Alzheimer’s disease is associated with increased levels of β-amyloid peptide (Aβ), which has been shown to mediate at least some of its cellular effects by activation of mitogen activated protein (MAP) kinases. Here we have examined the effects of Aβ and D-JNK1, an inhibitor of c-Jun-N-terminal kinase (JNK) on long-term potentiation (LTP) in the CA1 region in vivo. We also examined phosphorylation of JNK2, JNK3 and the transcription factor, c-jun.
Following experimental procedures animals were humanely killed. Tissue was prepared from the CA1 region of electrically stimulated hippocampi from urethane-anaesthetized (1.5g kg-1, I.P.) male Wistar rats injected intracerebroventricularly (I.C.V.) 2 h and 6 h previously with water or Ab[1-40] (1 nmol in 5ml). Phosphorylation of JNK2, JNK3 and c-jun was assessed by gel electrophoresis and immunoblotting using phospho-specific antibodies.
JNK2 phosphorylation was increased significantly 2 h post Aβ-injection (12.72 ± 1.0 n = 6) compared with controls (9.2 ± 0.2, n = 6) and at 6 h (43.6 ± 4.3) compared with controls (29.0 ± 1.6 n = 5) (arbitrary units; means ± S.E.M.; P < 0.05, Student’s unpaired t test). JNK3 phosphorylation was also increased significantly 2 h post Aβ-injection (10.13 ± 0.44) compared with control tissue (6.76 ± 0.37, n = 5) (P < 0.05), while 6 h post injection (27.82 ± 4.33) levels were similar to the control level (22.43 ± 2.58, n = 6). In addition, phosphorylation of the JNK substrate, c-jun, was increased significantly 2 h post Aβ injection (14.97 ± 2.64) compared with the control value (8.35 ± 1.19, n = 6; P < 0.05, unpaired t test).
To examine the effects of Aβ and D-JNK1 on LTP, water vehicle, Aβ [1-40] (1 nmol) and/ or D-JNK1 (1 or 2.5 nmol in 5ml) was injected 1 h prior to high frequency stimulation (HFS). HFS comprised 10 trains of 10 pulses at 200 Hz at 20 s intervals delivered 3 times. LTP was measured 60 min post HFS. Aβ[1-40] (1 nmol) significantly reduced LTP (115 ± 6 %, n = 12, P < 0.001) (mean ± S.E.M., ANOVA) compared with control (160 ± 9 %, n = 15). Following injection of D-JNK1 (1 nmol) there was no significant change in LTP (155 ± 10 %, n = 7). A higher concentration of D-JNK1 (2.5 nmol) caused a significant depression of LTP (135 ± 9 %, n = 7, P < 0.05), compared with control values (160 ± 9 %, n = 15). Combined injection of Aβ[1-40] and D-JNK1 at 1 or 2.5 nmol attenuated the Aβ-mediated depression of LTP observed previously; the mean percentage changes were 133 ± 7 % (n = 10) and 140 ± 11 % (n = 5), respectively. These values are significantly different from those recorded in the presence of Aβ alone (P < 0.05).
These results demonstrate that Aβ causes an increase in JNK2, JNK3 and c-jun phosphorylation, while changes in phospho-JNK3 are time dependent. The inhibitor D-JNK1 attenuated the depressive effects of Aβ[1-40] on LTP suggesting that the JNK signalling pathway may be involved in mechanisms associated with the Aβ mediated depression of LTP.
This work was supported by Enterprise Ireland (Grant SC/2001/470). A.M. is a TCD Ussher fellow.