Neuronal cells but not muscle cells are resistant to oxidative stress mediated protein misfolding and cell death: role of molecular chaperones.

Our recent study in a mouse model of familial-Amyotrophic Lateral Sclerosis (f-ALS) revealed that muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low mutant CuZn-superoxide dismutase, which is considered to be the key toxic element for initiation and progression of f-ALS. More importantly, we observed differential level of heat shock proteins (Hsp's) between skeletal muscle and spinal cord tissues prior to the onset and during disease progression; spinal cord maintains significantly higher level of Hsp's compared to skeletal muscle. In this study, we report two important observations; (i) muscle cells (but not neuronal cells) are extremely vulnerable to protein misfolding and cell death during challenge with oxidative stress and (ii) muscle cells fail to mount Hsp's during challenge unlike neuronal cells. These two findings can possibly explain why muscle atrophy precedes the death of motor neurons in f-ALS mice.

Elevated protein carbonylation and oxidative stress do not affect protein structure and function in the long-living naked-mole rat: a proteomic approach.

The 'oxidative stress theory of aging' predicts that aging is primarily regulated by progressive accumulation of oxidized macromolecules that cause deleterious effects to cellular homeostasis and induces a decline in physiological function. However, our reports on the detection of higher level of oxidized protein carbonyls in the soluble cellular fractions of long-living rodent naked-mole rats (NMRs, lifespan ~30yrs) compared to short-lived mice (lifespan ~3.5yrs) apparently contradicts a key tenet of the oxidative theory. As oxidation often inactivates enzyme function and induces higher-order soluble oligomers, we performed a comprehensive study to measure global protein carbonyl level in different tissues of age-matched NMRs and mice to determine if the traditional concept of oxidation mediated impairment of function and induction of higher-order structures of proteins are upheld in the NMRs. We made three intriguing observations with NMRs proteins: (1) protein carbonyl is significantly elevated across different tissues despite of its exceptional longevity, (2) enzyme function is restored despite of experiencing higher level of protein carbonylation, and (3) enzymes show lesser sensitivity to form higher-order non-reducible oligomers compared to short-living mouse proteins in response to oxidative stress. These observations were made based on the global analysis of protein carbonyl and identification of two heavily carbonylated proteins in the kidney, triosephosphate isomerase (TPI) and cytosolic peroxiredoxin (Prdx1). These un-expected intriguing observations thus strongly suggest that oxidative modification may not be the only criteria for impairment of protein and enzyme function; cellular environment is likely be the critical determining factor in this process and may be the underlying mechanism for exceptional longevity of NMR.

Differential effects of mutant SOD1 on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: role of chaperone network.

Protein misfolding is considered to be a potential contributing factor for motor neuron and muscle loss in diseases like Amyotrophic lateral sclerosis (ALS). Several independent studies have demonstrated using over-expressed mutated Cu/Zn-superoxide dismutase (mSOD1) transgenic mouse models which mimic familial ALS (f-ALS), that both muscle and motor neurons undergo degeneration during disease progression. However, it is unknown whether protein conformation of skeletal muscle and spinal cord is equally or differentially affected by mSOD1-induced toxicity. It is also unclear whether heat shock proteins (Hsp's) differentially modulate skeletal muscle and spinal cord protein structure during ALS disease progression. We report three intriguing observations utilizing the f-ALS mouse model and cell-free in vitro system; (i) muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low level of soluble and absence of insoluble G93A protein aggregate, unlike in spinal cord, (ii) Hsp's levels are lower in muscle compared to spinal cord at any stage of the disease, and (iii) G93ASOD1 enzyme-induced toxicity selectively affects muscle protein conformation over spinal cord proteins. Together, these findings strongly suggest that differential chaperone levels between skeletal muscle and spinal cord may be a critical determinant for G93A-induced protein misfolding in ALS.

Reduced mitochondrial ROS, enhanced antioxidant defense, and distinct age-related changes in oxidative damage in muscles of long-lived Peromyscus leucopus.

