Cell Line Neuro-2a Research Articles

The Neural Differentiation Potential of Limbal Stem Cells: A Model for Studying the Multipotency of Limbal Stem Cells.

To investigate the multipotency, especially the neural differentiation potential, of limbal stem cells (LSCs) using a coculture system and to provide a relevant literature review. Limbal tissue was harvested from adult New Zealand white rabbits and treated with collagenase A. Small pieces of the resulting limbal epithelial sheets were cocultured with a neuroblastoma cell line (Neuro-2A) in transwells. Morphological observation of the growing epithelial sheets was accomplished by microscopy, and marker expression was detected by immunocytochemistry for ßIII-tubulin and microtubule-associated protein 2. A literature review of associated studies was performed. In the coculture group, directly adherent colonies of neuron-like (DACN) cells were observed among the growing limbal epithelial sheets from day 3. The DACN cells exhibited neuron-like morphology. The control group comprising limbal cell sheets cultured alone showed a very small number of DACN cells at the end of the culture period (day 14). Immunocytochemical staining revealed that the DACN cells were positive for ßIII-tubulin and microtubule-associated protein 2, confirming the neuronal phenotype of the neuron-like cells. By contrast, the DACN cells in the control group produced negative results. In previous reports, LSCs and niches exhibited neural potential, but most differentiated neural cells were observed as floating spheres, in contrast to the DACN cells observed in the present study. We developed a coculture system of LSCs and Neuro-2A neuroblastoma cells and obtained DACN cells with neural differentiation potential. Our findings confirm the neural potential of LSCs, consistent with previous reports, but in a form other than floating spheres.

Exosomal DNA Aptamer Targeting α-Synuclein Aggregates Reduced Neuropathological Deficits in a Mouse Parkinson’s Disease Model

The α-synuclein aggregates are the main component of Lewy bodies in Parkinson’s disease (PD) brain, and they showed immunotherapy could be employed to alleviate α-synuclein aggregate pathology in PD. Recently we have generated DNA aptamers that specifically recognize α-synuclein. In this study, we further investigated the in vivo effect of these aptamers on the neuropathological deficits associated with PD. For efficient delivery of the aptamers into the mouse brain, we employed modified exosomes with the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface. We demonstrated that the aptamers were efficiently packaged into the RVG-exosomes and delivered into neurons in vitro and in vivo. Functionally, the aptamer-loaded RVG-exosomes significantly reduced the α-synuclein preformed fibril (PFF)-induced pathological aggregates, and rescued synaptic protein loss and neuronal death. Moreover, intraperitoneal administration of these exosomes into the mice with intra-striatally injected α-synuclein PFF reduced the pathological α-synuclein aggregates and improved motor impairments. In conclusion, we demonstrated that the aptamers targeting α-synuclein aggregates could be effectively delivered into the mouse brain by the RVG-exosomes and reduce the neuropathological and behavioral deficits in the mouse PD model. This study highlights the therapeutic potential of the RVG-exosome delivery of aptamer to alleviate the brain α-synuclein pathology.

Gingko biloba Extract (EGb) Inhibits Oxidative Stress in Neuro 2A Cells Overexpressing APPsw.

Alzheimer's disease (AD) is a common neurodegenerative disease. Abundant evidence demonstrates that oxidative stress may be not only an early event in this disease, but also a key factor in the pathogenesis of AD. Ginkgo biloba extract (EGb) has a strong ability to scavenge oxygen free radicals and supply hydrogen. The present study aims to investigate the effects of EGb on Neuro 2A cells transfected with Swedish mutant APP (APPsw). Stably transfected Neuro 2A cell lines expressing human wild-type APP (APP695), APPsw, or empty vector(neo) pEGFP-N2 were treated with 100 μg/ml EGb for 0, 2, 4, 6, 8, and 10 h. Oxidative stress was assessed by measuring free radicals and the activities of antioxidant enzymes. Our studies showed that EGb treatment reduced the production of reactive oxygen species (ROS) and the levels of malondialdehyde (MDA) significantly while total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were enhanced in Neuro 2A cells overexpressing APPsw. Meanwhile, Aβ levels in these cells were also reduced compared to the levels in untreated cells and control cells (empty vector(neo) pEGFP-N2). These findings suggest that EGb can reduce oxidative stress by decreasing free radical and enhancing antioxidant status, further leading to reduced Aβ aggregation; EGb might be a potential therapeutic agent for Alzheimer's disease (AD).

