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Biochimica et Biophysica Acta xxx (2009) xxx–xxx

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Review

The TOR pathway comes of age Monique N. Stanfel a, Lara S. Shamieh b, Matt Kaeberlein b,⁎, Brian K. Kennedy a,⁎ a b

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Department of Pathology, University of Washington, Seattle, WA 98195, USA

a r t i c l e

i n f o

Article history: Received 23 February 2009 Received in revised form 10 June 2009 Accepted 11 June 2009 Available online xxxx Keywords: Aging TOR S6 kinase Neurodegeneration Metabolic syndrome Cardiovascular disease Dietary restriction

a b s t r a c t Studies in a variety of model organisms indicate that nutrient signaling is tightly coupled to longevity. In nutrient replete conditions, organisms develop, grow, and age quickly. When nutrients become sparse as with dietary restriction, growth and development decline, stress response pathways become induced and organisms live longer. Considerable effort has been devoted to understanding the molecular events mediating lifespan extension by dietary restriction. One central focus has been on nutrient-responsive signal transduction pathways including insulin/IGF-1, AMP kinase, protein kinase A and the TOR pathway. Here we describe the increasingly prominent links between TOR signaling and aging in invertebrates. Longevity studies in mammals are not published to date. Instead, we highlight studies in mouse models, which indicate that dampening the TOR pathway leads to widespread protection from an array of age-related diseases. © 2009 Elsevier B.V. All rights reserved.

1. Introduction To say that caloric intake and aging are tightly coupled is not news. Calorie excess and an increasingly sedentary lifestyle have resulted in obesity on a grand scale, not only in first world countries but worldwide [1–4]. Obesity is a major risk factor for a range of ageassociated diseases including, but not limited to, type II diabetes, cardiovascular disease and many forms of cancer [5,6]. Conversely, dietary restriction, defined as a reduction in caloric intake without malnutrition, results in lifespan extension and protection from many of the same diseases. Why are excess calories bad? This question has been debated for decades and is still not satisfactorily answered. One thing to remember, at least in regions where the obesity epidemic is occurring, is that humans (as mimicked by laboratory animals on ad libitum diets) now have access to cheap high calorie foods [7,8]. This, coupled with a transition to a sedentary lifestyle, has likely placed caloric intake out of register with much of our evolutionary past, when long periods of consistent access to high calorie diets are thought to have been rare. The human body is optimized for one range of caloric intake and exercise, but it is encountering another. Although they are under intense scrutiny, the mechanisms that underlie obesity-induced pathology or lifespan extension by dietary restriction have remained stubbornly refractory. Mechanistically, it is not clear that excess calories and dietary restriction are two ends of the same register. Obesity research will not be discussed in detail here. ⁎ Corresponding authors. E-mail address: [email protected] (B.K. Kennedy).

Instead, readers are referred to recent reviews on the topic [9–12]. Here, we will focus on studies to define the pathways that sense nutrients and modulate lifespan extension by dietary restriction. Cells and tissues have myriad ways of detecting nutrient levels in the environment and, when engaged, mediate an array of overlapping downstream responses. For instance, the insulin pathway is responsive to glucose levels in the bloodstream, with islet cells producing and secreting insulin in response to glucose, and with peripheral tissues like skeletal muscle and fat responding directly to insulin. The TOR pathway is activated by several signals (see below), but amino acid levels may be the primary efferent. Other sensors, which may also activate the TOR pathway, detect cellular energy stores. For instance, AMP kinase activity is determined by cellular AMP/ATP ratios [13]. In both invertebrate and mammalian aging model organisms, dietary restriction leads to reduced insulin/IGF-1 and TOR signaling, but increased AMP kinase activity. Making molecular dissection more challenging, all of these pathways (and others) communicate on several levels presumably to bring about an integrated cellular response. In this review, we focus on recent evidence that the TOR pathway modulates aging, and that reduced TOR signaling may be a primary mechanism by which dietary restriction extends longevity and offsets age-related disease in aging model organisms. 2. The target of rapamycin kinase is a conserved regulator of growth The target of rapamycin (TOR) kinase acts as a central regulator of eukaryotic growth and cell division in response to nutrient and growth factor cues. TOR proteins are highly conserved from yeast to

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Table 1 Identified orthologous components of TOR complex 1 and TOR complex 2. S. cerevisiae

