Human NT2 Cells Research Articles

PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells

The Gαq/phospholipase C-β (PLCβ) signaling system mediates calcium responses to a variety of hormones and neurotransmitters. Recent studies suggest that PLCβ1 expression plays a role in the differentiation of two types of cultured neuronal cells (PC12 and SK-N-SH) through a mechanism independent of Gαq. Here, we show that, similar to that observed in PC12 and SK-N-SH cells, PLCβ1 expression increases when human NT2 cells are induced to differentiate either through cytosine-β-D-arabinofuranoside or retinoic acid. Preventing this increase, abolishes differentiation, and down-regulating PLCβ1 in rat primary astrocytes causes cells to adapt an undifferentiated morphology. Surprisingly, transfecting PLCβ1 into undifferentiated PC12 or NT2 cells induces differentiation without the need for differentiating agents. Studies to uncover the underlying mechanism focused on the transcription factor early growth response 1 (Egr-1) which mediates PLCβ1 expression early in differentiation. Over-expressing PLCβ1 in HEK293 cells enhances Egr-1 expression and induces morphological changes. We show that increased levels of cytosolic PLCβ1 in undifferentiated PC12 cells disrupts the association between Egr-1 and its cytosolic binding partner (Tar RNA binding protein), promoting relocalization of Egr-1 to the nucleus, which promotes transcription of proteins needed for differentiation. These studies show a novel mechanism through which differentiation can be modulated.

Synthesis and Biological Evaluation of Novel Benzylidene Thiazolo Pyrimidin-3(5H)-One Derivatives

Starting compound 1 was synthesized according to reference. 1 Benzylidene thiazole pyrimidin-3(5H)-ones were synthesized reactions of 1 with bromoacetic acid and various aryl-aldehydes in the same vessel via one-step, unlike studies in the literature. Quantum chemical parameters and full geometry optimizations for all compounds were computed using DFT based on B3LYP. Cytotoxic action potential of synthesized compounds was evaluated using trypan blue dye exclusion and MTT assays in different cell lines including adenocarcinoma alveolar basal epithelial-like adherent A549 cells, the colon adenocarcinoma HT-29 cells, prostate adenocarcinoma DU-145 cells, and diploid ARPE-19 retinal pigment epithelial cells. Embryotoxicity and genotoxicity assessments were performed on pluripotent human embryonal carcinoma NT2 and human lymphocyte cells, respectively. Compound A1 exhibited good anticancer activity on A549 and DU-145 cell lines, and the compounds including A3, 4, 6, and 9 induced cytotoxicity on A549 cells. The compounds A1-10 also showed a good biosafety profile at relatively lower concentrations.

Effects of Tolerance-Induced Preconditioning on Mitochondrial Biogenesis in Undifferentiated and Differentiated Neuronal Cells.

Mitochondrial biogenesis occurs in response to chronic stresses as an adaptation to the increased energy demands and often renders cells more refractive to subsequent injuries which is referred to as preconditioning. This phenomenon is observed in several non-neuronal cell types, but it is not yet fully established in neurons, although it is fundamentally important for neuroprotection and could be exploited for therapeutic purposes. This study was designed to examine whether the preconditioning treatment with hypoxia or nitric oxide could trigger biogenesis in undifferentiated and differentiated neuronal cells (rat PC12 and human NT2 cells) as well as in primary mouse cortical neurons. The results showed that both preconditioning paradigms induced mitochondrial biogenesis in undifferentiated cell lines, as indicated by an increase of mitochondrial mass (measured by flow cytometry of NAO fluorescence) and increased expression of genes required for mitochondrial biogenesis (Nrf1, Nrf2, Tfam, Nfκb1) and function (Cox3, Hk1). All these changes translated into an increase in the organelle copy number from an average of 20-40 to 40-60 mitochondria per cell. The preconditioning treatments also rendered the cells significantly less sensitive to the subsequent oxidative stress challenge brought about by oxygen/glucose deprivation, consistent with their improved cellular energy status. Mitochondrial biogenesis was abolished when preconditioning treatments were performed in the presence of antioxidants (vitamin E or CoQ10), indicating clearly that ROS-signaling pathway(s) played a critical role in the induction of this phenomenon in undifferentiated cells. However, mitochondrial biogenesis could not be re-initiated by preconditioning treatments in any of the post-mitotic neuronal cells tested, i.e., neither rat PC12 cells differentiated with NGF, human NT2 cells differentiated with retinoic acid nor mouse primary cortical neurons. Instead, differentiated neurons had a much higher organelle copy number per cell than their undifferentiated counterparts (100-130 mitochondria per neuron vs. 20-40 in proliferating cells), and this feature was not altered by preconditioning. Our study demonstrates that mitochondrial biogenesis occurred during the differentiation process resulting in more beneficial energy status and improved tolerance to oxidative stress in neurons, putting in doubt whether additional enhancement of this phenomenon could be achieved and successfully exploited as a way for better neuroprotection.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Evaluating the effect of siRNA on SOX2OT expression in the human neuron-committed teratocarcinoma NT2 cell line.

