NT2N Cell Research Articles

PPARα activation attenuates amyloid-β-dependent neurodegeneration by modulating Endo G and AIF translocation.

The accumulation of a large amount of amyloid-β (Aβ42) in brain neurons is one of the debilitating characteristics of Alzheimer's disease. In this study, we determined the effects of peroxisome proliferator-activated receptor alpha (PPARα) activation on neuronal degeneration using a model of Aβ42-induced cytotoxicity. We found that 0.5 μM Aβ42 induced DNA damage and apoptosis in NT2N cells after 6 h of treatment. Co-treatment of Aβ42-treated cells with Wy14643, a PPARα ligand, significantly increased cell viability after 24 h compared with cells treated with Aβ42 alone. There were no differences in the protein levels of caspase-3, Bcl-2/Bax or p53 between cells treated with Aβ42 alone and those treated with both Aβ42 and Wy14643. However, the addition of Wy14643 significantly suppressed the Aβ42-induced upregulation of Endo G and AIF protein levels. Immunohistochemical analyses further demonstrated that Wy14643 reduced the expression of Endo G and AIF translocated from the cytoplasm into the nucleus, which occurred concomitantly with the decrease in DNA damage in Aβ42-treated cells. Our data clearly show that PPARα activation prevents DNA damage and neuronal cell apoptosis by decreasing the expression and translocation of AIF/Endo G to the nucleus in a caspase-3- and p53-independent pathway in the NT2N cell model. This role of PPARα in promoting neuron survival suggests a possible clinical application in treating Aβ42-associated neurotoxicity in Alzheimer's disease.

CD59 Efficiently Protects Human NT2‐N Neurons Against Complement‐mediated Damage

The complement regulatory protein CD59 controls cell survival by the inhibition of C5b-9 formation on the cell membrane. Loss of CD59 increases the susceptibility of cells to complement-mediated damage and lysis. Deposition of IgM can induce complement activation with subsequent cell death. We have previously demonstrated the presence of CD59 on human NT2-N neurons. In this study, we investigated the functional role of CD59 for NT2-N cell survival after IgM-mediated complement activation. Complement activation was induced on NT2-N neurons with human serum following incubation with the IgM monoclonal antibody A2B5 reacting with a neuronal cell membrane epitope. Deposition of C1q and C5b-9 was detected on the cell membrane and sC5b-9 in the culture supernatant. Specific inhibition of complement was obtained by the C3 inhibitor compstatin, and by anti-C5/C5a MoAb. CD59 was blocked by the MoAb BRIC 229. Membrane damage of propidium iodide-stained NT2-N cells was confirmed by immunofluorescence microscopy and degeneration of neuronal processes was shown with crystal violet staining. A2B5, but not the irrelevant control IgM antibody, induced complement activation on NT2-N neurons after incubation with a human serum, as detected by the deposition of C1q. A marked membrane deposition of C5b-9 on NT2-N neurons with accompanying cell death and axonal degeneration was found after the blocking of CD59 with MoAb BRIC 229 but not with an isotype-matched control antibody. Compstatin and anti-C5 monoclonal antibodies which blocked C5 activation efficiently inhibited complement activation. In conclusion, CD59 is essential for protecting human NT2-N neurons against complement-mediated damage, which is known to occur in a number of clinical conditions including stroke.

Gene therapy, cell transplantation and stroke

The use of neuroteratocarcinoma cells for transplantation therapy in stroke has emerged as a strategy for cell replacement therapy that has begun its transition from basic science laboratories to a clinical setting. Procurement logistics and novel neuroprotective functions associated with these cells allow neuroteratocarcinoma cells to serve as efficacious alternatives to using fetal cells as donor cell grafts for stroke therapy, although the optimal transplantation regimen must still be determined. In particular, the limitations of current stroke treatments and management reveal an urgent need to examine the efficacy of experimental treatments, such as neural transplantation, in order to develop better treatment therapies. This chapter will discuss the characteristics of NT2N cells, the role of the host brain microenvironment and NT2N cell grafts, laboratory research and clinical trials for the intracerebral transplantation of NT2N cells in stroke, the mechanisms underlying the grafts' effects, and NT2N cell grafts and the need for immunosuppression. This chapter will also highlight some of the most recent findings regarding NT2N cells.

Human neuronal cells (NT2-N) express functional substance P and neurokinin-1 receptor coupled to MIP-1 beta expression.