Comparing biological processes in closely related species with divergent life spans is a powerful approach to study mechanisms of aging. The oxidative stress hypothesis of aging predicts that longer-lived species would have lower reactive oxygen species (ROS) generation and/or an increased antioxidant capacity, resulting in reduced oxidative damage with age than in shorter-lived species. In this study, we measured ROS generation in the young adult animals of the long-lived white-footed mouse, Peromyscus leucopus (maximal life span potential, MLSP = 8 yr) and the common laboratory mouse, Mus musculus (C57BL/6J strain; MLSP = 3.5 yr). Consistent with the hypothesis, our results show that skeletal muscle mitochondria from adult P. leucopus produce less ROS (superoxide and hydrogen peroxide) compared with M. musculus. Additionally, P. leucopus has an increase in the activity of antioxidant enzymes superoxide dismutase 1, catalase, and glutathione peroxidase 1 at young age. P. leucopus compared with M. musculus display low levels of lipid peroxidation (isoprostanes) throughout life; however, P. leucopus although having elevated protein carbonyls at a young age, the accrual of protein oxidation with age is minimal in contrast to the linear increase in M. musculus. Altogether, the results from young animals are in agreement with the predictions of the oxidative stress hypothesis of aging with the exception of protein carbonyls. Nonetheless, the age-dependent increase in protein carbonyls is more pronounced in short-lived M. musculus, which supports enhanced protein homeostasis in long-lived P. leucopus.

Protein misfolding, mitochondrial dysfunction and muscle loss are not directly dependent on soluble and aggregation state of mSOD1 protein in skeletal muscle of ALS.

Mutant superoxide dismutase 1 (mSOD1) is often found as aggregates at the outer-membrane of mitochondria in motor neurons of various mouse models and familial amyotrophic lateral sclerosis (f-ALS) patients. It has been postulated that disruption of mitochondrial function by physical association of misfolded mSOD1 aggregates may actually be the trigger for initiation of degeneration of motor neurons in ALS. However, it was not clear if the same mechanism is involved in muscle degeneration and mitochondrial dysfunction in skeletal muscles of ALS. Recent study from our laboratory show that two skeletal muscle proteins, namely creatine kinase (CK) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) undergo major conformational and functional changes in the f-ALS mouse model of ALS (G93A). In this paper, we report two intriguing observations which are as follows:(i) G93A protein does not form aggregates in skeletal muscle at any stages of disease process probably due to high chymotrypsin-like activity of proteasome and thus G93A protein aggregates have no direct effects on progressive loss of muscle mass and global changes in protein conformation in ALS, and (ii) the soluble G93A protein does not have direct effects on mitochondrial dysfunction as determined by quantifying the release of reactive oxygen species (ROS) in skeletal muscle mitochondria; instead, the proteins affected by G93A possibly affect mitochondrial ROS release. These data strongly suggest for the first time that unlike in motor neurons, the soluble and aggregation states of the G93A protein do not have direct effects on protein misfolding and mitochondrial dysfunction in skeletal muscle during ALS.

Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat.

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.

The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis.

Altered structure, and hence function, of cellular macromolecules caused by oxidation can contribute to loss of physiological function with age. Here, we tested whether the lifespan of bats, which generally live far longer than predicted by their size, could be explained by reduced protein damage relative to short-lived mice. We show significantly lower protein oxidation (carbonylation) in Mexican free-tailed bats (Tadarida brasiliensis) relative to mice, and a trend for lower oxidation in samples from cave myotis bats (Myotis velifer) relative to mice. Both species of bat show in vivo and in vitro resistance to protein oxidation under conditions of acute oxidative stress. These bat species also show low levels of protein ubiquitination in total protein lysates along with reduced proteasome activity, suggesting diminished protein damage and removal in bats. Lastly, we show that bat-derived protein fractions are resistant to urea-induced protein unfolding relative to the level of unfolding detected in fractions from mice. Together, these data suggest that long lifespan in some bat species might be regulated by very efficient maintenance of protein homeostasis.

Superoxide-mediated oxidative stress accelerates skeletal muscle atrophy by synchronous activation of proteolytic systems.