Non-coding RNA elongated (lncRNAs) have recently attracted as molecules that regulate gene expression of the pluripotent properties (pluripotency) of stem cells. Recently our colleagues examined the role of one of these RNAs called SOX2OT in esophageal squamous cell carcinoma, and found a concomitant increase in its expression with some regulatory genes of cell proliferation. In the present study, using the design of suitable primers from SOX2OT gene, we investigated the effect of siRNA on expression of SOX2OT.

The Superiority of Sucrose Cushion Centrifugation to Ultrafiltration and PEGylation in Generating High-Titer Lentivirus Particles and Transducing Stem Cells with Enhanced Efficiency

Viral gene delivery is hailed as a great milestone in gene-based therapeutic approaches. The human immunodeficiency virus-derived lentiviral vectors (LVs) are advantageous in infecting both dividing and non-dividing cells leading to continuous expression of transgenes. A variety of protocols are available for concentration of LVs. We primarily generated our internal ribosome entry site (IRES)-based LVs. Virus titration and transduction efficiency were compared between various strategies that included sucrose cushion centrifugation (SCC), protein column ultrafiltration and polyethylene glycol precipitation. Among these approaches, SCC resulted in concentration of high-titer EGFP-expressing lentivirus (1.4±0.3×109TU/ml) with the lowest protein impurities. Further, we examined transduction strengths of our three methods on two challenging stem cells. Both human NT2 and mouse bone marrow-derived mesenchymal stem cells demonstrated high transduction using SCC of 65±2.8 and 49±0.8%, respectively. Finally, lentivirus particles harboring IRES-based transfer vectors of specific genes, concentrated by SCC, integrated into host genome. Taken together, development of cost-effective and efficient concentration strategies such as our SCC method is yet highly demanded to broaden the horizons of lentivirus application in clinical and translational research.

Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity

Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO’s increasing number of differentiated neurons in OHSC. In conclusion, CO’s increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO’s effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Exogenous carbon monoxide improves neuronal differentiation: a near-death experience

Cerebral ischemic injuries and neurodegenerative disorders lead to death or impairment of neurons in the central nervous system. Application of stem cell based therapies, namely stimulation of endogenous neurogenesis or cell transplantation, are promising strategies and currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase. Although there is no published data reporting CO as a factor involved in stem cell differentiation, several evidences support this hypothesis. This gasotransmitter induces mitochondrial biogenesis, which is also broadly described to be involved in metabolic shifts during neuronal differentiation process. Likewise, CO-induced pathways can occur via generation of small amounts of ROS, which are also important signaling molecules in neuronal differentiation. The CO effect on modulation of neuronal differentiation is assessed in three different models with increasing complexity: human neuroblastome SH-S5Y5 cell line, human teratocarcinome NT2 cell line and hippocampal organotypic slice cultures (HOSC). CO does increase the final yield of post-mitotic neurons. During neuronal differentiation, CO promotes an increase on precursor cell proliferation and in parallel CO inhibits cell death. Furthermore, cell mitochondrial population is increased by CORM-A1 supplementation. Further work is needed for assessing the mechanisms underlying CO effects in neuronal differentiation, namely by targeting modulation of cellular metabolic pathways, redox alterations and autophagy related pathways. In conclusion, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Transcription of the Human Sodium Channel SCN1A Gene Is Repressed by a Scaffolding Protein RACK1