Substance P (SP), the most extensively studied and potent member of the tachykinin family, is a major modulator of inflammation and immunomodulatory activities within the central and peripheral nervous systems. We have examined the gene expression of SP and its receptor in a human neuronal cell line (NT2-N). Using reverse transcribed polymerase chain reaction (RT-PCR), the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma, and delta) were detected in the NT2-N. We also identified the presence of mRNA for neurokinin-1 receptor (NK-1R), a primary receptor for SP, in the NT2-N cells. Concomitant with NT2 cell differentiation into neurons, SP and NK-1R mRNA expression increased consistently. Intracellular SP and cell membrane NK-1R immunoreactivity were all observed in NT2-N cells. Most importantly, we demonstrated that SP and NK-1R presented in NT2-N cells are functionally involved in the regulation of macrophage inflammatory protein 1 beta (MIP-1beta), an important beta-chemokine participating in the activation and directional migration of immune cells to sites of central nervous systems (CNS) inflammation. Thus, SP and its receptor may play an important role in modulation of neuronal functions related to regulation of immune activities within the CNS. The NT2-N cell line is well suited for in vitro investigations of the SP-NK-1R pathway in immune responses and inflammation in the CNS.

Cloning and mutation analysis of the human potassium channel KCNQ2 gene promoter.

Benign familial neonatal convulsions (BFNC) have been previously found to be associated with mutations within the coding region of KCNQ2. We have now cloned and analyzed the promoter region of the human KCNQ2 gene. 5'-RACE identified a transcription start site (TSS) located 200 bp upstream of the ATG start codon. The TSS is located close to a repetitive region containing seven copies of a degenerate 42-mer repeat. Several different luciferase (LUC) reporter plas- mids containing fragments from the KCNQ2 5'-flanking region were constructed and expressed in NT2N and SH-SY5Y cell lines. A core promoter region was found to be located between bp 20 and bp 74 upstream of the TSS. Neither the promoter region nor the repetitive region showed any mutations in 13 index patients from unrelated BFNC families.

Evidence for neuroprotective effects of acidic fibroblast growth factor in Alzheimer disease.

Recent studies indicate that fibroblast growth factors (FGFs) might confer neuroprotection against excitotoxicity. Therefore, the fact that acidic FGF (aFGF) is more abundant in motoneurons than in the hippocampal formation suggests that aFGF contributes to the selective vulnerability of neurons in entorhinal cortex (EC) in Alzheimer disease (AD). In order to understand the role of aFGF in AD, patterns of aFGF FGF receptor (FGFR), and N-methyl-D-aspartate (NMDA) receptor (NMDAR) expression in the EC and hippocampus of AD and control cases were investigated, and effects of aFGF on excitotoxicity were examined in vitro. In AD, the number of aFGF immunolabeled neurons was decreased in EC, while the remaining neurons showed significantly higher aFGF immunoreactivity. This latter group of neurons did not show cytoskeletal abnormalities. Acidic FGF and FGFR immunoreactivity were positively correlated, whereas a negative correlation was found between aFGF and NMDAR expression. These results were confirmed in vitro utilizing NT2N cells. Higher levels of FGFR protein were expressed in aFGF-treated cells, while less NMDAR protein was found compared with untreated cells. Furthermore, exposure of treated and untreated NT2N cell to glutamate revealed that aFGF can prevent glutamate induced cell death. Taken together these data suggest that aFGF regulates the expression of NMDAR and FGFR and thereby contributes to neuroprotection against glutamate excitotoxicity. Therefore, altered patterns of aFGF immunoreactivity in EC in AD are an important marker for selective vulnerability of EC neurons.

Survival and integration of transplanted postmitotic human neurons following experimental brain injury in immunocompetent rats.

