Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

Abstract

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).

Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2

“…not the endoproteolytically-cleaved, γ-secretase-active form of PSEN1) was required for normal acidification of lysosomes by promoting N-glycosylation of the V0a1 subunit of v-ATPase that is required for its correct localization to the lysosome. (Note that it has not yet been demonstrated formally that PSEN2 plays a similar role although, in work yet to be published formally, we claim that homozygosity for psen2 S4Ter causes decreased autophagic flux in zebrafish larvae [67]). Recently, Yambire et al demonstrated that deficient lysosomal acidification causes a cellular deficiency of ferrous iron leading to brain mitochondrial dysfunction and inflammatory responses [68].…”