The maintenance of skeletal muscle mass depends on the overall balance between the rates of protein synthesis and degradation. Thus, age-related muscle atrophy and function, commonly known as sarcopenia, may result from decreased protein synthesis, increased proteolysis, or simultaneous changes in both processes governed by complex multifactorial mechanisms. Growing evidence implicates oxidative stress and reactive oxygen species (ROS) as an essential regulator of proteolysis. Our previous studies have shown that genetic deletion of CuZn superoxide dismutase (CuZnSOD, Sod1) in mice leads to elevated oxidative stress, muscle atrophy and weakness, and an acceleration in age-related phenotypes associated with sarcopenia. The goal of this study is to determine whether oxidative stress directly influences the acceleration of proteolysis in skeletal muscle of Sod1-/- mice as a function of age. Compared to control, Sod1-/- muscle showed a significant elevation in protein carbonyls and 3-nitrotyrosine levels, suggesting high oxidative and nitrosative protein modifications were present. In addition, age-dependent muscle atrophy in Sod1-/- muscle was accompanied by an upregulation of the cysteine proteases, calpain, and caspase-3, which are known to play a key role in the initial breakdown of sarcomeres during atrophic conditions. Furthermore, an increase in oxidative stress-induced muscle atrophy was also strongly coupled with simultaneous activation of two major proteolytic systems, the ubiquitin-proteasome and lysosomal autophagy pathways. Collectively, our data suggest that chronic oxidative stress in Sod1-/- mice accelerates age-dependent muscle atrophy by enhancing coordinated activation of the proteolytic systems, thereby resulting in overall protein degradation.

Thioredoxin 2 haploinsufficiency in mice results in impaired mitochondrial function and increased oxidative stress.

The mitochondrial form of thioredoxin, thioredoxin 2 (Txn2), plays an important role in redox control and protection against ROS-induced mitochondrial damage. To evaluate the effect of reduced levels of Txn2 in vivo, we measured oxidative damage and mitochondrial function using mice heterozygous for the Txn2 gene (Txn2(+/-)). The Txn2(+/-) mice showed approximately 50% decrease in Trx-2 protein expression in all tissues without upregulating the other major components of the antioxidant defense system. Reduced levels of Txn2 resulted in decreased mitochondrial function as shown by reduced ATP production by isolated mitochondria and reduced activity of electron transport chain complexes (ETCs). Mitochondria isolated from Txn2(+/-) mice also showed increased ROS production compared to wild type mice. The Txn2(+/-) mice showed increased oxidative damage to nuclear DNA, lipids, and proteins in liver. In addition, we observed an increase in apoptosis in liver from Txn2(+/-) mice compared with wild type mice after diquat treatment. Our results suggest that Txn2 plays an important role in protecting the mitochondria against oxidative stress and in sensitizing the cells to ROS-induced apoptosis.

Oxidation and structural perturbation of redox-sensitive enzymes in injured skeletal muscle.

Molecular events that control skeletal muscle injury and regeneration are poorly understood. However, inflammation associated with oxidative stress is considered a key player in modulating this process. To understand the consequences of oxidative stress associated with muscle injury, inflammation, and regeneration, hind-limb muscles of C57Bl/6J mice were studied after injection of cardiotoxin (CT). Within 1 day post-CT injection, polymorphonuclear neutrophilic leukocyte accumulation was extensive. Compared to baseline, tissue myeloperoxidase (MPO) activity was elevated eight- and fivefold at 1 and 7 days post-CT, respectively. Ubiquitinylated protein was elevated 1 day postinjury and returned to baseline by 21 days. Cysteine residues of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were irreversibly oxidized within 1 day post-CT injection and were associated with protein conformational changes that fully recovered after 21 days. Importantly, protein structural alterations occurred in conjunction with significant decreases in CK activity at 1, 3, and 7 days post-CT injury. Interestingly, elevations in tissue MPO activity paralleled the time course of conformational changes in CK and GAPDH. In combination, these results demonstrate that muscle proteins in vivo are structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after muscle injury and preceding muscle regeneration.

Caspase-2 deficiency enhances aging-related traits in mice.

Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.

High oxidative damage levels in the longest-living rodent, the naked mole-rat.

Oxidative stress is reputed to be a significant contributor to the aging process and a key factor affecting species longevity. The tremendous natural variation in maximum species lifespan may be due to interspecific differences in reactive oxygen species generation, antioxidant defenses and/or levels of accrued oxidative damage to cellular macromolecules (such as DNA, lipids and proteins). The present study tests if the exceptional longevity of the longest living (> 28.3 years) rodent species known, the naked mole-rat (NMR, Heterocephalus glaber), is associated with attenuated levels of oxidative stress. We compare antioxidant defenses (reduced glutathione, GSH), redox status (GSH/GSSG), as well as lipid (malondialdehyde and isoprostanes), DNA (8-OHdG), and protein (carbonyls) oxidation levels in urine and various tissues from both mole-rats and similar-sized mice. Significantly lower GSH and GSH/GSSG in mole-rats indicate poorer antioxidant capacity and a surprisingly more pro-oxidative cellular environment, manifested by 10-fold higher levels of in vivo lipid peroxidation. Furthermore, mole-rats exhibit greater levels of accrued oxidative damage to lipids (twofold), DNA (approximately two to eight times) and proteins (1.5 to 2-fold) than physiologically age-matched mice, and equal to that of same-aged mice. Given that NMRs live an order of magnitude longer than predicted based on their body size, our findings strongly suggest that mechanisms other than attenuated oxidative stress explain the impressive longevity of this species.