Limitations regarding cell homogeneity and survivability do not affect neuronlike hNT cells, which are derived from a human teratocarcinoma cell line (Ntera2) that differentiates into postmitotic neurons with exposure to retinoic acid. Because NT2N neurons survive longer than 1 year after transplantation into nude mice brains, the authors grafted these cells into the brains of immunocompetent rats following lateral fluid-percussion brain injury to determine the long-term survivability of NT2N cell grafts in cortices damaged by traumatic brain injury (TBI) and the therapeutic effect of NT2N neurons on cognitive and motor deficits. Seventy-two adult male Sprague-Dawley rats, each weighing between 340 and 370 g, were given an anesthetic agent and subjected to lateral fluid percussion brain injury of moderate severity (2.2-2.5 atm in 46 rats) or to surgery without TBI (shamoperation, 26 rats). Twenty-four hours postinjury, 10(5) NT2N cells (24 injured animals) or 3 microl of vehicle (22 injured and 14 control animals) was stereotactically implanted into the periinjured or control cerebral cortex. Motor function was assessed at weekly intervals and all animals were killed at 2 or 4 weeks after their posttraumatic learning ability was assessed using a Morris water maze paradigm. Viable NT2N grafts were routinely observed to extend human neural cell adhesion molecule-(MOC-1)immunoreactive processes into the periinjured cortex at 2 and 4 weeks posttransplantation, although no significant improvement in motor or cognitive function was noted. Inflammation identified around the transplant at both time points was assessed by immunohistochemical identification of macrophages (ED-1) and microglia (isolectin B4). Long-term survival and integration of NT2N cells in the periinjured cortex of immunocompetent rats provides the researcher with an important cellular system that can be used to study maturation, regulation, and neurite outgrowth of transplanted neurons following TBI.

Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells.

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs-HEPES buffer containing 2.5 mM CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and D(-)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 +/- 14 nM to 59 +/- 8 nM. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 microM) or of the N-type inhibitor omega-conotoxin GVIA (5 microM) significantly, although incompletely, suppressed Ca2+ oscillations, while omega-conotoxin MVIIC (5 microM), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 microM, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2-5 mM) or Co2+ (1 mM) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 microM), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (alpha1D subunit) and N-type (alpha1B subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (alpha1-subunit) was not present in the NT2N cells as shown by reverse transcription-polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.

Cytotoxic Effect of β‐Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation

The effects of synthetic beta-amyloid (A beta1-42) on cell viability and cellular Ca2+ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. A beta1-42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 microM, and the strongest effect was produced by 40 microM. Control NT2N cells produced spontaneous cytosolic Ca2+ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4-40 microM) by A beta1-42 that was present in the cell culture for 1 or 4 days. Ca2+ wave frequency was decreased from 0.21 +/- 0.02 to 0.05 +/- 0.02/min, amplitude from 88 +/- 8 to 13 +/- 4 nM, and average Ca2+ level from 130 +/- 8 to 58 +/- 3 nM. The Ca2+ responses to 30 mM K+ and 100 microM glutamate were not different between control and A beta-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca2+ accumulation mediates A beta-induced NT2N cell death.

Transplantation of Cryopreserved Human Embryonal Carcinoma-Derived Neurons (NT2N Cells) Promotes Functional Recovery in Ischemic Rats

This study was designed to explore the efficacy of a human clone cell line as an alternative neural graft source and to validate the practice of cryopreservation and xenografting as logistical approaches toward conducting neural transplantation. We investigated the biological effects of transplanting cultured human neurons (NT2N cells) derived from a well-characterized embryonal carcinoma cell line into the brains of rats subjected to transient, focal cerebral ischemia induced by embolic occlusion of the middle cerebral artery. At 1 month and extending throughout the 6-month posttransplantation test period, ischemic animals that were transplanted with NT2N cells and treated with an immunosuppressive drug displayed a significant improvement in a passive avoidance task as well as a normalization of asymmetrical motor behavior compared to ischemic animals that received rat fetal cerebellar cell grafts or vehicle alone. Remarkably, cryopreserved NT2N cell grafts, compared with fresh NT2N cell grafts, remained viable in the immunosuppressed rat brain and effective in producing behavioral recovery in immunosuppressed ischemic animals. The long-term viability of cryopreserved NT2N cell xenograftsin vivoand their sustained effectiveness in promoting behavioral recovery suggest potential utilization of xenografting and cryopreservation as useful protocols for establishing clone cell lines as graft source in neural transplantation therapies for central nervous system disorders.

Opposing regulation of tau protein levels by ionotropic and metabotropic glutamate receptors in human NT2 neurons

Human NT2-N neurons derived from retinoic acid treatment of the NTera 2 cell line were used to determine the consequences of ionotropic glutamate receptor (iGluR) hyperstimulation and possible modulatory role(s) exerted by metabotropic glutamate receptor (mGluR) activation. We found that NT2-N neurons express the NR1 subunit of N-methyl- d-aspartate (NMDA) iGluRs and mRNA encoding the 1a isoform of mGluRs. A 15 min pulse with 100 μM NMDA induced an increase in the levels of tau proteins in NT2-N cells. This effect was prevented by incubating NT2-N neurons in the presence of the mGluR agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection against NMDA-induced NT2-N cell death. Our findings suggest that iGluRs and mGluRs might participate in the control of human neuron viability by differentially affecting the expression of tau proteins.

