Quantification of autophagosome/lysosome fusion in cardiomyocytes and brains of biallelic SPRED2-KO mice reveals a defect in autophagy
Authors: Hepbasli Denis , Gredy Sina , Niedermeier Anne , Schuh Kai 
Published: May 2023

Dysfunction of autophagy has been associated with cardiac arrhythmias and cardiomyopathies, mainly because failure of the system leads to intracellular depositions. One important signaling cascade controlling myocardial cell homeostasis and important steps of autophagy, is the ERK-MAPK pathway. SPRED2 is an important inhibitor of this pathway and plays a role in a variety of processes, e.g. in cardiovascular organ development and brain development. SPRED2 also interacts with LC3, which is essential for autophagy to occur. LC3-II, which is present in all autophagic vacuoles, is the most commonly used protein to determine autophagic flux. Biallelic SPRED2-KO mice develop impaired autophagy, heart failure, and a shortened lifespan. SPRED2 is an essential regulator of cardiac autophagy, and its absence leads to cardiac dysfunction and life-threatening arrhythmias. The objective of this study was to verify if SPRED2 influences the fusion process of autophagosomes with lysosomes and thus disrupts autophagy.To prove our hypothesis, we crossed SPRED2-KO mice with transgenic CAG-RFP-EGFP-LC3 mice (JaxMice, strain 027139), which express double-labeled LC3 ubiquitously. This allows both quantification of autophagosomes, characterized by double EGFP/RFP fluorescence and quantification of lysosomes, characterized by disappearance of EGFP fluorescence due to low pH in lysosomes.Using structured illumination microscopy, we revealed that in cardiomyocytes the number of sites of lysosome fusion was reduced in SPRED2-KO mice. We also observed that in WT cardiomyocytes more acid-insensitive RFP signal remained, which can be explained by the lower pH after fusion with lysosomes. To investigate the influence of SPRED2 on autophagy in other organs, we measured the expression of tagged LC3 in SPRED2-KO brain slices using a confocal microscope. In the hippocampus, we observed an increased signal of EGFP in SPRED-KO mice. This difference was evident in both the average size of the signal as well as the number of EGFP signals. These data indicate that the fusion of autophagosomes and lysosomes is disturbed in SPRED2-KO mice.Our findings demonstrate that SPRED2 is involved in fusion or transport of autophagosomes and lysosomes, and that autophagy is disrupted in mice lacking SPRED2. With the decreased fusion of lysosomes intracellular recycling gets interrupted, which may lead to substrate overload in the cell. Recently, the first humans carrying mutations in the SPRED2 gene have been identified. To diagnose and treat such very rare diseases in the future, investigation of such molecular and physiological functions of SPRED2 is indispensable. Funded by DFG, German research foundation. (Public, Funding application: Schu1600/6-1) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.