Figure 7.

Increased TrkB-FL expression together with TrkB-T1 interference confers neuroprotection against excitotoxicity. (a) Schematic diagram of lentivirus LV-HA-TrkB-FL/2xmiT1, which was designed for the neuro-specific expression of recombinant HA-TrkB-FL simultaneously with the double interference of TrkB-T1 by miT1(2178) and miT1(2310) pre-miRNAs (purple and violet boxes, respectively). (b) Time course of NMDA treatment in cultures transduced with LV-HA-TrkB-FL/2xmiT1 (m.o.i.=2) showing higher levels of total TrkB-FL and strong interference of TrkB-T1 both in basal and stimulated conditions compared with LV-DsRed/GFP infections. (c) Relative neuronal viability (mean±S.E.M., n=3) of cultures transduced with different viruses that modified the TrkB-FL/TrkB-T1 balance and stimulated with NMDA (2 h) are expressed as percentages of the values obtained in untreated cells infected with the same virus (arbitrarily established as 100%). Cultures were infected with LV-HA-TrkB-FL/2xmiT1, LV-DsRed/GFP, LV-HA-TrkB-FL/GFP, LV-miC/GFP (m.o.i.=2), or LV-miT1(2178)/GFP, together with LV-miT1(2310)/GFP (m.o.i.=1 for each virus). Student's unpaired t-test demonstrated statistically significant differences between cells infected with LV-HA-TrkB-FL/2xmiT1 or LV-DsRed/GFP, and treated with NMDA (^***P<0.001). (d) Time-course analysis of the effect of NMDA on neuronal viability after LV-HA-TrkB-FL/2xmiT1 infection (m.o.i.=2). Relative neuronal viability (mean±S.E.M., n=3) is expressed as percentage of the value obtained in untreated LV-DsRed/GFP infected cells (arbitrarily established as 100%). Student's unpaired t-test showed significant differences between cells infected with LV-HA-TrkB-FL/2xmiT1 or LV-DsRed/GFP at different times of NMDA treatment (^***P<0.001), which demonstrates a neuroprotective effect of the simultaneous increase in TrkB-FL expression and the interference with TrkB-T1 synthesis