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. 2018 Sep 10;8:13545. doi: 10.1038/s41598-018-31787-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Lunasin downregulates JNK signaling to block Aβ42 mediated neurodegeneration. (A) Schematic presentation of JNK signaling pathway. (B) Levels of phospho-JNK (pJNK) in a semi-quantitative Western Blot can provide the status of JNK signaling. The higher levels of JNK signaling in GMR > Aβ42 as compared to the wild-type background were significantly downregulated in GMR > Aβ42 + Lun background. The tubulin bands served as controls to normalize the levels of total protein loaded in all three conditions. The p-JNK band staining intensity was calculated by ImageJ. In comparison to the wild-type (C) eye imaginal discs and (D) adult eyes, activation of JNK signaling in GMR domain using (E,F) Djun^aspv7 (GMR > jun) and (K,L) constitutively active hep^Act (GMR > hep) result in strong neurodegenerative phenotype. Furthermore, activation of JNK signaling in GMR > Aβ42 background (G,H) GMR > Aβ42 + jun (M,N) GMR > Aβ42 + hep exhibits stronger neurodegenerative phenotype which are not rescued by misexpression of Lunasin (I,J) GMR > Aβ42 + Lun + jun and (O,P) GMR > Aβ42 + Lun + hep. (Q,R) Downregulation of JNK signaling by misexpression of puc, a dual phosphatase, results in near wild-type (Q) eye imaginal discs and (R) adult eyes. (S–V) Misexpression of puc in (S,T) GMR > Aβ42 (GMR > Aβ42 + puc) and (U,V) GMR > Aβ42 + Lun (GMR > Aβ42 + Lun + puc) results in significant rescue as seen in eye imaginal discs and the adult eyes. The puc-lacZ reporter is used as a functional read out of JNK signaling pathway. Expression of puc-lacZ reporter (Green) in (W,W’) Wild-type, (X,X’) GMR > Aβ42 and (Y,Y’) GMR > Aβ42 + Lun eye imaginal discs. (W,W’) Note that puc has weak expression in the developing photoreceptor neurons in the wild-type eye imaginal discs. However, (X,X’) puc expression is dramatically upregulated in GMR > Aβ42 background. (Y,Y’) Misexpression of Lunasin (Lun) along with Aβ42 (GMR > Aβ42 + Lun) can significantly downregulate puc expression in the developing third instar eye disc. Magnification of all eye discs is 20X.