Abstract TP110: Dissecting the Optimized Cytoprotective Responses of Arctic Ground Squirrels With CRISPR

Abstract

Arctic ground squirrels (AGS) have evolved to survive harsh environmental conditions through hibernation. AGS retain resistance to stroke and ischemic injury independent of hibernation, suggesting that genetic cell autonomous changes are also critical to AGS resilience. The mechanistic details of these cytoprotective adaptations are largely unknown. A functional cDNA expression screen identified AGS candidate genes facilitating stress resilience and a bioinformatics pipeline identified AGS protein sequence changes in regions of high conservation. These unique AGS amino acid substitutions were edited using a combination of CRISPR base-editing and Cas9 knock-out approaches to generate AGS cell lines with loss-of-function variants. We found that AGS neural cells exhibit marked resilience to diverse metabolic stressors in assays of in vitro survival, function, and mitochondrial morphology. The functional genetic screen identified a network of evolutionarily-conserved AGS transcripts imparting cytoprotection, particularly in endoplasmic reticulum (ER) stress and oxidative phosphorylation (OXPHOS) pathways. CRISPR-based genome editing creating AGS cell lines with loss-of-function mutations indicated that editing of key candidates, such as ATP5G1, ameliorates features of the AGS resilient phenotype including increasing susceptibility to metabolic stressors, decreasing spare respiratory capacity, and altering mitochondrial dynamic organization. We gained key functional insights into how specific amino acid substitutions in the machinery of the OXPHOS and ER stress responses systems alter mitochondrial function to impart cytoprotection against metabolic insults. This detailed dissection of the AGS optimized adaptive stress response pathway will serve as a template for the development of new neuroprotective treatments.