c-Jun N-terminal Kinase (JNK) induces phosphorylation of amyloid precursor protein (APP) at Thr668, in okadaic acid-induced neurodegeneration

Abstract

Several lines of evidence have revealed that phosphorylation of amyloid precursor protein (APP) at Thr668 is involved in the pathogenesis of Alzheimer's disease (AD). Okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used in AD research models to increase tau phosphorylation and induce neuronal death. We previously showed that OA increased levels of APP and induced accumulation of APP in axonal swellings. In this study, we found that in OA-treated neurons, phosphorylation of APP at Thr668 increased and accumulated in axonal swellings by c-jun N-terminal kinase (JNK), and not by Cdk5 or ERK/MAPK. These results suggest that JNK may be one of therapeutic targets for the treatment of AD. [BMB Reports 2016; 49(7): 376-381].

Fig. 1.. Increased level of phosphorylated APP in OA-treated neuron cultures. (A) Western blot analysis with phosphorylated-amyloid precursor protein (APP) antibody shows that APP phosphorylation meaningfully increased in okadaic acid-treated neurons compared with control neurons. (B) Temporal changes in level of phosphorylated APP in the neurite swellings. Control neurons show reduced immunoreactivity for APP. APP immunoreactivity was increased in the neurite swellings within 4 hr post treatment of OA; APP immunoreactivity was found to be accumulated in cell bodies at 16 hr post-treatment. Scale bar represents 20 µm. (C) APP (+) swellings in neurites does not colocalize with MAP2 (+) dendrites. Scale bar represents 20 µm.

Fig. 2.. Representative images showing colocalization of APP with JNK in the axonal swellings of OA-treated neurons. (A) p-JNK increases in the axonal swellings of OA-treated neurons, where APP immunoreactivity also increases (arrowheads) (B), (C) Similar to the consequences of p-JNK, both JIP-1 and JIP-3 indicates co-localization with APP phorphorylation in axonal swellings of OA-treated neurons. Scale bar represents 20 µm.

Fig. 3.. Inhibition of OA-induced APP phosphorylation with various kinase inhibitors. (A) Western blot analysis with OA-treated neurons and several kinase inhibitors, including JNK inhibitor, shows that only JNK inhibitor-SP600125 meaningfully decreased APP phosphorylation. (B) Decrease of APP phosphorylation is generally proportional to concentration of JNK inhibitor.

Fig. 4.. Reduced level of phosphorylated APP with SP600125-induced JNK inhibition. (A) APP immunoreactivity in OA-treated neuron at 8 h post treatment showed diminished axonal accumulations with SP600125 treatment. Scale bar represents 20 µm. (B) A quantitative LDH-release assay showed that treatment with OA for 48 hr increased LDH release by about 150% compared to untreated controls (CTL). Pretreatment with SP600125 reduced OA-induced LDH release by about 50% (*P < 0.05 vs. OA). MTT assay also shows a protective effect of SP600125.