Anegawa NJ, Guttmann RP, Grant ER, Anand R, Lindstrom J, Lynch DR.. N-Methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines: characterization using fluorescent measures of cell viability and reactive oxygen species production. Brain Res Mol Brain Res 77: 163-175
Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtypes undergo cell death that mimics glutamate-induced excitotoxicity pharmacologically. We have further characterized the mechanisms of cell death resulting from NMDA receptor activation in such cells through development of cell counting methods based on co-transfection with green fluorescent protein. When co-transfected with NMDA receptors, GFP expression was limited to live cells as indicated by the observation that GFP was only detected in cells which were positive for markers of live cells, and was found in no cells which were trypan blue or propidium iodide positive. Using co-transfection with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific profile of NMDA receptor-mediated cell death in cells transfected with NMDA receptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2B receptor, and least in NR1A/2C receptors. Cell death also differed pharmacologically between subunit combinations. Cell death in cells transfected with NR 1A/2A was blocked by amino-phosphonovaleric acid at lower concentrations than in cells transfected with NR 1A/2B. In cells transfected with the NR1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associated with production of reactive oxygen species. In addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell death. The use of GFP based cell counting provides a sensitive mechanism for assessing the mechanism of excitotoxicity in transfected cell models.
"In addition, several authors reported that GFP, stably integrated into the genome of test cells, provides an excellent and simple tool to measure apoptosis or necrosis by flow cytometry (Strebel et al., 2001) and that GFP is an useful ''live-cell biosensor'' for many cellular activities, representing an accurate measure of viable cells (Anegawa et al., 2000). Because the transfection efficiency and GFP intensity depend upon several factors, such as the culture time of tissue biopsies after transfection, lipofectamine ratios, and capacitance for EP, we have tried to optimize the transfection conditions testing different peri- Fig. 3. "
[Show abstract][Hide abstract] ABSTRACT: In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.
Microscopy Research and Technique 01/2009; 73(3):229-33. DOI:10.1002/jemt.20779 · 1.15 Impact Factor
"Firstly, it is not clear whether NSC components can be disrupted without affecting pro-survival or plasticity signaling. Secondly, NMDAR-dependent cell death can be reconstituted in non-neuronal cells lacking the NSC simply by expressing NMDARs (Cik et al., 1993; Anegawa et al., 2000) which shows that certain NMDAR-induced death pathways do not require the NSC. "
[Show abstract][Hide abstract] ABSTRACT: NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca(2+), and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2008; 28(42):10696-710. DOI:10.1523/JNEUROSCI.1207-08.2008 · 6.34 Impact Factor
"Importantly , similar to young wild-type cultures, excitotoxicity can be mediated via the NR2B subunit also in older mice if NR2A receptor signalling is impaired. The importance of the C-terminal domain of the NR2AR subunit was also shown in HEK293 cells in which truncation of the last 406 amino acids (NR2A D1050 ) reduced NMDAR dependent toxicity (Anegawa et al., 2000). In contrast to this, truncation of the C-terminal 190 amino acids (NR2A D1266 ) of NR2A does not alter NMDA-mediated toxicity in HEK293 cells (Wagey et al., 2001), indicating that amino acids 1051-1266 of this subunit might be involved in mediating the toxic NMDA effect. "
[Show abstract][Hide abstract] ABSTRACT: Excitotoxicity, exacerbating acute brain damage from brain trauma or stroke, is mediated in part by excessive Ca(2+)-influx from prolonged NMDA receptor activation. However, the contribution to excitotoxicity by each of the main NMDAR subtypes in glutamatergic forebrain neurons, the NR2A- and NR2B-types, has remained enigmatic. Here, we investigated this issue by use of pharmacological and genetic tools in cultured cortical neurons. In wild-type neurons the contribution of the NMDA receptor subtypes to excitotoxicity changed with the age of the cultures. The blockade of NR2B-containing NMDA receptors prevented NMDA-mediated toxicity in young cultures after 14days in vitro (DIV14), but both subtypes triggered excitotoxicity in older (DIV21) cultures. Notably, blocking either of the two subtypes failed to prevent NMDA-elicited cell death, indicating that the remaining subtype triggers cell demise. Intriguingly, a neuroprotective aspect of the NR2A subtype became apparent at submaximal NMDA concentration only at DIV21. The NR2A subtype mediated NMDA toxicity as well as partial protection only if it carried a functional C-terminal domain. Upon deletion of this domain in the NR2A subtype, excitotoxicity was mediated entirely via the NR2B subtype, both at DIV14 and DIV21. Our findings predict that successful therapeutic intervention in stroke based on currently available NMDA receptor subtype-selective blockers is unlikely.
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