Protective effects of noradrenaline on benzo[a]pyrene-induced oxidative stress responses in brain tumor cell lines.

Benzo[a]pyrene (B[a]P) is an ubiquitous environmental pollutant that is generated during combustion of fossil fuels. We examine the effect of noradrenaline (NA) on B[a]P-induced neurotoxicity in brain tumor cell lines like neuroblastoma (Neuro2a) and glioma (C6). We pre-treated tumor cells with NA for 6h, followed by addition of B[a]P for additional 24h. Cell viability was measured using trypan blue dye-exclusion assay and comet assay was performed to measure DNA damage. Cell cycle status was analyzed using flow cytometry and oxidative DNA damage (8-oxodG) production was examined by immunostaining. The intracellular Ca2+ concentration was analyzed using Fura-2AM. Our results showed viability of Neuro2a and C6 cells declined (24% and 20%) in B[a]P-treated groups. However, pre-treating with NA increased viability of cells by reducing percentage of cell death in both. Furthermore, B[a]P-induced deregulation of cell cycle (G2/M and S phase cell arrest) was significantly restored by pre-treatment with NA in Neuro2a cells as compared to C6 cells. We further observed increased 8-oxodG production in B[a]P-treated cells; however, NA pre-treatment significantly (p < 0.05) reduced the 8-oxodG production in Neuro2a, while C6 cells were less affected possibly due to better protective machinery. B[a]P-induced intracellular Ca2+ influx was significantly reduced in both the cell lines due to co-treatment of NA possibly by reducing Ca2+ influx. NA protects brain tumor cells against B[a]P-induced neurotoxicity may be by decreasing percentage of G2 cell arrest, oxidative DNA damage, and reducing intracellular Ca2+ influx. These findings suggested that NA may be considered as a natural potential protective agent against B[a]P-induced neurotoxicity. Graphical abstract Graphical abstract showing differential protective mechanism of NA against B[a]P-induced toxicity through antioxidant mechanism maintaining homeostasis for oxidative stress in Neuro2a and C6 cell lines. The schematic graph showed the biological significance of the NA that regulates the induction of metabolic processes of cell cycle after exposure to the environmental pollutants. B[a]P increases the intracellular levels of Ca2+, but also induces damage to cellular molecules including DNA causing cell cycle arrest. The B[a]P-induced DNA damage due to base lesions generated in the genome, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the most abundant because of guanine's lowest redox potential among DNA bases through intracellular calcium homoeostasis.

Thiamine and benfotiamine protect neuroblastoma cells against paraquat and β-amyloid toxicity by a coenzyme-independent mechanism

BackgroundBenfotiamine (BFT) is a synthetic thiamine precursor with high bioavailability. It is efficient in treating complications of type 2 diabetes and has beneficial effects in mouse models of neurodegenerative diseases. The mechanism of action of BFT remains unknown, though it is sometimes suggested that it may be linked to increased thiamine diphosphate (ThDP) coenzyme function. MethodsWe used a mouse neuroblastoma cell line (Neuro2a) grown in thiamine-restricted medium. The cells were stressed by exposure to paraquat (PQ) or amyloid β1-42 peptide in the presence or absence of BFT and the cell survival was measured using the MTT method. In each case, BFT was compared with sulbutiamine (SuBT), an unrelated thiamine precursor, and thiamine. Metabolites of BFT were determined by HPLC and mass spectrometry. ResultsAt 50 μM, BFT protects the cells against PQ and amyloid β1-42 peptide-induced toxicity with the same efficacy. Protective effects were also observed with SuBT and with higher concentrations of thiamine. The main metabolites of BFT were thiamine and S-benzoylthiamine (S-BT). Treatment with both precursors induces a strong increase in intracellular content of thiamine. Protective effects of BFT and SuBT are directly related to thiamine (but not ThDP) levels in Neuro2a cells. ConclusionsBFT, SuBT and thiamine all protect the cells against oxidative stress, suggesting an antioxidant effect of thiamine. Our results are not in favor of a direct ROS scavenging effect of thiamine but rather an indirect effect possibly mediated by some antioxidant signaling pathway. It is however not clear whether this effect is due to thiamine itself, its thiol form or an unknown metabolite. General significanceOur results suggest a role of thiamine in protection against oxidative stress, independent of the coenzyme function of thiamine diphosphate.

