Project Summary/Abstract Oxidative stress, mitochondrial dysfunction and neuroinflammation are inter-related processes central to the pathogenesis and progression of Alzheimer's disease (AD). Mitochondrial dysfunction increases levels of reactive oxygen species (ROS), and together these increase pro-inflammatory cytokines. Pro-inflammatory cytokines, along with the increased ROS, cause further damage to mitochondria perpetuating a destructive cycle that contributes to cell death and cognitive decline. This underlying biology suggests that a multi-targeted agent could be an effective treatment strategy for AD. The traditional use of Centella asiatica herb to improve cognitive function is supported by a substantial body of preclinical literature. We have shown that a water extract of Centella asiatica (CAW) enhances synaptic density, activates the antioxidant response pathway and improves mitochondrial deficits in isolated neurons as well as in mouse models of aging and AD, in which CAW also improved cognitive performance. Our in vitro preliminary data also suggest anti-inflammatory effects of CAW; however, this remains to be confirmed in vivo. The 5xFAD mouse model of Aβ accumulation shows elevated neuroinflammation compared to wild type (WT) mice. This project will examine changes in markers of neuroinflammation in stored brains from male and female 5xFAD mice whose cognition improved following treatment with CAW in their drinking water. We have also found also triterpenes (TTs) and caffeoylquinic acids (CQAs) present in CAW affect antioxidant, mitochondrial and synaptic health in vitro and can improve cognition in 5xFAD mice. However, the specific associations of these compounds with the neuroprotective activities of CAW remain to be elucidated. Using additional stored brain samples, we will investigate the effects CAW or equivalent amounts of TTs and CQAs mixed in the diet, on markers of antioxidant response, mitochondrial activity, synaptic density, neuroinflammation and Aβ pathology in male and female 5xFAD mice. We will measure CQA and TT levels in the plasma following each treatment, both to corelate to their biological effects and to explore reasons for the differential behavioral response seen when CAW was administered in the drinking water versus in the diet. This project will inform future clinical trials of CAW by evaluating the role of neuroinflammatory markers as biological signatures of target engagement by CA, the validity of using TT and CQA as chemical markers to standardize CAW preparations, and the optimum mode of administration of CAW. This work complements the parent grant investigating resilience promoting effects of CAW, CQAs and TTs in healthy aged mice, where oxidative stress, declining mitochondrial function and neuroinflammation may occur.