Protection from cyanide‐induced brain injury by the Nrf2 transcriptional activator carnosic acid

Acute cyanide poisoning is life threatening, blocking cellular respiration by inhibiting the mitochondrial enzyme cytochrome c oxidase. The result is cardiopulmonary failure and death within minutes (Hamel 2011; Nelson 2006). Cyanide is also a potential bioterroristic agent, e.g., that can be toxic via subacute intake in a contaminated water supply. If recognized quickly, systemic cyanide poisoning is treatable by agents binding to cyanide in order to protect cytochrome c oxidase, and efforts devoted to developing more effective antidotes are ongoing (Hamel 2011; Brenner et al. 2010; Hall et al. 2009). In a 2007 annual report from the American Association of Poison Control Centers, most of the 242 cyanide-poisoning cases survived; there were only 5 fatalities (Bronstein et al. 2008). Complete protection from acute cyanide poisoning depends on rapid administration and effective penetration into various tissues. However, current systemic antidotes, such as hydroxocobalamin, may not adequately cross the blood-brain-barrier (BBB), so the brain may remain vulnerable even in humans surviving acute cyanide poisoning. In fact, in over a dozen human suicide cases of cyanide intoxication, a delayed neurological syndrome has been documented that includes dystonia and parkinsonian signs and symptoms, even in patients who had initially appeared to have had a full recovery after the exposure (Rachinger et al. 2002; Riudavets et al. 2005; Rosenow et al. 1995; Uitti et al. 1985; Valenzuela et al. 1992; Borgohain et al. 1995; Rosenberg et al. 1989; Carella et al. 1988). Typically, these patients begin to manifest symptoms and signs after a few weeks or months, with progressive rigidity accompanied by flexed upper limbs and extended lower limbs. CT and MRI examinations of the brain consistently revealed lesions in the basal ganglia, including the globus pallidus and putamen. Damage in the substantia nigra, cerebellum, and cerebral cortex has also been reported in several human cases (Rosenow et al. 1995; Uitti et al. 1985; Carella et al. 1988). This imaging evidence for damage in the human brain has been confirmed at autopsy in some cases (Uitti et al. 1985; Riudavets et al. 2005). Similar observations have been reported in human patients after subchronic, lower dose, non-lethal cyanide exposure (Di Filippo et al. 2008), as might occur in a bioterroristic attempt at contaminating a city’s water supply. Multiple mechanisms are thought to underlie cyanide-induced neuronal damage. For example, in addition to binding to and poisoning hemoglobin, cyanide inhibits cytochrome c oxidase, thus blocking electron transport in the mitochondrial respiratory chain and disrupting oxidative phosphorylation (OX-PHOS) (Hamel 2011). Such global cessation of aerobic cell metabolism results in severe consequences for active cellular processes requiring ATP. Additionally, increased levels of reactive oxygen species (ROS) generated in response to cyanide poisoning initiate lipid peroxidation that is toxic to neurons (Johnson et al. 1987). Particularly harmful to neurons is the malfunction of glutamate transport and sodium/potassium ion exchangers, contributing to excitotoxicity due to excessive extracellular glutamate (Persson et al. 1985; Patel et al. 1991). Under these conditions, glutamate overstimulates a variety of receptors, including N-methyl-D-aspartate-type glutamate receptors (NMDARs), to literally excite neurons to death. Additionally, the release of normal Mg2+ blockade of NMDARs because of membrane depolarization, with consequent inrush of cations, further overactivates NMDARs (Nowak et al. 1984). Therefore, neurons are particularly vulnerable to cyanide poisoning, although different brain regions show differential sensitivity to cyanide (Prabhakaran et al. 2002). Since cyanide poisoning blocks mitochondrial respiration, which produces an increase in ROS in the brain with consequent severe oxidative damage in neurons, one possible strategy for the development of neuroprotective drugs is to use low-molecular-weight compounds that can cross the blood-brain-barrier and counter oxidative damage. In this regard, our group is developing pro-electrophilic drugs (PEDs) to treat neurological disorders due in part to excessive ROS; these drugs become active electrophilic compounds (EPs) when they encounter oxidative insults that chemically convert them to the active form (Satoh and Lipton 2007). By using pro-electrophiles that are themselves innocuous, we increase the chances that a compound will be a clinically-tolerated therapeutic. In contrast, compounds that are electrophilic themselves (often represented by quinone structures) may be toxic, in part, by reacting with and depleting glutathione (GSH) in normal areas of the brain (Satoh et al. 2013). We found that one such pro-electrophilic compound is carnosic acid (CA), a natural substance found in the herb rosemary (Satoh et al. 2008a; Satoh et al. 2008b; Satoh et al. 2011). Previously, we showed that CA readily crosses the blood-brain-barrier and exerts its protective effect after conversion from its catechol form to its quinone form through redox stress-induced oxidation/activation (Satoh et al. 2008a; Satoh et al. 2008b). This conversion makes CA an electrophile that then binds to the protein Keap1 in the cytoplasm of neural cells, probably in astrocytes to a larger extent than neurons, to release the transcription factor Nrf2. Nrf2 is thus ‘activated’ and enters the nucleus to stimulate transcription of an endogenous antioxidant enzyme system known as phase 2 enzymes (Satoh et al. 2008b) (Fig. 1). Here, we show that CA protects a variety of neuronal types from different brain regions from sublethal cyanide poisoning both in vitro and in vivo when administered repeatedly around the time of a subchronic exposure. Importantly, for the first time we are able to test the effects of a potential bioterroristic agent like cyanide in a human context by using human induced pluripotent stem cell (hiPSC)-derived neurons in our in vitro work. Additionally, this approach allows us to test the neuroprotective effect of CA in a human context. The results suggest that CA is a prospective therapeutic agent for preventing brain pathophysiology caused by cyanide poisoning, in which oxidative stress plays an important role. Fig. 1 Neuroprotective mechanism of carnosic acid (CA). CA is a pro-electrophilic drug (PED) for treatment of neurological disorders in part due to excessive ROS; these drugs become active electrophilic compounds when they encounter oxidative insults that chemically ...