Cytotoxicity screening of supercritical fluid extracted seaweeds and phenylpropanoids.

Detached leaves of Posidonia oceanica and Zostera marina creating nuisance at the shores were extracted by means of supercritical CO2 enriched with a co-solvent, compared with that of soxhlet extraction. The extracts and their active compounds which are phenylpropanoids (chicoric, p-coumaric, rosmarinic, benzoic, ferulic and caffeic acids) were screened for cytotoxicity in cancer cell lines including human breast adenocarcinoma (MCF-7, MDA-MB-231, SK-BR-3), human colon adenocarcinoma (HT-29), human cervix adenocarcinoma (HeLa), human prostate adenocarcinoma (PC-3), Mus musculus neuroblastoma (Neuro 2A) cell lines and African green monkey kidney (VERO) as healthy cell line. Supercritical CO2 extracts proved to be more active than soxhlet counterparts. Particularly, Zostera marina extract obtained by supercritical CO2 at 250bar, 80°C, 20% co-solvent and a total flow rate of 15g/min revealed the best IC50 values of 25, 20, 8μg/ml in neuroblastoma, colon and cervix cancer cell lines. Among the major compounds tested, p-coumaric acid exhibited the highest cytotoxic against colon and cervix cell lines by with IC50 values of 25, 11μg/ml. As for the effects on healthy cells, the extract was not cytotoxic indicating a selective cytotoxicity. Obtained supercritical CO2 extracts can be utilized as a supplement for preventive purposes.

Electrochemical Potential-Biological Activity Relationships of Cyclic Sulfur-Containing Molecules Against Steinernema feltiae, Botrytis cinerea, and Neuro 2a Cell Line

This article provides a brief overview of electrochemical potential-biological activity relationships of natural and synthetic cyclic sulfur-containing molecules against Steinernema feltiae, Botrytis cinerea, and Neuro 2a cell line (from murine neuroblastoma). This article finds natural cyclic sulfur-containing molecules and their synthetic analogues were more reducing than glutathione (GSH) and therefore apparently did not react with GSH. The nematicidal assay indicated that cyclic disulfide compound of 1 (3-vinyl-4H-1,2-dithiin, 1,2-VDT) was more active against Steinernema feltiae with the LD50 value 151.93 ± 1.3 μM, while dithiole thione group compounds showed moderate activity against this nematode. The article also finds compound 7 (3H-1,2-dithiole-3-thione or dithiolethione, DT) has a strong activity against all different strains of Botrytis cinerea in the range concentration of 0.1–0.5 mM. This article also finds that compounds 3 (1,2-dithiane, 1,2-DT), 4 (1,5-dithiacyclooctane, 1,5-DTCO), and 7 (3H-1,2-dithiole-3-thione or dithiolethione, DT) possess some moderate activity on Neuro 2a cell lines. Antinematode, antifungal, and anticancer activity of cyclic sulfur-containing molecules indicated that they could be promising candidates for “green pesticides” or phytoprotectans and for cancer prevention.

Vitronectin is Involved in the Morphological Transition of Neurites in Retinoic Acid-Induced Neurogenesis of Neuroblastoma Cell Line Neuro2a.

