ACETYL-L-CARNITINE (ALCAR) PREVENTS HYPOBARIC HYPOXIA–INDUCED SPATIAL MEMORY IMPAIRMENT THROUGH EXTRACELLULAR RELATED KINASE–MEDIATED NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 PHOSPHORYLATION K. BARHWAL, S. K. HOTA, V. JAIN, D. PRASAD, S. B. SINGH* AND G. ILAVAZHAGAN Defence Institute of Physiology and Allied Sciences, Defence Re- search and Development Organization, Lucknow Road, Timarpur, Delhi 110054, India Abstract—Exposure to hypobaric hypoxia, a condition in- volving decreased availability of oxygen is known to be as- sociated with oxidative stress, neurodegeneration and mem- ory impairment. The multifactorial response of the brain and the complex signaling pathways involved therewith limits the therapeutic efficacy of several antioxidants in ameliorat- ing hypobaric hypoxia–induced memory impairment. The present study was therefore aimed at investigating the po- tential of acetyl-L-carnitine (ALCAR), a known antioxidant that has been reported to augment neurotrophin-mediated sur- vival mechanisms, in ameliorating hypoxia-induced neurode- generation and memory impairment. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor involved in the cellular defense mechanism against oxidative stress related to brain injury and neurological disorders. The study was designed to understand the mechanisms involving Nrf2 stabilization following exposure to hypobaric hypoxia. The results displayed reference memory impairment in Sprague– Dawley rats exposed to hypobaric hypoxia (7620 m) for 14 consecutive days which however improved on administration of ALCAR during hypoxic exposure. The study also revealed Nrf2 regulated augmented antioxidant response on adminis- tration of ALCAR which was through a novel tyrosine kinase A (TrkA) receptor-mediated mechanism. A decrease in free radical generation, lipid peroxidation and protein oxidation was also observed along with a concomitant increase in thioredoxin and reduced glutathione levels on administration of ALCAR during exposure to hypobaric hypoxia. The present study therefore reveals the therapeutic potential of ALCAR under conditions of hypobaric hypoxia and eluci- dates a novel mechanism of action of the drug. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hypobaric hypoxia, memory, acetyl-L-carnitine, nerve growth factor, extracellular related kinase, nuclear fac- tor erythroid 2-related factor 2. Decrease in partial pressure of oxygen associated with ascent to high altitude, a situation referred to as hypobaric hypoxia, limits oxygen availability to tissues. The brain in particular is highly vulnerable to this oxygen scarcity. There are several reports on occurrence of cognitive dys- functions in human subjects exposed to hypobaric hypoxia (Shukkit-Hale et al., 1994, 1996). fMRI studies have also revealed decrease in hippocampal size in mountaineers (Shukkit-Hale et al., 1998). The causes behind memory impairment following exposure to hypobaric hypoxia how- ever still remain elusive. Though there have been several studies on the role of oxidative stress and excitotoxicity in mediating cognitive dysfunctions at high altitude, administration of compounds quenching free radicals like N-acetyl cysteine provided limited protection against hypoxic neuronal damage (Jay- alakshmi et al., 2007). Another antioxidant, acetyl-L-carni- tine (ALCAR) has however been reported to provide better neuroprotection in in vitro model of ischemia (Picconi et al., 2006). The better efficacy of ALCAR in protecting neurons in hypoxic stress can be attributed to its multifactorial mode of action that includes quenching free radicals, augmenting the energy status, promoting nerve growth factor (NGF)– mediated survival signals and altering expression of pro- teins (Zanelli et al., 2005; Poon et al., 2006; Barhwal et al., 2008; Bagetta et al., 2008). We have previously reported on the therapeutic efficacy of ALCAR in ameliorating cog- nitive impairment in animals exposed to simulated hypo- baric hypoxia for shorter durations of 3 days. Since previ- ous studies show extensive neuronal damage in the CA1 region of hippocampus on 14 days of hypoxic exposure when compared to shorter durations of 3 and 7 days’ exposure, the ability of ALCAR to prevent these changes on chronic exposure needed to be studied to qualify its use as a therapeutic agent during exposure to hypobaric hyp- oxia (Barhwal et al., 2007). ALCAR has been previously reported to augment ty- rosine kinase A (TrkA) receptor expression and enhance Elk-1 (Ets-related transcription factor) phosphorylation in vitro (Barhwal et al., 2008). ALCAR administration has also been reported to enhance the antioxidant status and de- crease oxidative damage in neuronal cells. However, not much is known on the effect of ALCAR administration on *Corresponding author. Tel: +11-23946526; fax: +11-23914790. E-mail address: sbs_1958@yahoo.co.in (S. B. Singh). Abbreviations: ALCAR, acetyl-L-carnitine; ARE, antioxidant responsive element; BSA, bovine serum albumin; Cul3, Cullin-3; DCFHDA, 2=,7=- dichlorofluorescein-diacetate; ERK1/2, extracellular related kinase; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, re- duced glutathione; GSSG, oxidized glutathione; HIF, hypoxia-induc- ible factor; Keap1, Kelch-like-ECH-associated protein 1; MAPK, mito- gen activated protein kinase; MDA, malondialdehyde; NGF, nerve growth factor; Nrf2, nuclear factor erythroid 2-related factor 2; PBS, phosphate buffer saline; ROS, reactive oxygen species; SOD, super- oxide dismutase; TBA, thiobarbituric acid; TrkA, tyrosine kinase A; Trx, thioredoxin-1. Neuroscience 161 (2009) 501–514 0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.02.086 501