Characterisation of short-hair pin RNA (shRNA) interference targeting the excitatory amino acid transporter 2 (EAAT2) in Neuro 2A cells

Nisha Kurian, Madeleine King, Kevin Fone, David Kendall, Charles Marsden, Tyson Sharp
University of Nottingham, Nottingham, United Kingdom

Glutamate is the principal excitatory neurotransmitter within the mammalian brain, and tight regulation of extracellular concentrations is essential to prevent excitotoxicity. Glutamate reuptake occurs via a family of five Na+ dependent transporters, with the glial-type EAAT2 contributing over 90% of transport capacity (Shigeri et al., 2004). The present study used shRNA interference, with lentiviral-based delivery, to target the expression of EAAT2 in a mouse neuroblastoma cell line (Neuro 2A) as a prelude to using the approach to manipulate extracellular glutamate in vivo.

Lentiviral vectors targeting EAAT2 (ES1, ES2, ES3, ES4 and ES5), and appropriate non-target vector control, were purchased from Mission® TRC shRNA library (Sigma). Lentiviral virions were produced by transfection of these into HEK293T cells using Mission® lentiviral packaging mix (Sigma) and Genejuice (Merck Biosciences). Viral particles were collected 48h post-transfection and stably transduced into 50-60% confluent Neuro 2A cell cultures, with 5μg ml-1 puromycin selection 48h post-transduction. EAAT2 expression was assessed by Western blot analysis, using an anti-EAAT2 (Alpha Diagnostics) or monoclonal β-actin antibody (Sigma). Expression levels are presented as percentages of EAAT2 expression in untransduced Neuro 2A cells (mean ± s.e.m., n=5). Glutamate uptake in these cells was assessed by incubating for 2mins with 100μM [3H]-L-glutamate (1μCi ml-1; Amersham). Uptake was terminated and cells lysed with 1.5N NaOH prior to scintillation spectrophotometry. Data are presented as DPM μg-1 of cell protein (mean ± s.e.m., n=4).

A range of EAAT2 knockdowns was observed in cells transduced with lentivirus (ES1 74.7±8; ES2 77.8±12; ES3 84.8±7; ES4 84.5±9 and ES5 80±8% of parental), but not non target vector (97±7%, n=5) control. [3H]-L-glutamate uptake at 37°C appeared to be markedly decreased in four of the five EAAT2 knockdowns (parental 51.1±15.4; ES1 21.9±1.8; ES2 27.2±6.2; ES4 22.7±4.0 and ES5 22.6±4.9DPM μg-1; knockdowns 42.8 – 53.2% of parental), but not ES3 (46.8±13.6; 91.5% of parental) or the non target vector (54.2±11.3; 106%). However, the effect of cell line just failed to reach statistical significance (P=0.0625; one-way ANOVA). Incubation at 4°C significantly reduced uptake in all cell lines (3.7±0.8 – 7.5±3.1DPM μg-1; 10.1 – 17.7% of levels at 37°C). In summary, we report the knockdown of EAAT2 expression and activity in Neuro 2A cells using shRNA interference with lentiviral-based delivery. Future studies will test the behavioural efficacy of the shRNAs after local delivery into mouse brain.



The authors are grateful to the European Commission (through the Sixth Framework Programme for Research and Development), for financial support.

Shigeri. Y et al. (2004) Brain Res. Rev. 45, 250-265