Vitronectin (Vtn), one of the extracellular matrix proteins, has been reported to result in cell cycle exit, neurite formation, and polarization of neural progenitor cells during neurogenesis. The underlying mechanism, however, has not been fully understood. In this study, we investigated the roles of Vtn and its integrin receptors, during the transition of neurites from multipolar to bipolar morphology, accompanying the cell cycle exit in neural progenitor cells. We used mouse neuroblastoma cell line Neuro2a as a model of neural progenitor cells which can induce cell cycle exit and the morphological transition of neurites by retinoic acid (RA)-stimulation. Treatment with an antibody for Vtn suppressed the RA-induced cell cycle exit and multipolar-to-bipolar transition. Furthermore, immunostaining results showed that in the cells displaying multipolar morphology Vtn was partially localized at the tips of neurites and in cells displaying bipolar morphology at both tips. This Vtn localization and multipolar-to-bipolar transition was perturbed by the transfection of a dominant negative mutant of cell polarity regulator Par6. In addition, a knockdown of β5 integrin, which is a receptor candidate for Vtn, affected the multipolar-to-bipolar transition. Taken together, these results suggest that Vtn regulates the multipolar-to-bipolar morphological transition via αvβ5 integrin.

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.

PO-228 Regulation of GDE2 membrane localization and trafficking

IntroductionGlycerophosphodiester phosphodiesterase 2 (GDE2) is a multi-pass membrane protein that promotes neuronal differentiation through the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins at the cell surface. High GDE2 expression is associated with favourable outcome in neuroblastoma, while loss of GDE2 in mice leads to neuronal pathologies similar to human neurodegenerative diseases. Thus, enhancing GDE2 activity could be an attractive therapeutic strategy for neuroblastoma and related pathologies. However, the regulation of GDE2 is poorly understood.Material and methodsWe employed TIRF microscopy to study GDE2 subcellular localization in neuronal cell lines. Membrane internalisation of GDE2 was detected by confocal microscopy, and confirmed biochemically by biotin labelling assays. To determine the nature of the GDE2-containg intracellular compartments, we examined co-localization of GDE2 with endosome markers by both confocal microscopy and immunoprecipitation assays.Results and discussionsWhen expressed at relatively low levels in N1E-115, Neuro2A and SH-SY5Y neuronal cell lines, GDE2 localises to discrete membrane microdomains, as well as in high-turnover intracellular vesicles. We corroborated this intracellular trafficking by biotin labelling assays, which showed that GDE2 undergoes constitutive endocytosis and recycling back to the plasma membrane in a serum-independent manner.In addition, GDE2 was found to co-localise with well-established early-endosome markers, namely EEA1 and Rab5, indicating that GDE2 internalises from the plasma membrane. GDE2 also localised partially to Rab7-positive late endosomes, but was hardly detected in lysosomes (LAMP1, LysoTracker). Furthermore, GDE2 localised to Rab11-positive recycling endosomes, pinpointing the long recycling pathway.When co-expressed with labelled ubiquitin, GDE2 was heavily poly-ubiquitinated, which takes place non-specifically in the four cytosolic lysine residues of GDE2. Lastly, sequential truncations in the N- and C-terminal cytosolic tails of GDE2 highlighted a 10-amino acid stretch that potentially regulates intracellular trafficking.ConclusionHere we report that, in neuronal cells, GDE2 is constitutively internalised and undergoes endocytic recycling along both the short and, in particular, the long recycling pathways. This process appears to be regulated by a stretch of 10 residues in the cytosolic C-terminal tail, which could be related to non-specific ubiquitin ligation.

Loss of the basic helix-loop-helix transcription factor Bhlhe41 induces cell death and impairs neurite outgrowth in Neuro2a cells

The basic helix-loop-helix (bHLH) superfamily of transcription factors have been implicated in a wide range of cellular functions such as proliferation, differentiation, tumorigenesis, and circadian rhythms. In a previous siRNA-based screen, bHLH family member e41 (BHLHE41) had been identified as a putative regulator of neuronal differentiation; however, its function remains largely elusive. To this end, using the CRISPR-Cas9 system, we established an isogenic Neuro2a (N2a) cell line with biallelic targeting of Bhlhe41 gene (Bhlhe41-/-). In undifferentiated N2a cells, complete knockout of Bhlhe41 resulted in marked proliferation inhibition, together with accumulation of apoptotic cells. Furthermore, retinoic acid (RA)-induced neurite outgrowth and expression of neuronal markers are significantly weakened in Bhlhe41-/- cells. We also showed that the activity of ERK1/2 signaling, a key regulator of neuronal differentiation, is likewise impaired in knockout cells. Together, these results suggest that Bhlhe41 plays critical roles in regulating cell death and neurite outgrowth in N2a cells.

