may serve as a novel node for therapeutic intervention. Using an in silico approach, I identified promising candidate kinases, namely: glycogen synthase kinase 3 beta (GSK3β), cyclin‐dependent kinase 5 (cdk5), extracellular signal‐regulated kinase 5 (ERK5), and ERK1/2. I utilized the Neuro2A mouse neuroblastoma cell line and established an induction protocol that robustly increases both SGK1 S78 phosphorylation and catalytic activity, as measured by phosphorylation of N‐myc downstream regulated gene (NDGR) protein, the only known SGK1‐specific substrate. I then evaluated the role of candidate kinases using specific pharmacological inhibitors: roscovitine (cdk5), GSK3β inhibitor XXVII (GSK3β kinase), U0126 inhibitor (ERK1/2) and BIX02189 (ERK5). I found that inhibition of any of upstream candidate kinases can dose‐dependently decrease SGK1 S78 phosphorylation without affecting the SGK1 catalytic activity, while SGK1‐specific inhibition (GSK650394) downregulates both S78 phosphorylation and SGK1 catalytic activity. These data suggest that our in silico candidates are valid and S78 phosphorylation is not necessary for kinase activity, but that changes in catalytic activity may influence S78 phosphorylation. We have now developed a series of mutant constructs to directly test the relationship between S78 phosphorylation and catalytic activity. Additionally, given our data that VTA overexpression of an SGK1 mutant that lacks S78 phosphorylation decreases morphine and cocaine reward, I will next test whether intra‐VTA infusion of pharmacological inhibitors is sufficient to prevent the cocaine‐ and morphine‐induced increase in SGK1 S78 phosphorylation and associated behavioral changes. Together, my work seeks to characterize the regulation of SGK1 S78 phosphorylation in drug‐related behaviors to identify a potential novel pharmacological target for improved treatment of addiction.Support or Funding InformationPhRMA Foundation Research Starter GrantR01 DA039895, NIDAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterizing PTGER4 as a Target Gene of Autism Protein E6AP

BackgroundAutism spectrum disorder (ASD) has been found to be associated with duplication or triplication of the UBE3A gene, which encodes for E6‐associated protein (E6AP, an E3 ubiquitin ligase and transcriptional coactivator). Mice with three copies of UBE3A exhibit core ASD features and humans with Dup15q share many symptoms with ASD. However, the few E6AP ubiquitination substrates found do not explain ASD pathology. Our lab has identified and characterized E6AP as a coactivator of steroid hormone receptor signaling. Estrogens (E2) affect learning, memory and many other brain processes via estrogen receptors, which are transcription factors.ObjectivesThis study tests the hypothesis that deregulation of E6AP‐mediated steroid hormone receptor transcriptional signaling in the brain leads to the development of ASD. The project aims are to identify steroid hormone‐dependent E6AP target genes in neurons and to study the role of these target genes in the pathogenesis of ASD. Identification of new molecular pathways that are transcriptionally regulated by E6AP will broaden our understanding of ASD.MethodsPotential E6AP target genes were identified by microarray of MCF‐7 breast cancer cells. Cells from the mouse neuroblastoma Neuro2a cell line were cultured. Cells were transfected with E6AP or had E6AP knocked down by siRNA and then were treated with physiologically relevant doses of estrogen, the estrogen receptor antagonist tamoxifen, or vehicle. Assays included western blot, co‐immunoprecipitation, qRT‐PCR, and microscopy.Results1) E6‐AP and ER colocalize in mouse HPC neurons. 2) E6‐AP and ERα translocate to the nucleus of Neuro2a (N2a) cells upon E2 treatment. 3) E6‐AP and ERα complex in N2a. 4) The learning and memory gene for prostaglandin E receptor 4, PTGER4, is an E6AP‐dependent target gene that is downregulated in the presence of E6AP or estrogen.ConclusionsWe have identified a memory and learning gene that is regulated by E6AP and E2‐dependent: PTGER4. This is evidence that PTGER4 may be altered in ASD, leading to learning and memory symptoms. PTGER4 allows phosphorylation of glycogen synthase kinase 3 (GSK3). GSK3 has a large role in apoptosis and has been implicated in neuropsychiatric disorders such as Alzheimer's disease and bipolar disorder. Given that GSK3 is amyloidogenic and ASD patients exhibit increased beta amyloid deposition, increased E6AP leading to decreased PTGER4 may lead to decreased inhibitory phosphorylation of GSK3. In a case study, the GSK3 inhibitor ketamine actually improved an adult ASD patient's symptoms, supporting this theory. Further experiments will be necessary to confirm these promising findings.Support or Funding InformationUniversity of Miami

