Figure 2.

EGF neuroprotection against excitotoxicity is abolished in PS1-deficient neurons. A) WT, PS1^+/−, and PS1^−/− mouse PCNC were grown in 24-well plates in Neurobasal medium plus B27 supplement. At 9 DIV, neurons were treated with 20 ng/ml Hb-EGF or EGF or 35 nM progranulin (PGRN; a.k.a. GRN) overnight. The next day, the medium was switched to HBSS containing Hb-EGF, EGF, or PGRN for 30 min, followed by 50 μM glutamate incubation for 3 h, and cell viability was evaluated by MTT assay and normalized to nontreated cells, as previously described (3, 29). No significant effect compared to nontreated was observed when growth factors alone were added to cultures. B) WT PCNC in 6-well plates were treated at 9 DIV either with ERK inhibitor U0126 (U0, 5 μM) or PI3K/AKT inhibitor wortmannin (W, 50 nM) for 30 min before addition of 20 ng/ml Hb-EGF for 15 min in Neurobasal medium plus B27 supplement. After incubation, lysates were collected and assayed on WBs for the indicated proteins. C) Mouse PCNC grown as above [as in (A)] were treated at 9 DIV either with ERK inhibitor U0126 (5 μM) or PI3K/AKT inhibitor wortmannin (50 nM) for 30 min before addition of 20 ng/ml Hb-EGF. Three hours later, medium was switched to HBSS plus Hb-EGF and inhibitors for 30 min, followed by 50 μM glutamate incubation for 3 h. Cell viability was evaluated by MTT assay and normalized to nontreated cells. Results (means ± sem) were summarized from at least 4 independent experiments. In each experiment, each condition is the average of 4 identical wells. *P < 0.05 comparing between cultures treated with glutamate in the presence or absence of Hb-EGF and/or inhibitors (paired Student’s t test).