Voltage-gated sodium channel α subunit type I (Nav1.1, encoded by SCN1A gene) plays a critical role in the initiation of action potential in the central nervous system. Downregulated expression of SCN1A is believed to be associated with epilepsy. Here, we found that the SCN1A promoter (P1c), located at the 5' untranslated exon 1c, drove the reporter gene expression in human NT2 cells, and a region between nt +53 and +62 downstream of the P1c promoter repressed the promoter activity. Further analyses showed that a scaffolding protein RACK1 (receptor for activated C kinase 1) was involved in binding to this silencer. Knockdown of RACK1 expression in NT2 cells deprived the repressive role of the silencer on the P1c promoter and increased SCN1A transcription, suggesting the potential involvement of RACK1 in negatively regulating SCN1A transcription via interaction with the silencer. Furthermore, we demonstrated that the binding of the protein complex including RACK1 to the SCN1A promoter motif was decreased in neuron-like differentiation of the NT2 cells induced by retinoic acid and resulted in the upregulation of SCN1A transcription. Taken together, this study reports a novel role of RACK1 in regulating SCN1A expression that participates in retinoic acid-induced neuronal differentiation of NT2 cells.

A melatonin‐based fluorescence method for the measurement of mitochondrial complex III function in intact cells

Mitochondrial complex III (MC-3) plays a pivotal role in electron transfer and oxidative phosphorylation. Impaired MC-3 functions may contribute to a variety of diseases by interrupting normal bioenergetics and increasing reactive oxygen production and oxidative stress. Currently, MC-3 function is assessed by measuring the cytochrome c reductase activity spectrophotometrically in isolated mitochondria or MC-3. The cytoplasmic microenvironment critical for mitochondrial complex functions may be depleted during these isolation processes. The development of a reliable method to measure MC-3 activities in intact cells or tissues is highly desirable. This report describes a novel fluorescence-based method to assess MC-3 functions, i.e., Qi site electron transfer, in the intact cells. Human mesangial and teratocarcinoma NT2 cells were used to demonstrate that melatonin-induced oxidation of 2',7'-dichlorodihydrofluorescein (H2 DCF) was inhibited by antimycin A, the MC-3 Qi site-specific inhibitor, but not by myxothiazol, the MC-3 Qo site-specific inhibitor, nor rotenone, the mitochondrial complex I inhibitor. These results indicate that melatonin-induced oxidation of H2 DCF is reflecting MC-3 Qi site electron transfer activities. Modifying structures of the side groups at the R3 and R5 positions of the indole ring of melatonin diminished its efficacy for inducing H2 DCF oxidation, suggesting a specific interaction of melatonin with the MC-3 Qi site. These results suggest that the fluorogenic property of melatonin-induced H2 DCF oxidation provides a MC-3 Qi site electron transfer-specific measurement in intact cells. Interestingly, using this method, the Qi site electron transfer activity in transformed or immortalized cells was found to be significantly higher than the nontransformed cells.

Cul4B regulates neural progenitor cell growth

BackgroundCullin ubiquitin ligases are activated via the covalent modification of Cullins by the small ubiquitin-like protein nedd8 in a process called neddylation. Genetic mutations of cullin-4b (cul4b) cause a prevalent type of X-linked intellectual disability (XLID) in males, but the physiological function of Cul4B in neuronal cells remains unclear.ResultsThere are three major isoforms of Cul4B (1, 2, and 3) in human and rodent tissues. By examining the endogenous Cul4B isoforms in the brain, this study demonstrates that Cul4B-1 and Cul4B-2 isoforms are unneddylated and more abundant in the brain whereas the lesser species Cul4B-3 that misses the N-terminus present in the other two isoforms is neddylated. The data suggest that the N-terminus of Cul4B inhibits neddylation in the larger isoforms. Immunostaining of human NT-2 cells also shows that most Cul4B is unneddylated, especially when it is localized in the process in G0-synchronized cells. This study demonstrates that Cul4B accumulates during mitosis and downregulation of Cul4B arrests NPCs and NT-2 cells in the G2/M phase of the cell cycle. In both human and rodent brain tissues, Cul4B-positive cells accumulate β-catenin in the dentate subgranular zone and the subventricular zone. These Cul4B-positive cells also co-express the MPM-2 mitotic epitope, suggesting that Cul4B is also necessary for mitosis progression in vivo.ConclusionsThis study provides first evidence that unneddylated Cul4B isoforms exist in the brain and are necessary for mitosis progression in NPCs. The data suggest that unneddylated Cul4B isoforms specifically inhibits β-catenin degradation during mitosis. Furthermore, unneddylated Cul4B may play a role in addition to cell cycle since it is exclusively localized to the processes in starved NT-2 cells. Further analyses of the different isoforms of Cul4B will help understand the cognitive deficits in Cul4B-linked XLID and give insights into drug and biomarker discoveries.

