Abstract
Purpose:
Although autophagy is associated with cell death in neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease, it is unclear whether autophagy contributes to cell death, protects cells from dying, or is an epiphenomenon driven by a pathway perturbed by or contributing to cell death. We have previously observed autophagy associated with cell death in transgenic X. laevis models of retinitis pigmentosa. Here we report the use of a transgenic marker to facilitate the study of autophagy in normal and disease states.
Methods:
LC3 is a ubiquitin-like protein that is conjugated to the membranes of autophagosomes, and is a standard marker for autophagy. We made transgene constructs consisting of a tandemly linked fluorescent protein mRFP-eGFP-LC3 driven by either the hsp70 heat shock promoter (hsp70-lc3) or the rod opsin promoter (xop-lc3), and used these transgenes to generate transgenic X. laevis tadpoles.<br />
Results:
Both transgenes were successfully expressed in tadpole rod photoreceptors with little or no toxicity. Upon heat shock induction, hsp70-LC3 was expressed in a variety of retinal cell types, including rods, although we noted preferential expression in the peripheral retina. The XOP-LC3 transgene was expressed only in rod photoreceptors. The dually fluorescent LC3 fusion was incorporated into newly formed autophagosomes that appeared as green or dually green and red dots in fluorescent micrographs, which gradually become exclusively red due to quenching of the GFP signal by acid hydrolases in lysozomes. Thus, expression of these fusions can be used to track the dynamic process of autophagy in photoreceptors.
Conclusions:
We can track the process of autophagy in photoreceptors of transgenic tadpoles. Further investigations will characterize the process of autophagy, including numbers of autophagosomes and their rate of turnover, in normal and disease states.