C. elegans

D. melanogaster

Mammals

TORC1 TOR1/TOR2 KOG1 LST8 TCO89

let-363 daf-15 C10H11.8 –

TOR-PA Raptor-PA CG3004-PA –

mTOR Raptor mLST8 –

TORC2 TOR2 AVO1 AVO2 BIT61 LST8 SLM1 SLM2 AVO3/TSC11

let-363 – – – C10H11.8 – – F29C12.2

TOR-PA – – – CG3004-PA – – Rictor-PA

mTOR – – – mLST8 – – Rictor

Components of the TORC1 and TORC2 complexes in C. elegans, D. melanogaster, and mammals were identified based on orthology mapping from the yeast protein sequences available on the Saccharomyces cerevisiae Genome Database http://www. yeastgenome.org.

humans, and were first identified (and named) from studies of the growth inhibiting properties of the anti-fungal compound rapamycin [14]. Yeast have two TOR paralogs, TOR1 and TOR2, while other eukaryotes have but one (mTOR in mammals) [15–17]. TOR kinases exist in two complexes termed TORC1 and TORC2. TORC1 and TORC2 have distinct cellular functions, but both are essential for viability [18,19]. In yeast, Tor1 is exclusively found in TORC1, while Tor2 functions in both complexes. In mammals, mTOR functions in both complexes. TOR binds to specific accessory proteins in each complex; among these are Raptor (yeast Kog1) in TORC1 and Rictor (yeast Avo3) in TORC2 (Table 1). TOR kinases act as signal integrators that modulate permissive signals for cell growth by responding to the status of nutrients, energy signals, growth factors, and cellular stresses. TORC1, when activated by nutrient availability (particularly amino acids), coordinates protein synthesis and degradation to promote growth when nutrients are plentiful. TORC2 is responsive to growth factors, such as insulin and insulin-like growth factors, and promotes stress responses necessary for cell survival. In addition, TORC2 plays an important role in organizing the actin cytoskeleton [20,21]. TORC2 also mediates cross-talk between the insulin signaling pathways and TOR signaling by phosphorylating Akt, a phosphatidyl 3-OH kinase that acts downstream of the insulin receptor [22]. Insulin signaling and Akt, in turn, also modulate TORC1 function (see below) [23], providing a potential link between TORC1 and TORC2 activity. Many features of the upstream regulation and downstream processes controlled by both TOR complexes remain poorly understood. The elucidation of these events is an ongoing subject of intense study. Rapamycin is produced by the bacterium Streptomyces hygroscopicus [24] and specifically inhibits the function of the two TOR proteins

in yeast through a mechanism which is still not completely understood. Rather than competing for substrate binding directly, as is the case with many kinase inhibitors, rapamycin serves as the lynchpin in a trimolecular interaction that also includes TOR and FKBP12 [25,26]. This complex inhibits TOR-mediated phosphorylation of downstream targets in vivo, perhaps by disrupting protein–protein interactions within TORC1. The non-competitive nature of rapamycin action may underlie its unusual specificity among kinase inhibitors. TORC2, unlike TORC1, is thought to be rapamycin-insensitive [19,21]. However, in certain cell types, long-term exposure of cells in culture to rapamycin leads to inhibition of TORC2 assembly and reduced TORC2 activity [27]. The upstream regulatory features of TORC1 activity is better understood than those of TORC2. TORC1 is activated by Akt in response to insulin and other growth factors [23]. Activation of TORC1 by Akt is mediated by inhibition of the tuberous sclerosis complex 2 (TSC2), which inhibits the TORC1-activator Rheb [28–30]. TORC1 is also repressed by the energy sensing AMP-activated protein kinase (AMPK) [31–33]. In addition to its ability to respond directly to nutrient abundance, many other input pathways place TORC1 at a key regulatory nexus in responding to nutrients, growth cues, and cellular energy status. 3. Modulation of longevity by TOR signaling in invertebrate organisms The role of TOR as an important longevity pathway has been firmly established from studies performed in three invertebrate model systems: the budding yeast Saccharomyces cerevisiae, nematode Caenorhabditis elegans, and fruit fly Drosophila melanogaster. In each of these organisms, a reduction in TORC1 activity has been shown to be sufficient to increase lifespan significantly. The genetic and pharmacological models of reduced TOR signaling associated with enhanced longevity in each of these organisms are shown in Table 2. In the budding yeast, aging can be studied by two assays: replicative and chronological [34,35]. Replicative lifespan is defined by the proliferative capacity of a mother cell and is measured by counting the number of daughter cells a mother cell produces prior to senescence. Chronological lifespan is defined by the length of time that a cell can survive in a quiescent state, and is typically measured by maintaining cells in stationary phase while periodically monitoring their ability to reenter the cell cycle in response to appropriate cues. Both aging paradigms have been studied extensively and a variety of genetic modifiers of replicative or chronological lifespan have been identified [34,35]. TOR signaling has been shown to be a particularly potent regulator of both replicative and chronological aging of yeast cells. Replicative and chronological lifespan can both be extended by deletion of TOR1 [36,37], by pharmacologic inhibition of TORC1 [36,38], or by deletion of the gene coding for the yeast S6 kinase homolog, SCH9 [37,39]. Additionally, replicative lifespan can also be increased by mutations of downstream targets of TOR signaling