Absence of CuZn superoxide dismutase leads to elevated oxidative stress and acceleration of age-dependent skeletal muscle atrophy.

We describe a novel phenotype in mice lacking the major antioxidant enzyme, CuZn-superoxide dismutase (Sod1(-/-) mice), namely a dramatic acceleration of age-related loss of skeletal muscle mass. Sod1(-/-) mice are 17 to 20% smaller and have a significantly lower muscle mass than wild-type mice as early as 3 to 4 months of age. Muscle mass in the Sod1(-/-) mice is further reduced with age and by 20 months, the hind-limb muscle mass in Sod1(-/-) mice is nearly 50% lower than in age-matched wild-type mice. Skeletal muscle tissue from young Sod1(-/-) mice has elevated oxidative damage to proteins, lipids, and DNA compared to muscle from young wild-type mice. The reduction in muscle mass and elevated oxidative damage are accompanied by a 40% decrease in voluntary wheel running by 6 months of age and decreased performance on the Rota-rod test at 13 months of age, but are not associated with a decline in overall spontaneous activity. In some of the old Sod1(-/-) mice, the loss in muscle mass is also associated with the presence of tremors and gait disturbances. Thus, the absence of CuZnSOD imposes elevated oxidative stress, loss of muscle mass, and physiological consequences that resemble an acceleration of normal age-related sarcopenia.

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach.

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.

Oxidative damage to myelin proteins accompanies peripheral nerve motor dysfunction in aging C57BL/6 male mice.

Aging is associated with a decline in peripheral nerve function of both motor and sensory nerves. The decline in function of peripheral sensorimotor nerves with aging has been linked to sarcopenia, the age-related decline in muscle mass and function that significantly compromises the quality of life in older humans. In this study, we report a significant increase in oxidized fatty acids and insoluble protein carbonyls in sciatic nerves of aged C57BL/6 male mice (28-30mo) that exhibit a profound decline in motor nerve function and degenerative changes in both axon and myelin structure, compared to young mice (6-8mo). Our data further suggests that this age-related loss of function of peripheral motor nerves is likely precipitated by changes in mechanisms that protect and/or repair oxidative damage. We predict that interventions that target these mechanisms may protect against age-related decline in peripheral sensorimotor nerve function and likely improve the debilitating outcome of sarcopenia in older humans.

Moderate modulation of disease in the G93A model of ALS by the compound 2-(2-hydroxyphenyl)-benzoxazole (HBX).

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease characterized by degeneration and death of motor neurons. Aberrant protein aggregation and oxidative stress are implicated in the etiology of ALS; thus preventing propagation of early aggregation events and oxidative damage could be an effective therapy. We tested the effect of dietary supplementation (initiated 40 days of age) with 2-(2-hydroxyphenyl)-benzoxazole (HBX), a compound with metal chelator and anti-aggregation properties, on disease onset, progression and lifespan in the G93A mouse model of ALS. Tests were not sufficiently powerful to detect any change to survival distribution of mice treated with HBX. However, the disease onset was delayed and max lifespan was increased in the treatment group. Additionally, disease progression was moderated as shown by reduced neuromuscular denervation measured by repetitive nerve stimulation. F2-isoprostanes, a marker of oxidative damage, are elevated in skeletal muscle from G93A mice at onset and this increase is prevented in HBX fed G93A mice. Furthermore, HBX treatment reduced mutant SOD1 protein aggregation in whole spinal cord of G93A mice at disease onset. Overall, our data suggests that HBX may be able to improve the degenerative symptoms of ALS through the prevention of oxidative damage and protein aggregation. Further studies are needed to uncover the mechanistic effects of HBX in ameliorating ALS pathology.

Longevity and GAPDH Stability in Bivalves and Mammals: A Convenient Marker for Comparative Gerontology and Proteostasis.