The human testis‐determining factor SRY localizes in midbrain dopamine neurons and regulates multiple components of catecholamine synthesis and metabolism

The male gender is determined by the sex-determining region on the Y chromosome (SRY) transcription factor. The unexpected action of SRY in the control of voluntary movement in male rodents suggests a role in the regulation of dopamine transmission and dopamine-related disorders with gender bias, such as Parkinson's disease. We investigated SRY expression in the human brain and function in vitro. SRY immunoreactivity was detected in the human male, but not female substantia nigra pars compacta, within a sub-population of tyrosine hydroxylase (TH) positive neurons. SRY protein also co-localized with TH positive neurons in the ventral tegmental area, and with GAD-positive neurons in the substantia nigra pars reticulata. Retinoic acid-induced differentiation of human precursor NT2 cells into dopaminergic cells increased expression of TH, NURR1, D2 R and SRY. In the human neuroblastoma cell line, M17, SRY knockdown resulted in a reduction in TH, DDC, DBH and MAO-A expression; enzymes which control dopamine synthesis and metabolism. Conversely, SRY over-expression increased TH, DDC, DBH, D2 R and MAO-A levels, accompanied by increased extracellular dopamine levels. A luciferase assay demonstrated that SRY activated a 4.6 kb 5' upstream regulatory region of the human TH promoter/nigral enhancer. Combined, these results suggest that SRY plays a role as a positive regulator of catecholamine synthesis and metabolism in the human male midbrain. This ancillary genetic mechanism might contribute to gender bias in fight-flight behaviours in men or their increased susceptibility to dopamine disorders, such as Parkinson's disease and schizophrenia.

Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines.

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Rare Drosha Splice Variants Are Deficient in MicroRNA Processing but Do Not Affect General MicroRNA Expression in Cancer Cells

Drosha is a key enzyme in microRNA biogenesis, generating the precursor miRNA (pre-miRNA) by excising the stem-loop embedded in the primary transcripts (pri-miRNA). The specificity for the pri-miRNAs and determination of the cleavage site are provided by its binding partner DGCR8, which is necessary for efficient processing. The crucial Drosha domains for pri-miRNA cleavage are the middle part, the two enzymatic RNase III domains (RIIID), and the dsRNA binding domain (dsRBD) in the C-terminus. Here, we identify alternatively spliced transcripts in human melanoma and NT2 cell lines, encoding C-terminally truncated Drosha proteins lacking part of the RIIIDb and the entire dsRBD. Proteins generated from these alternative splice variants fail to bind to DGCR8 but still interact with Ewing sarcoma protein (EWS). In vitro as well as in vivo, the Drosha splice variants are deficient in pri-miRNA processing. However, the aberrant transcripts in melanoma cells do not consistently reduce mature miRNA levels compared with melanoma cell lines lacking those splice variants, possibly owing to their limited abundance. Our findings show that alternative processing-deficient Drosha splice variants exist in melanoma cells. In elevated amounts, these alternatively spliced transcripts could provide one potential mechanism accounting for the deregulation of miRNAs in cancer cells. On the basis of our results, the search for alternative inactive splice variants might be fruitful in different tumor entities to unravel the molecular basis of the previously observed decreased microRNA processing efficiency in cancer.

Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A.

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.