Table 2 Cross species comparison of interventions that reduce TOR signaling, increase lifespan, and affect aging. Intervention

S. cerevisiae CLS

S. cerevisiae RLS

C. elegans

D. melanogaster

M. musculus

Dietary restriction TOR mutation/knock-down Raptor mutation/knock-down Activation of AMP kinase Activation of Tsc1/2 Pharmacological inhibition of TORC1 (e.g. rapamycin) S6 kinase mutation/knock-down Ribosomal protein mutation/knock-down Translation initiation factor mutation/knock-down Mutations reducing amino acid uptake

✓ ✓ – – – ✓ ✓ – – ✓

✓ ✓ – – – ✓ ✓ ✓ ✓ ✓

✓ ✓ ✓ ✓ – – ✓ ✓ ✓ ✓

✓ ✓ – – ✓ – ✓ – – –

✓ – – – – – – – – –

Multiple interventions that reduce TORC1 signaling have been reported to increase lifespan and affect aging in different model organisms.


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involved in protein synthesis, including multiple ribosomal proteins, rRNA processing factors, and translation initiation factors [37,40]. As with yeast, worm adult lifespan can be extended by RNAi knockdown or mutation of the gene coding for TOR (let-363), the S6 kinase homolog (rsks-1), several ribosomal protein genes, and a number of genes coding for mRNA translation initiation factors [41–45]. Additional evidence for the importance of TORC1 specifically is provided by the observation that mutation of the gene coding for raptor (daf-15) also increases lifespan [46], as does knock-down of an intestinal peptide transporter (pep-2) thought to act upstream of TORC1 in C. elegans [47]. In flies, dominant negative alleles of TOR or S6 kinase have been reported to increase lifespan, as has overexpression the fly homologs of TSC1 and TSC2, which act as negative regulators of TORC1. A hypomorphic allele of TOR has also been shown to increase lifespan in flies [48]. Interestingly, reducing TOR activity specifically in fat bodies is sufficient to provide lifespan extension in this organism [49]. Dietary restriction (DR) is the best-characterized paradigm for slowing aging across multiple eukaryotic species, including both yeast aging models, worms, flies, fish, spiders, mice, and rats [50–52]. The link between TORC1 activity and nutrient response suggests the hypothesis that DR promotes longevity by reducing TOR activity. This hypothesis is supported by the observations that TOR activity is inhibited by DR and, like DR, reduced TOR signaling is sufficient to slow aging in each of the invertebrate models where DR has been extensively studied. Epistasis studies in all three organisms have further strengthened the model that DR is mediated, at least in part, by reduced TOR signaling [37,42,48,53]. 4. Possible mechanisms by which TOR modulates aging in invertebrate organisms There has been much interest in understanding potential molecular mechanisms by which inhibition of TORC1 can slow aging across such disparate species as yeast, worms, and flies. Dissecting out specific downstream targets of TOR signaling that are causally involved in modulating longevity has been difficult, however, because of the particularly complex (and often poorly understood) network through which TOR signaling influences multiple aspects of cellular physiology. Current experimental data suggest that at least four distinct outputs of TOR signaling influence aging in invertebrates: regulation of mRNA translation, autophagy, stress response pathways, and metabolic changes associated with mitochondrial function. TORC1 activity promotes mRNA translation through at least two different downstream targets: S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding proteins (4E-BP) [54]. Activation of S6K1, in turn, promotes the activity of other translation initiation factors, such as eukaryotic initiation factor 4B (eIF4B), and stimulates production of ribosomal proteins and ribosome biogenesis [55]. As mentioned above, decreased S6K1 activity, translation initiation factor activity, or ribosomal protein expression has been found to promote longevity in yeast, worms, and flies. Thus, perturbing mRNA translation in a manner similar to the effect of reducing TOR activity is sufficient to phenocopy the lifespan extension associated with reduced TOR signaling in yeast, worms, and flies. In the yeast replicative aging model, further evidence supports the idea that much of the lifespan extension from TOR inhibition can be directly attributed to altered mRNA translation. Epistasis studies place DR, Tor1, Sch9, and multiple ribosomal proteins in a single epistasis group with respect to replicative lifespan [37,40]. A global reduction in translation in response to DR or in long-lived mutants in this epistasis group is associated with a relative increase in translation of GCN4 mRNA [56,57]. GCN4 codes for a starvation-responsive transcription factor [58] and its deletion prevents full lifespan extension in these mutants [40]. Thus, one model for how TOR signaling and regulation of mRNA translation could modulate aging in multicellular eukaryotes