Mechanism of nitric oxide and peroxynitrite induced NT2N cell death: [formula omitted], Andrew J. Gow, Marios Themistocleous, Virginia M-Y Lee and Harry Ischiropoulos. Children's Hospital of Philadelphia

The reaction of the S-nitrosothiol compound N-acetyl-1-amino-2-methylpropyl-2-thionitrite (RSNO) with the model metalloporphyrin complex RuII(OEP)(CO) (OEP = octaethylporphyrinato dianion) gives the addition product trans-RuII(OEP)(NO)(SR). Here we report the details of a stopped flow kinetics investigation which demonstrates the rapid equilibrium formation of an intermediate concluded to be S-bound RSNO complex RuII(OEP)(RSNO)(CO), which undergoes a rate-limiting step, presumably S-NO bond cleavage to give a second intermediate RuIII(OEP)(SR)(CO) too short lived for direct observation. Notably, this is different from the nitrogen coordination pathway often proposed and represents an alternative mechanism by which S-nitrosothiols may be formed or decomposed in the presence of redox active metal centers. Also reported is a brief study of the quantitative photochemistry of RSNO, the photodecomposition of which complicates the kinetics studies by spectroscopic techniques.

Amyloids and Are Generated Intracellularly in Cultured Human Neurons and Their Secretion Increases with Maturation

Previous studies have demonstrated the presence of amyloid beta (Abeta) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Abeta is present intracellularly since trypsin digestion of intact NT2N cells at 4 degrees C did not eliminate the Abeta recovered in cell lysates. To determine whether both Abeta40 and Abeta42 are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Abeta monoclonal antibodies. Sandwich ELISA detected intracellular Abeta40 and A++beta42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Abeta40 and Abeta42 were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Abeta40/Abeta42 in the medium, suggesting that Abeta is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Abeta40 and Abeta42 increases more than 5-fold over 7 weeks. This increase in the secretion of Abeta40/Abeta42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.

Toxicity of TNF alpha and platelet activating factor for human NT2N neurons: a tissue culture model for human immunodeficiency virus dementia.

A significant proportion of HIV-1 infected individuals develop a symptom complex consisting of dementia and motor deficits termed HIV Dementia (HIVD) or the AIDS Dementia complex (ADC). The pathophysiology of this neurologic complication is unclear, but neuronal injury and death may occur as a direct result of the release of cytokines from HIV-1 infected microglial cells (Everall et al, 1991). To evaluate the utility of a human neuronal cell line, NT2N, for studies of HIV-related neuronal cytotoxicity, we studied cellular viability after exposure to HIV-1 gp120, tumor necrosis factor alpha (TFN alpha), platelet activating factor (PAF), interleukin 1 beta (IL-1 beta), and interferon gamma (IFN gamma), all of which have been implicated in previous publications as having a role in HIVD (Brenneman et al, 1988; Dreyer et al, 1990; Merrill et al, 1992; Gelbard et al, 1993). Neither gp 120 nor the cytokines IL-1 beta and IFN gamma resulted in significant NT2N cell death. However, TNF alpha and PAF were highly neurotoxic in this assay. Pentoxifylline, which inhibits the effects of TNF alpha, had a significant protective effect. This system provides an excellent substrate for the evaluation of neurotoxicity and for the development of pharmacologic agents that may be useful in HIV dementia.

Chapter 14 Expression of Foreign Proteins in a Human Neuronal System

The NT2N cell system offers an attractive way to overcome some of the technical limitations inherent in working with primary neuronal cultures. In particular, it is possible to obtain large quantities of neurons with which to perform biochemical experiments, and the growth of neurites can be synchronized and controlled by varying the substrate on which the cells grow. In addition, because the differentiated NT2N neurons are derived from a mitotically active precursor cell line in vitro, it is possible to employ a variety of techniques, that are not otherwise available when working directly with postmitotic neurons, to obtain expression of foreign proteins. Because they are fully polarized, NT2N cells offer a way to study protein sorting to axons and dendrites at both the biochemical and the morphological level. Further characterization of NT2N cells is underway, and more efficient ways to obtain expression of foreign proteins will no doubt be found.