Involvement of impaired autophagy and mitophagy in Neuro-2a cell damage under hypoxic and/or high-glucose conditions

Chronic cerebral hypoperfusion (CCH) plays an insidious role in the development of cognitive impairment. Considerable evidence suggests that Diabetes Mellitus (DM) as a vascular risk factor may exacerbate CCH and is closely related to cognitive decline. Dysregulation of autophagy is known to be associated with the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease. To elucidate the role of autophagy in CCH- and/or DM-related pathogenesis, mouse neuroblastoma Neuro-2a cells were exposed to hypoxia and/or high glucose for 48 h, mimicking CCH complicated with DM pathologies. Chronic hypoxia reduced cell proliferation and increased levels of cleaved caspase-3, whereas high glucose had no obvious synergistic toxic effect. Accumulation of autophagic vacuoles under hypoxia may be due to both autophagy impairment and induction, with the former accounting for Neuro-2a cell death. Additionally, aberrant accumulation of mitochondria in Neuro-2a cells may be attributed to insufficient BNIP3-mediated mitophagy due to poor interaction between BNIP3 and LC3-II. Despite the lack of a significant cytotoxic effect of high glucose under our experimental conditions, our data indicated for the first time that impaired autophagy degradation and inefficient BNIP3-mediated mitophagy may constitute mechanisms underlying neuronal cell damage during chronic hypoxia.

Heat Shock Protein B8 (HSPB8) Reduces Oxygen-Glucose Deprivation/Reperfusion Injury via the Induction of Mitophagy

Background/Aims: We have reported the neuroprotective properties of Heat shock protein B8(HSPB8) against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury by inhibiting the mitochondrial apoptotic pathway. However, the exact underlying mechanism of its protective effect on mitochondrial function remains unknown. Here we examined whether the beneficial effect of HSPB8 on OGD/R-induced cell death is associated with mitophagy in mouse neuroblastoma Neuro2a (N2a) cells. Methods: Using the mouse transient middle cerebral artery occlusion (tMCAO) model and mouse neuroblastoma Neuro2a (N2a) cell cultures subjected to OGD/R, we employed western-blot, RT-PCR and immunostaining to analyze the change of expression pattern of HSPB8 and mitophagic flux after brain I/R both in vivo and in vitro. Moreover, via overexpressing HSPB8 or knocking down HSPB8 expression with siRNA in N2a cell, we evaluated the effect of HSPB8 on mitochondrial function during OGD/R. The impact of HSPB8 on mitophagic pathway was also assessed. Finally, mitotophagy inhibitors (CQ and Mdivi-1) were adopted to verify the involvement of mitophagy in HSPB8- induced neuroprotection. Results: HSPB8 could be up-regulated by brain I/R both in vivo and in vitro. Mitophagy enhancement coincided with induction of HSPB8 during I/R. Overexpression of HSPB8 reinforced I/R-induced mitophagy in OGD/R-treated mouse N2a cells and HSPB8 silence suppressed mitophagy process. Inhibition of mitophagy compromised neuroprotection conferred by HSPB8 overexpression. Conclusions: HSPB8 promoted OGD/R-induced mitophagy, which restored the mitochondrial function and contributed to the decrease in cell apoptosis after OGD/R. Therefore, HSPB8 could be a favorable neuroprotective agent for cerebral I/R related disorders.