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is through differential translation of specific target messages, such as GCN4. Functional homologs of Gcn4 are present and are translationally regulated; however it remains to be determined whether they also modulate longevity in these organisms. Regulation of carbon metabolism and stress resistance seem to be the primary mechanisms by which TOR signaling modulates yeast chronological lifespan. In addition to significantly increasing lifespan, deletion of either TOR1 or SCH9 causes a change in carbon metabolism away from alcoholic fermentation toward mitochondrial respiration [59,60] and an induction of stress response genes [39,61]. These changes are necessary for increased lifespan, suggesting that they play a causal role in the chronological aging process. Reduced fermentation may play a relatively direct role in lifespan extension since a recent manuscript has shown that organic acids produced and secreted during fermentation act as limiting agents to longevity later when cells enter a post-replicative state [62]. Deletion of SCH9 also provides direct resistant to what may be the primary culprit: acetic acid. To date, there is little evidence that reduced expression of translation initiation factors or ribosomal proteins significantly extend chronological lifespan, which differentiates this aging model from other invertebrate systems. In worms, evidence suggests that both mRNA translation and regulation of autophagy are important TOR-regulated modifiers of longevity. Enhanced autophagy, a process involved in recycling damaged macromolecules, is a potential avenue to lifespan extension. As discussed above, knock-down of S6 kinase, translation initiation factors, and ribosomal proteins is known to increase adult lifespan in worms [41–45]. In response to DR or TOR inhibition, autophagy is also induced and this increase in autophagy is required for lifespan extension [53,63,64]. It remains unclear whether induction of autophagy promotes longer lifespan or is simply necessary for the extension of lifespan associated with these interventions. 5. Does mTOR activity promote aging in mammals The data from invertebrate organisms unquestionably demonstrates that TOR signaling plays an important role in aging across multiple evolutionarily divergent species. Does TOR activity modulate mammalian aging as well? Although no definitive answer to this question is yet available, there is a growing body of evidence that TOR signaling does influence a variety of aging-related processes in rodents and possibly hints of similar effects in people (Fig. 1). DR is known to both dramatically increase lifespan and retard the onset of a variety of age-associated pathologies in rodents [50–52]. The invertebrate studies indicate that reduced TOR signaling is likely

Fig 1. TOR signaling and age-associated disease. Schematic depicts age-related pathologies for which there is a clear link between TOR signaling and disease progression.