Formation of New High Density Glycogen-Microtubule Structures Is Induced by Cardiac Steroids

Cardiac steroids (CS), an important class of naturally occurring compounds, are synthesized in plants and animals. The only established receptor for CS is the ubiquitous Na(+),K(+)-ATPase, a major plasma membrane transporter. The binding of CS to Na(+),K(+)-ATPase causes the inhibition of Na(+) and K(+) transport and elicits cell-specific activation of several intracellular signaling mechanisms. It is well documented that the interaction of CS with Na(+),K(+)-ATPase is responsible for numerous changes in basic cellular physiological properties, such as electrical plasma membrane potential, cell volume, intracellular [Ca(2+)] and pH, endocytosed membrane traffic, and the transport of other solutes. In the present study we show that CS induces the formation of dark structures adjacent to the nucleus in human NT2 and ACHN cells. These structures, which are not surrounded by membranes, are clusters of glycogen and a distorted microtubule network. Formation of these clusters results from a relocation of glycogen and microtubules in the cells, two processes that are independent of one another. The molecular mechanisms underlying the formation of the clusters are mediated by the Na(+),K(+)-ATPase, ERK1/2 signaling pathway, and an additional unknown factor. Similar glycogen clusters are induced by hypoxia, suggesting that the CS-induced structural change, described in this study, may be part of a new type of cellular stress response.

Autophagy is involved in the early step of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry−LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.

Neurotoxic factors released by stimulated human monocytes and THP-1 cells

Activated monocytes/macrophages are known to release toxic materials. Identification of these materials is important for developing more effective treatments for inflammatory disorders where self attack occurs. We stimulated human monocytes and THP-1 cells with LPS/IFNγ and measured the toxic effects of their conditioned media against differentiated human NT-2 cells. Their cytotoxicity, as measured by LDH release, was reduced by half when their conditioned media was passed through a 3 kDa cutoff filter, indicating an equal division between high and low molecular weight materials. When the high molecular weight components tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and IL-6 were removed from the conditioned medium by specific antibodies, the toxicity was reduced by 37–38%. When prostaglandin production was blocked by treatment with the COX inhibitors acetylsalicylic acid and ibuprofen, toxicity was reduced by 15–16%. When oxygen free radical production was blocked by the NADPH inhibitor diphenylene iodonium (DPI) the toxicity was reduced by 17–18%. Treatment with the nitric oxide scavenger carboxy-phenyl-tetramethylimidazolineoxyl-oxide, or the NOS inhibitor N G-monomethylene- l-arginine, attenuated the toxicity by about 20%. Removal of released glutamate by glutamate decarboxylase also attenuated the toxicity by 12–13%. In combination, these treatments reduced the toxicity by approximately 50% accounting for the low molecular weight component toxicity. About 10% of the overall toxicity, which was associated with the high molecular weight component, was not identified. Optimal antiinflammatory therapy may require combined suppression of these identified toxin-generating pathways as well as relatively minor pathways yet to be identified.

GABAergic pathway in a rat model of chronic neuropathic pain: Modulation after intrathecal transplantation of a human neuronal cell line

Current understanding of chronic pain points a decrease in level of the inhibitory neurotransmitter GABA, in the spinal dorsal horn, leading to an imbalance between excitatory and inhibitory pathways. A subcloned derivative of the human NT2 cell line (hNT2.17) which, after neuronal differentiation, secretes different inhibitory neurotransmitters such as GABA and glycine has been recently isolated. In this study, we have investigated the effect of this new cell line on peripheral nerve injury induced by chronic constriction (CCI) and notably the effect on the cellular GABAergic pathway. Our data show that the decrease in GABA expression in the spinal dorsal horn of injured animals is concomitant with a decline of its synthetic enzyme GAD67 -Ir and mRNA but not GAD65. Interestingly, in transplanted animals we observed a strong induction of GAD67 mRNA with one week after graft, which is followed by a recovery of GAD67 and GABA Ir. This effect paralleled a reduction of hindpaw hypersensitivity and thermal hyperalgesia induced by CCI. These results suggest that hNT2.17 GABA cells can modulate neuropathic pain after CCI certainly by minimizing the imbalance and restoring the cellular GABAergic pathway.