Ratiometric Imaging of Mitochondrial Hydrogen Peroxide in Aβ42-Mediated Neurotoxicity

Oxidative stress is important for the etiology andpathogenesis of Alzheimer's disease (AD). Research tools that canconveniently evaluate oxidative stress in AD models are expected tocatalyze and accelerate research on AD. This study explored the useof genetically encodedfluorescent indicators (GEFIs) to detectmitochondrial oxidative stress in organotypic brain slices and ADmouse models. To enable ratiometric normalization and avoid tissueautofluorescence, we genetically fused a greenfluorescent hydrogenperoxide (H2O2) indicator, HyPer7, with each of two selected, brightredfluorescent proteins (RFPs), mScarlet-I and tdTomato. Theresultant indicators, namely, HyPerGRSand HyPerGRT, were taggedwith mitochondrial targeting sequences and examined for localizationand function in cultured HeLa cells and primary mouse neurons. Wefurther utilized HyPerGRT, which is a genetic fusion of HyPer7 with tdTomato, to monitor mitochondrial H2O2in response to thehuman beta-amyloid 1-42 isoform (A beta 42) in cultured brain slices and an AD mouse model. Owing to the high sensitivity and lowautofluorescence interference resulting from HyPerGRT, we successfully detected A beta 42-mediated mitochondrial H2O2in these ADmodels. The results suggest that HyPerGRTis a valuable tool for studying mitochondrial oxidative stress in tissues and animals.