Figure 9. Aβ-induced cell compromise increases the amount of truncated tau in medium from primary rat hippocampal neurons. A, Aggregation of SEC-isolated Aβ(1–42) was followed using a continuous thioflavin T (ThT)-binding assay and fluorescence values are expressed in relative fluorescence units and plotted versus time. The ½tmax point at which sample was collected and used for toxicity experiments is indicated by the red arrow and the inset shows a negatively stained transmission EM of ½tmax Aβ(1–42). Scale bar, 100 nm. B, LDH activity in PRN CM after 1, 3, 5, and 7 d of treatment with 20 μm ½tmax Aβ(1–42); (■), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in LDH levels between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate caused an elevation in tau on all days (p < 0.01), whereas Aβ treatment increased tau only on days 5 and 7 (p < 0.01). Insets show MAP2-stained neurons after 7 d of treatment with 20 μm ½tmax Aβ(1–42) and 10.9 mm HEPES, pH 7.8. C, Tau detected by BT2-Tau5 (mid-region) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 20 μm ½tmax Aβ(1–42); (■), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in tau concentration between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.01), whereas Aβ(1–42) (■) caused a significant increase only on days 5 and 7 (p < 0.01). D, E, Tau detected by the CT1 (K9JA-K9JA; D) and CT2 (K9JA-Tau46; E) ELISAs evinced elevation on days 1, 3, 5, and 7 in medium from cells treated with glutamate (▴; p < 0.05) and on days 5 and 7 when cells were treated with Aβ(1–42) (■; p < 0.02). F, Tau detected in PRN CM by the FL (HJ9.4-Tau46) ELISA showed no difference between cultures treated with vehicle or glutamate on any day, whereas the levels of FL tau were elevated in CM from neurons treated with Aβ(1–42) (■) compared with vehicle (10.9 mm HEPES, pH 7.8; ●) on day 7 (p < 0.05). G, ADDLs were prepared as described in the Materials and Methods and characterized by aSEC (void volume indicated with an arrow) and negative contrast EM. Scale bar, 100 nm. H, Tau detected by BT2-Tau5 (mid-region) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 0.5 μm ADDLs (♦), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in tau concentration between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.05), whereas ADDLs (♦) did not cause a significant increase on any day (p > 0.05). I, Tau detected by the CT1 (K9JA-K9JA) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 0.5 μm ADDLs (♦), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.01), whereas ADDLs (♦) did not cause a significant increase on any day (p > 0.05). The data shown are derived from a minimum of six wells per treatment.