| Oxidative stress, mitochondrial dysfunction, and high levels of extracellular glutamate are underlying mechanisms associated with the progressive neuronal loss that occurs with neurodegenerative diseases and injuries affecting the central nervous system. Recent studies have indicated that Vitamin K (VK), an essential lipophilic vitamin that plays well known roles in blood coagulation and bone formation, may also play important roles in brain function, including its ability to protect neurons and oligodendrocytes from oxidative cell death. The mechanism by which it mediates this effect is unknown, however, and in this study we use the HT22 mouse neuronal cell line to investigate the protective effects of VK against oxidative glutamate toxicity. Using fluorescent dyes, enzyme assays, and specific antibodies, we found that VK prevents mitochondria) ROS generation and maintains mitochondria) membrane potential in cells exposed to oxidative stress. Using confocal microscopy, fluorescent dyes, and Seahorse respirometry, we also found that VK treatment improves several parameters of cellular and mitochondria) bioenergetic status, including increased ATP and NAD(P)H levels, increased mitochondria) membrane potential, and decreased oxygen consumption. In isolated brain mitochondria, we used substrate regulation and selective pharmacological inhibitors to assess the site-specific effects of VK on mitochondrial ROS generation. We found that VK specifically prevents the production of reactive oxygen species (ROS) from Complex Ill in the electron transport chain, which appears to be critical for the initiation of oxidative cell death.;Furthermore, several studies have implicated defects in the transport and metabolism of VK as being contributing factors to the development of neurodegenerative diseases. Therefore, we conducted a structure activity relationship study of VK using the HT22 oxidative glutamate toxicity model. This study resulted in a line of VK analogs that mimic its protective functions but that have increased neuroprotective potency and possess structural properties enabling them to penetrate the brain without the need for active transport or metabolic processing, which we assessed by analyzing brain extracts of treated mice using HPLC-MS/MS.. These analogs are indicated to be non-toxic and capable of preventing seizures in multiple epilepsy models. |
