Study of the JNK1 pathway as a selective target for the treatment of cognitive metabolic disorder: ADAM10 protein analysis

Abstract

Although the brain had been once considered an insulin-insensitive organ, several in vivo and in vitro studies have shown that insulin regulates neuronal survival, circuit function and plasticity, and it also acts as a brain growth factor. Moreover, the hippocampus, a key brain region in memory and learning processes, exhibits high levels of insulin receptors and has been identified as an important target for insulin. These data suggest that insulin could play a role in mechanisms related with brain activity and, specifically, with memory formation. In this way, alterations in insulin pathway have been associated with brain disorders, including Alzheimer's disease (AD), and may affect the pathways of this illness, including their acting players, as ADAM10 protein. In fact, chronic consumption of HFD can promote brain insulin resistance, triggering the onset of AD. Indeed, patients with diabetes type 1 and 2 show structural brain alterations and cognitive impairment, with high risk to develop AD. In light of these findings, AD has been proposed to be denominated "type 3 diabetes" (T3DM) or "cerebral diabetes". Concerning this point, it has been proposed that an aberrant TNF-± signaling leads to activation of the stress c-Jun N terminal kinase (mainly JNK-1). Activated JNK-1 phosphorylates insulin receptor substrate 1 (IRS-1) at serine residues (IRS-1pSer), blocking downstream insulin signaling and thus triggering peripheral and brain insulin resistance. To inhibit this pathway, we propose the use of chalcones compounds, concretely Licochalcone A (Lic-A), which has been identified as an inhibitor of PTP1B (protein tyrosine phosphatase 1B), an enzyme responsible for the negative regulation of the insulin signaling pathway, and it is therefore a potential drug target for the treatment of type 2 diabetes. Likewise, recent studies have demonstrated that Lic-A targets JNK1 and JNK2, although it inhibits selectively JNK1 kinase activity. Therefore, we hypothesize that JNK1 inhibition by Lic-A will prevent the phosphorylation of IRS-1 receptors and will enhance insulin-related signaling pathways, which might provide a safe and effective strategy to prevent or treat AD/T3DM. The main aims of this project are to study the JNK1 kinase activity during the cognitive metabolic syndrome progression (AD/type 3 diabetes) and evaluate and characterize the selective inhibitor of JNK1 Lic-A and ADAM10 protein levels in APPsw/PS1E9 mice, as pre-clinical target for AD treatment. Wild type strain C57/BL6 animals, JNK1 (-/-) and CamKII-Cre mice MKK4 flox/flox MKK7 flox/flox R26 flox/flox STOP-YFP flox/flox will be used in this project, subjected to chronic intake of high fat diet (HFD) or conventional diet, during 8 months. For the second aim, C57 / BL6 and APPsw/PS1E9 male mice will be fed with conventional or HFD diet plus Lic-A. Immunocytochemical techniques will be performed, following by western blotting, spine density analysis, RT-qPCR and ELISA of homogenate cortex and hippocampus for a range of proteins and genes, as anti-insulin receptor, anti substrates insulin 1 and 2, phosphorylated on serine or tyrosine, to assess the activity of RIP receptor (Tyr 1150), pIRS1 Ser 612, pIRS2 (Ser 731), anti-PGC1-± and anti-PPARy, anti-phospho-CREB Ser133, AMPK anti-mTOR and anti-ADAM10. In the project under development in the country, the prevalence of metabolic alterations (hypertension, diabetes mellitus and dyslipidemia) reaches more than 80% of patients with AD, vascular, Parkinson and frontotemporal dementia. Thus, the analysis of these alterations and their respective activity routes, as well as their possible interferences with ADAM10 protein levels are of extreme relevance for the research in progress. These studies may help us in interpretation of possible changes in ADAM10 not predicted between sample groups, as well as in the search for new peripheral biomarkers that are directly or indirectly related to ADAM10 between different dementia types. (AU)