SOD1 stimulates lamellipodial protrusions in Neuro 2A cell lines

ABSTRACTWe here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants.

Enhancement of biocompatibility on aligned electrospun poly(3‐hydroxybutyrate) scaffold immobilized with laminin towards murine neuroblastoma Neuro2a cell line and rat brain‐derived neural stem cells (mNSCs)

Electrospinning has been extensively used to construct tissue‐engineered scaffolds because of its ability to provide the fibrous scaffold with structurally analogous to the naturally occurring protein in the extracellular matrix of native tissues. In addition, the modification of scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation, and differentiation. In the present contribution, we prepared and investigated the potential used of aligned electrospun poly(3‐hydroxybutyrate) (PHB) scaffold immobilized with bioactive molecule to serve as nervous scaffold. Laminin was successfully immobilized on the surface using covalent binding between functional groups of modified scaffolds and protein. The ability to use for neural regeneration was evaluated in vitro towards murine neuroblastoma Neuro2a cell line and mouse brain‐derived neural stem cells. The surface modification with laminin immobilized on the PHB fibrous scaffolds supported the attachment and promoted the proliferation of Neuro2a very wells. Despite the good attachment and proliferation of Neuro2a and mouse brain‐derived neural stem cells were not able to proliferate on the neat PHB, hydrolyzed PHB and laminin immobilized on hydrolyzed PHB fibrous scaffold.

Gold nanoparticle-mediated laser stimulation induces a complex stress response in neuronal cells

Stimulation of neuronal cells generally resorts to electric signals. Recent advances in laser-based stimulation methods could present an alternative with superior spatiotemporal resolution. The avoidance of electronic crosstalk makes these methods attractive for in vivo therapeutic application. In particular, nano-mediators, such as gold nanoparticles, can be used to transfer the energy from a laser pulse to the cell membrane and subsequently activate excitable cells. Although the underlying mechanisms of neuronal activation have been widely unraveled, the overall effect on the targeted cell is not understood. Little is known about the physiological and pathophysiological impact of a laser pulse targeted onto nanoabsorbers on the cell membrane. Here, we analyzed the reaction of the neuronal murine cell line Neuro-2A and murine primary cortical neurons to gold nanoparticle mediated laser stimulation. Our study reveals a severe, complex and cell-type independent stress response after laser irradiation, emphasizing the need for a thorough assessment of this approach’s efficacy and safety.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

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

Antimicrobial and anticancer potential of low molecular weight polypeptides extracted and characterized from leaves of Azadirachta indica

Low molecular weight antimicrobial polypeptides were extracted and purified from the young fresh leaves of Azadirachta indica (neem). The total protein extracted was precipitated with 15% TCA-Acetone. The total purified proteins yielded from the two extraction methods were 122.33±2.21 and 115.09±1.88mg/g of the total fresh weight. The SDS-PAGE analysis identified the presence of eight low molecular weight polypeptide bands. The antimicrobial activity of the resolved bands was detected by Polyacrylamide gel-Agar overlay diffusion assay (PAG-ADA). Their broad-spectrum bactericidal activity was confirmed using the same technique and found three low molecular weight bands from 11 to 14kDa collectively exhibiting superior bactericidal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermis, Enterococcus faecalis, Pseudomonas aeruginosa and fungicidal activity against Candida tropicalis. The FTIR spectrum of the protein bands depicted the presence of hydroxyl and carbonyl groups in the protein bands. These polypeptides were characterized by MALDI-TOF/TOF analysis. Further, the purified protein extract was found to be active against HELA, BT-549 and Neuro-2a cell lines with IC50 value of 74.03±2.31, 64.82±1.64, 238.32±2.12 and 109.94±2.96, 59.61±0.75 for 24h and 48h, respectively. The results of present study indicate that these polypeptides exhibit broad spectrum antimicrobial and anticancer activity and can therefore be explored for their therapeutic potential.

Isolation, Synthesis and AChE Inhibitory Potential of Some Novel Cinnamyl Esters of Taraxerol, the Major Metabolite of the Mangrove Bruguiera cylindrica.

