Abstract TP251: CRISPR-engineered Mouse Neural Cells Expressing An Optimized Manf Variant Reveals Mechanisms Of MANF Neuroprotection

Abstract

Introduction: Hibernating mammals have evolved physiological strategies to cope with harsh winters by markedly reducing their metabolic rate and core temperature. Interestingly, neural stem cells derived from Arctic ground squirrels (AGS) also possess resilient cell biological processes that preserve cell integrity during hypoxia and exposure mitochondrial toxins. We identified AGS mesencephalic astrocyte-derived neurotrophic factor (MANF) as a key cytoprotective gene in diverse survival screens of mouse neural cells expressing an AGS cDNA library. Hypothesis: We hypothesize that adaptively evolved amino acid substitution in AGS MANF confers metabolic stress resilience by modulating actions of MANF including sulfatide-binding, reducing endoplasmic reticulum (ER) stress and altering mitochondrial metabolism. Methodology: CRISPR-Cas9 knock-in (KI) gene editing was utilized to generate mouse neural cells harboring the AGS Manf allele to allow for analysis of the beneficial effects of the AGS amino acid substitutions in cell survival to metabolic stress, mitochondrial metabolism, MANF protein stability, and sulfatide binding.. We also tested AGS compared to human MANF with regard to reducing ER stress under hypoxia, hypothermia, and rotenone. Results: AGS MANF expression reduced ER stress markers compared to human MANF, and demonstrated increased stability following rotenone treatment further pointing to its important role regulating cellular adaptations to metabolic stress and imparting neuroprotection in our in vitro model. We gained key functional insights into how specific amino acid substitutions improve MANF stability to impart cytoprotection to metabolic insults. This detailed dissection of the AGS optimized adaptive stress response pathway can serve as a template for the development of new neuroprotective treatments.