TY - JOUR T1 - Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations JF - bioRxiv DO - 10.1101/272351 SP - 272351 AU - Haowei Jiang AU - Morgan Newman AU - Dhanushika Ratnayake AU - Michael Lardelli Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/02/26/272351.abstract N2 - Protein aggregates such as those formed in neurodegenerative diseases can be degraded via autophagy. To assess changes in autophagic flux in zebrafish models of familial Alzheimer’s disease (fAD) mutations, we first developed a transgene, polyQ80-GFP-v2A-GFP, expressing equimolar amounts of aggregating polyQ80-GFP and a free GFP internal control in zebrafish embryos and larvae. This assay detects changes in autophagic flux by comparing the relative strength of polyQ80-GFP and free GFP moiety signals on western immunoblots probed with an antibody detecting GFP. However, the assay’s application is limited by the toxicity of polyQ80-GFP, and because aggregation of this protein may, itself, induce autophagy. To overcome these issues, we subsequently developed a similar ratiometric assay where expression of a GFP-Lc3a-GFP transgene generates initially equimolar amounts of GFP-Lc3a (directed to autophagic degradation) and a free GFP internal control. The sensitivity of this latter assay is reduced by a cellular protease activity that separates Lc3a from GFP-Lc3a, thus contributing to the apparent free GFP signal and somewhat masking decreases in autophagic flux. Nevertheless, the assay demonstrates significantly decreased autophagic flux in zebrafish lacking presenilin2 gene activity supporting that the Presenilin2 protein, like human PRESENILIN1, plays a role(s) in autophagy. Zebrafish heterozygous for a typical fAD-like, reading-frame-preserving mutation in psen1 show decreased autophagic flux consistent with observations in mammalian systems. Unexpectedly, a zebrafish model of the only confirmed reading-frame-truncating fAD mutation in a human PRESENILIN gene, the K115Efs mutation of human PSEN2, shows possibly increased autophagic flux in young zebrafish (larvae).AbbreviationsAβamyloid-βADAlzheimer’s DiseaseAPPAmyloid beta A4 proteinAtgautophagy-related geneAVautophagic vacuoleCQchloroquineGFPgreen fluorescent proteinLC3microtubule-associated protein 1 light chain 3PEphosphatidylethanolaminpolyQpoly-glutaminePSENpresenilin; Rapa, rapamycin ER -