Systematic chemical screening of the leaves of Bruguiera cylindrica, the tree mangrove of Rhizophoraceae family, afforded five single and pure compounds. The structures of the isolated compounds were established by their spectroscopic data as taraxerol (1), 3β-(E)-coumaroyltaraxerol (2), 3β-(Z)-coumaroyltaraxerol (3), β-sitosterol (4), and eicosanol (5). In view of significant accumulation and interesting biological activities, taraxerol (1) was chemically transformed to synthesize a series of ten cinnamyl esters in very good to excellent yields. The synthesized analogues along with the parent compound were evaluated for their AChE inhibitory potential, BBB permeability and cytotoxicity against Neuro 2A cell line. Among the tested samples, compound 9 showed promising AChE inhibition with significantly low IC50 values, low cytotoxicity and high BBB permeability. Hence, compound 9 can be considered as a lead molecule for further development as potent AChE inhibitor.

Green facile synthesis of low-toxic superparamagnetic iron oxide nanoparticles (SPIONs) and their cytotoxicity effects toward Neuro2A and HUVEC cell lines

Superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) were synthesized by co-precipitation using polyvinyl alcohol (PVA) as a capping agent under alkaline condition. The produced X-ray diffraction (XRD) pattern evidenced the presence of peaks corresponding to the inverse spinel structure of the prepared SPIONs. Debye-Scherrer and field emission scanning microscopy (FESEM) showed the prepared SPIONs to be well-defined with about < 50 nm size. Likewise, the superparamagnetic properties of the SPIONs measured by Vibrating Sample Magnetometer (VSM) showed high saturation magnetization (~ 65.36 emu/g). The in vitro cytotoxicity studies on Neuro2A and HUVEC cells have mentioned low toxic and non-toxic SPIONs, respectively in a range of concentrations (1.17–150 μg/ml), thus, we reckon that the synthesized SPIONs will have persistent utilization in different fields of medical applications.

Extracellular Vesicles Derived from Wharton’s Jelly Mesenchymal Stem Cells Prevent and Resolve Programmed Cell Death Mediated by Perinatal Hypoxia-Ischemia in Neuronal Cells

Hypoxic-ischemic (HI) insult in the perinatal phase harbors a high risk of encephalopathy in the neonate. Brain cells undergo apoptosis, initiating neurodegeneration. So far, therapeutic approaches such as cooling remain limited. Transplantation of mesenchymal stem cells (MSCs) exhibits therapeutic success despite the short-time survival in the host brain, providing strong evidence that their beneficial effects are largely based on secreted factors, including extracellular vesicles (EVs). The aim of this study was to investigate the effects of human Wharton’s jelly MSC (hWJ-MSC)-derived EVs on neuroprotection and neuroregeneration, using an in vitro model of oxygen–glucose deprivation/reoxygenation (OGD/R) mimicking HI injury in the mouse neuroblastoma cell line neuro2a (N2a). hWJ-MSC-derived EVs were isolated from cell culture supernatants by multistep centrifugation and identified by endosomal marker expression and electron microscopy. OGD/R significantly increased DNA fragmentation and caspase 3 (Casp3) transcription in N2a cells relative to undamaged cells. OGD/R-mediated DNA fragmentation and Casp3 expression could be prevented as well as resolved by the addition of hWJ-MSC-derived EV before and after OGD, respectively. hWJ-MSC-derived EV also tended to increase the phosphorylation of the B cell lymphoma 2 (Bcl2) family member Bcl-2-antagonist of cell death (BAD) in N2a cells, when added prior or post OGD, thereby inactivating the proapoptotic function of BAD. Fluorescence confocal microscopy revealed the close localization of hWJ-MSC-derived EVs to the nuclei of N2a cells. Furthermore, EVs released their RNA content into the cells. The expression levels of the microRNAs (miRs) let-7a and let-7e, known regulators of Casp3, were inversely correlated to Casp3. Our data suggest that hWJ-MSC-derived EVs have the potential to prevent and resolve HI-induced apoptosis in neuronal cells in the immature neonatal brain. Their antiapoptotic effect seems to be mediated by the transfer of EV-derived let-7-5p miR.