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to account for the lifespan extension associated with DR. In addition, the long-lived Ames dwarf mouse has reduced TOR signaling [65], similar to normal mice that are subjected to DR. However, it remains to be determined whether reduced TOR signaling will be a characteristic of many other long-lived rodent models. Studies are currently underway as part of the National Institute on Aging (NIA) Interventions Testing Program to determine whether inhibition of mTOR by feeding mice rapamycin is sufficient to mimic DR and increase lifespan in a mammal [66,67]. In the absence of such data, however, it is informative to examine whether reduced TOR signaling can delay a spectrum of diseases that are influenced by DR. A complicating issue in mammals is that TOR has different functions in different tissues, and pharmacological inhibition of the TORC1 complex may be beneficial in some tissues and detrimental in others. It is beyond the scope of this review to discuss in detail specific functions of TOR in different tissues. Here, we focus on studies examining the efficacy of TOR inhibition for diseases in which aging is considered a major risk factor. To summarize, many mouse models of age-associated disease, including cancer, Huntington's and cardiovascular diseases, display improvement upon pharmacological inhibition of TOR [54]. 6. TOR and cancer A primary hallmark of DR in rodents is a dramatic reduction in ageassociated cancer incidence and growth rate [52,68–70]. Indeed, it has been speculated that reduced cancer occurrence may account for a large fraction of the longevity benefit associated with DR in rodents [71]. Mutations of mTOR pathway components (although not mTOR itself) and/or elevated mTOR activity have been discovered in many cancers including lymphomas, melanomas, gliomas, and central nervous system malignancies as well as carcinomas of lung, bladder, kidney, ovary, breast, prostate, stomach, pancreas, and head and neck [72]. Upregulation of mTOR signaling due to mutation in its upstream negative regulators, TSC1 or TSC2, results in an increased risk of renal cell carcinoma [73,74]. Additionally, upregulation of mTOR signaling is correlated with poor tumor prognosis for melanoma [75], prostate [76], pancreas [77], thyroid [78], breast [79] and cervical cancers [80]. These findings have prompted extensive development and clinical testing of rapamycin and its derivatives (termed rapalogs) in a wide variety of tumors. The anti-proliferative, anti-angiogenetic, apoptosis-sensitizing effects of rapamycin make it an attractive anti-cancer therapy. Treating cells with rapamycin leads to cell cycle arrest at G1 due insufficient cell mass and size [14,81–83]. Administration of rapamycin was also shown to enhance autophagy, thereby inhibiting malignant glioma cell growth [84]. The anti-proliferative effect of rapamycin in cancers is being further evaluated in clinical trials, which are showing that this and other mTOR inhibitors have good efficacy with mild side effects [85–87]. Temsirolimus, a rapamycin analog, has received FDA approval for treatment of metastatic renal cell carcinoma, while clinical trials of another rapalog, everolimus, are showing promise [88]. The major value of rapamycin may be its application in cancer therapies in combination with other anti-tumor agents. Sensitivity to rapamycin appears to be cancer-type specific. Similarly, different tumors vary in their response to DR [69,70,89–93]. Tumor responsiveness to DR has been correlated with P13K pathway activity, which influences signaling through the AKT pathway [94]. Typically only b10% of patients respond to rapamycin therapy; however, clinical trials are underway to evaluate the effects of using mTOR inhibitors in combination with irradiation, chemotherapy, or other anti-angiogenic agents [86]. For example, when in combination with the caspase-3 inhibitor Z-DEVE, rapamycin increases radiation sensitivity in lung cancer cells [95]. Various other combinatorial therapy trials are also currently underway.

