Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1⌬aat1⌬ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.[Keywords: Calorie restriction (CR); Sir2; aging; NADH shuttles; respiration; metabolism] Supplemental material is available at http://www.genesdev.org.

Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes. Calorie restriction (CR)2 is the most effective intervention known to extend life span in a variety of species, including mammals (1, 2). CR has also been shown to delay the onset or reduce the incidence of many age-related diseases (1). Although it has been suggested that CR may work by reducing the levels of reactive oxygen species due to a slowing in metabolism (1, 3), the mechanism by which CR extends life span and ameliorates age-associated diseases is still uncertain.Moderate CR can be imposed in the budding yeast Saccharomyces cerevisiae by reducing the glucose concentration from 2 to 0.5% in rich media (4 -9). Under this CR condition, the growth rate remains robust, and yeast mother cells show an extended replicative life span (division potential) of about 20 -30%. Variations in CR protocols have been described where limitation of amino acids and other nutrients accompanies carbon source limitation (10, 11). These regimens may represent another possible longevity pathway that functions in parallel to CR in rich media. Genetic models of CR have also been identified and studied in multiple strain backgrounds, such as PSY316 (4, 5, 7), W303 (4, 8, 9, 12), and BY4742 (13). These CR mimics include a hexokinase mutant (hxk2⌬) and mutations that down-regulate the glucose-sensing cyclic-AMP/protein kinase A pathway: the temperature-sensitive alleles of the adenylate cyclase (cdc35-1) or the RAS nucleotide exchange protein (cdc25-10) and deletions of the glucose-sensing protein Gpa2 and Gpr1. Additional CR genetic models, the tor1⌬ and sch9⌬ mutants, have recently been reported to extend yeast life span (14, 15). The nutrient-sensing Target of Rapamycin (TOR) pathway and Sch9 kinase (a homolog of the...

Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from agedependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1D-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1D mutant also displays a decreased reactive oxygen species level and increased heatshock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR-and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

Calorie restriction (CR) in microorganisms such as budding and fission yeasts has a robust and well-documented impact on longevity. In order to efficiently utilize the limited energy during CR, these organisms shift from primarily fermentative metabolism to mitochondrial respiration. Respiration activates certain conserved longevity factors such as sirtuins and is associated with widespread physiological changes that contribute to increased survival. However, the importance of respiration during CR-mediated longevity has remained controversial. The emergence of several novel metabolically distinct microbial models for longevity has enabled CR to be studied from new perspectives. The majority of CR and life span studies have been conducted in the primarily fermentative Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, but studies in primarily respiratory Crabtree-negative yeast and obligate aerobes can offer complementary insight into the more complex mammalian response to CR. Not only are microorganisms helping characterize a conserved cellular mechanism for CR-mediated longevity, but they can also directly impact mammalian metabolism as part of the natural gut flora. Here, we discuss the contributions of microorganisms to our knowledge of CR and longevity at the level of both the cell and the organism.

Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate α-1 and α-2 adrenergic receptors in combination with NMDA and tested expression of the immediate-early gene c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, MAP kinase and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by PKC and MAPK. Keywords glutamate; norepinephrine; protein kinase C; MAP kinase; immediate-early gene; c-FosThe accessory olfactory bulb (AOB), for the past several years, has been the subject of investigations attempting to understand the role it plays in pheromone signal processing and pheromone memory formation. The AOB shares some similarities with the main olfactory bulb (MOB), such as having similar lamination patterns and similar neuronal types within the laminae. Much work has been carried out in order to better understand the signaling between neurons that underlies sensory processing in the AOB. The majority of this work has been restricted to behavioral and in vivo studies as well as some electrophysiological studies (Brennan and Keverne, 1997). Many of the important questions about the signal transduction within the AOB neurons, however, remain unanswered.Previous studies have elucidated the neurotransmitter systems that are likely to play a role in activating the signaling mechanisms in the AOB. Several studies have infused pharmacological agents directly into the AOB in order to disrupt the normal signaling and thereby identify Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Kaba and Keverne, 1988;Kaba et al., 1989). These infusion studies have established a role for glutamate and norepinephrine (NE) in mediating signaling in the AOB. Also, the behavioral studies in mice have established that expression of imm...

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