BACKGROUND: Comparative aging studies, particularly those that include species of exceptional resistance to aging processes, can potentially illuminate novel senescence-retarding mechanisms. In recent years, protein homeostasis (proteostasis) has been implicated in fundamental aging processes. Here we further evaluate the relationship between proteostasis and longevity in a selection of bivalve mollusks and mammals with maximum longevities ranging from 3 to 507 years. METHODS & RESULTS: We experimentally examined proteostasis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter, as it is ubiquitously expressed, highly conserved, and conveniently assayed. The ability to maintain this enzymatic function was tested with increasing concentrations of the chaotropic agent urea, revealing a robust relationship with longevity in bivalves and mice. While our shortest-lived mollusk and mouse lost all activity by 2.5 and 3.5 M urea respectively, the longest-lived mollusk species, Arctica islandica, still preserved 45% of its basal function even at 6 M urea. To confirm that GAPDH proteostasis has a broad association with longevity, we also investigated a selection of primate species ranging in maximum longevity from 22 to 122 years. They outperformed the mouse at all concentrations, but among the primates results were variable at low urea doses. Still, at 6 M urea baboon and human samples retained 10% of their activity while both mouse and marmoset samples had no activity. MECHANISM OF EXCEPTIONAL STRESS RESISTANCE: To explore possible mechanisms of the exceptional stress resistance of A. islandica GAPDH we enzymatically removed post-translational glycosylation, but observed no decrease in stability. We also removed molecules smaller than 30 kDa, which includes most small heat shock proteins, but again did not compromise the exceptional stress resistance of Arctica GAPDH. CONCLUSION: While the mechanism underlying A. islandica's exceptional stress resistance remains elusive, this research identifies an experimental system that may reveal hitherto unknown mechanisms of protein homeostasis.

Fluorescence-Based Approaches for Quantitative Assessment of Protein Carbonylation, Protein Disulfides, and Protein Conformation in Biological Tissues.

Protein oxidation and misfolding have been considered as key players for progression of aging and etiology of various pathological conditions. However, few attempts have been made to develop sensitive and reproducible assays to quantify the changes in protein oxidation and alteration in structure. Here we describe three distinct fluorescence-based assays to quantify changes in protein oxidation, namely carbonylation and disulfides and alteration in protein surface hydrophobicity as a reporter for protein conformation. These techniques will provide investigators the opportunity to address important biological questions in their experimental models.

The role of oxidative damage and stress in aging.

The Free Radical/Oxidative Stress Theory of Aging, which was first proposed in 1956, is currently one of the most popular explanations for how aging occurs at the biochemical/molecular level. However, most of the evidence in support of this theory is correlative, e.g., oxidative damage to various biomolecules increases with age, and caloric restriction, which increases life span and retards aging, reduces the age-related increase in oxidative damage to biomolecules. The most direct test of the Free Radical/Oxidative Stress Theory of Aging is to specifically alter the age-related increase in oxidative damage and determine how this alteration affects life span. For the first time, investigators can use genetically altered animals to test directly the role of oxidative damage in aging. In this manuscript, we critically review the past research in this area and discuss potential future research directions in testing the Free Radical/Oxidative Theory of Aging.

Genetic ablation of 12/15-lipoxygenase but not 5-lipoxygenase protects against denervation-induced muscle atrophy.

Skeletal muscle atrophy is a debilitating outcome of a number of chronic diseases and conditions associated with loss of muscle innervation by motor neurons, such as aging and neurodegenerative diseases. We previously reported that denervation-induced loss of muscle mass is associated with activation of cytosolic phospholipase A2 (cPLA2), the rate-limiting step for the release of arachidonic acid from membrane phospholipids, which then acts as a substrate for metabolic pathways that generate bioactive lipid mediators. In this study, we asked whether 5- and 12/15-lipoxygenase (LO) lipid metabolic pathways downstream of cPLA2 mediate denervation-induced muscle atrophy in mice. Both 5- and 12/15-LO were activated in response to surgical denervation; however, 12/15-LO activity was increased ~2.5-fold versus an ~1.5-fold increase in activity of 5-LO. Genetic and pharmacological inhibition of 12/15-LO (but not 5-LO) significantly protected against denervation-induced muscle atrophy, suggesting a selective role for the 12/15-LO pathway in neurogenic muscle atrophy. The activation of the 12/15-LO pathway (but not 5-LO) during muscle atrophy increased NADPH oxidase activity, protein ubiquitination, and ubiquitin-proteasome-mediated proteolytic degradation. In conclusion, this study reveals a novel pathway for neurogenic muscle atrophy and suggests that 12/15-LO may be a potential therapeutic target in diseases associated with loss of innervation and muscle atrophy.