Figure 3.
Neurites of N2a cells overexpressing tau are highly susceptible to oxidative stress. (A, top) Control N2a cells (mock-transfected with vector) differentiated for 2 d and exposed to 250 μM H2O2 for up to 2 h. At 60 min, long neurites (>30 μm) stained for microtubules are less frequent but still clearly visible, and even at 120 min some thin neurites can be discerned. (A, bottom) Tau-stable N2a cells treated with H2O2. Neurites (>30 μm) disappear quickly and are no longer visible at 60 min. (B) Quantitation of oxidative stress by H2O2 and protection by catalase. Differentiated N2a cells (2 d) were treated with 30 μM H2O2, and neurites longer than 30 μm (twice the cell body diameter) were scored. In mock-transfected cells (left group of bars), the fraction of cells with long neurites drops from an initial value of 12.3 to 5.9% (∼ 2-fold) after 40 min of H2O2. In tau-transfected cells, (right group of bars) this fraction drops from an initial 9.9% down to 0.3% (∼33-fold). Adding catalase (0.02 U/μl) but no H2O2 to the medium does not change the number of long neurites (black bars). When catalase is added immediately after H2O2 exposure in control cells, it protects the cells so that 8.7% still have long neurites after 40 min H2O2 treatment compared with only 5.9% without catalase. In the case of tau-overexpressing cells, the fraction of cells with long neurites drops from 9.9 to 0.3% after H2O2 treatment but only to 5.6% with catalase. Thus, the higher vulnerability of neurites in tau-expressing cells can be explained by the lack of peroxisomes carrying catalase. Experiments were done in triplicate, 3 × 150 cells were counted for each condition. Error bars show SEM. (C) Inhibition of catalase exacerbates vulnerability of neurites against H2O2 in controls but not in tau-transfected cells. (Left) Mock-transfected cells differentiated for 2 d: the fraction of cells with long neurites (>30 μm) drops from 11.5% (untreated) to 5.2% after H2O2 treatment (30 μM, 40 min). Inhibition of catalase with 10 mM 3-AT for 60 min causes some degeneration (to 9.0%), but treatment first with 10 mM 3-AT for 60 min and then 30 μm H2O2 for 40 min causes a drop to 2.7%. Thus, the inhibition of catalase enhances the toxic effect of H2O2. (Right) N2a cells stably transfected with tau have a lower initial number of long neurites (8.4%). They are diminished somewhat by 3-AT treatment (to 6.6%) but almost disappear after H2O2 treatment (0.7%). In these conditions, a similar value (0.9%) is observed with 3-AT + H2O2 treatment because the peroxisomes are already excluded from the neurites (which is equivalent to catalase inhibition). Error bars show SEM; n = 300.