7. TOR and vascular disease Dietary restriction lowers the risk factors associated with ageassociated cardiovascular disease, such as blood pressure, cholesterol, and hypertension. Similarly, inhibition of the mTOR signaling pathway by rapamycin administration has cardioprotective benefits in various mouse models of cardiac hypertrophy and heart failure [96]. Cardiac hypertrophy is characterized by enlargement of the heart as a consequence of increased cell size and enhanced protein synthesis [97]. Elevated PI3K/Akt/mTOR pathway activity has been implicated in this inappropriate enlargement of the heart [98]. mTOR inhibition by rapamycin treatment regresses cardiac hypertrophy induced by pressure overload [98–100] and inhibits angiotensin II-induced increases in protein synthesis in cardiac myocytes [101]. Rapamycin improved parameters of cardiac hypertrophy, such as increased left ventricular end-systolic dimensions, depressed fractional shortening, ejection fraction, as well as regressing left ventricular fibrosis in mice with hypertrophy and heart failure [100,102], thus suggesting that rapamycin may be a tool to improve cardiac function and repair cardiac hypertrophy. These effects are accompanied by suppression of ribosomal S6 protein and eIF4E phosphorylation which occurs due to pressure overload [100]. Rapamycin also attenuates hypoxia-induced pulmonary vascular remodeling and right ventricular hypertrophy in mice [103], and rapamycin-coated stents are approved for use to inhibit growth of vascular smooth muscle cell growth in patients with vascular disease after coronary angioplasty [104–106]. These data indicate that mTOR plays a key role in cardiac and vascular growth and homeostasis. With age there is an increase in the prevalence of cardiovascular diseases, such as congestive heart failure and coronary artery disease. Studies are demonstrating that modulation of mTOR activity might be a cardioprotective strategy that could be used to decrease the mortality associated with age-related cardiovascular disease. TOR signaling has also been implicated in a variety of ophthalmological conditions. Rodent models of corneal neovascularization as well as retinal and choroidal angiogensis showed improvement upon treatment with rapamycin [107–109] and clinical trials are currently underway to evaluate the efficacy of rapamycin in humans with choroidal neovascularization and neovascular age-related macular degeneration [110]. 8. TOR and metabolic disease Not surprisingly, age-related obesity and metabolic disease are offset by DR. Although it remains unclear how much of the lifespan extension from DR in rodents is due to the absence of obesityassociated metabolic effects, in people diabetes and obesity are major health risks among the elderly. A number of long-lived mouse models have phenotypes consistent with improved metabolic function. Examples include Ames and Snell dwarf mice which have reduced metabolic rate [111]; GHR/BP−/− mice which are small, have reduced fasting insulin and glucose levels and delayed sexual maturity [112]; and FIRKO (insulin receptor knockout in adipose) mice which are small and have reduced fat mass and lower fasting insulin [113]. Investigators have taken advantage of these observations by challenging many potentially long-lived models with the stress of nutrient overload to accelerate metabolic disease in young mice. Many longlived genetic models exhibit resistance to this high calorie challenge. For instance, p66shc−/− mice show reduced atherogenesis [114], S6K1−/− (S6 kinase 1) mice are protected against obesity [115], and mice overexpressing Sirt1 show better glucose tolerance among other phenotypes [116]. These findings make short-term nutrition overload experiments a strategy to increase the likelihood that a mouse genetic model in question might have enhanced longevity. The mTOR signaling pathway has been implicated in both Type 1 and Type 2 diabetes. The hallmark of Type 1 diabetes is reduced


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insulin production due to destruction of pancreatic β-cells, while Type 2 diabetes arises initially from an insulin resistance. Insulin-induced protein translation and proliferation [117] as well as growth induced by glucose and branched amino acids [118] is dependent on mTOR signaling in pancreatic β-cells. Meanwhile, desensitization of cells to insulin has been linked in part to S6K-dependent phosphorylation and inhibition of the insulin receptor substrate proteins (IRS1 and 2) [119,120]. Thus chronic TOR activation results in insulin resistance (i.e. hyperglycemia and Type 2 diabetes). Treatment of mouse models with rapamycin reduces specific features of diabetic nephropathy, including inhibition of renal or glomerular hypertrophy [121,122]. In addition mTOR plays a role in lipid metabolism. mTOR expression is increased during adipogenesis [123,124]. Administration of rapamycin results in inhibition of adipocyte differentiation as well as reduced adipogenesis and lipogenesis. mTORC1 has recently been shown to regulate SREBP (sterol-regulatory-element-binding protein), a transcription factor involved in coordinating expression of lipogenic genes [125]. In this study, SREBP activity was also found to be required for efficient Akt-dependent cell growth in mammalian and Drosophila cells. Evidence of involvement of the TOR signaling pathway in metabolism is further supported by studies of mice defective downstream components of mTOR signaling. An adiposespecific knockout of Raptor results in lean mice that are resistant to diet-induced obesity and insulin resistance [126]. These mice also exhibit increased energy expenditure due to enhanced mitochondrial uncoupling in adipose tissue. Changes in S6K-dependent IRS phosphorylation were not assessed. Interestingly, the consequences of knocking out Raptor expression vary dependent on the chosen tissue; a complete knockout results in developmental lethality [127]. Whereas the adipose-specific deletion of Raptor may have beneficial consequences, skeletal muscle-specific Raptor knockout mice have a muscular dystrophy phenotype [128]. S6K1-deficient mice have smaller islets and exhibit hypersensitivity to insulin as well as glucose intolerance [129]. These mice also display reduced weight gain and resistance to obesity due to age or challenge with a high calorie [115]. Similarly disruption of 4E-BP1, another downstream component of mTOR signaling, results in mice with reduced adipose tissue [130]. Furthermore, in Drosophila, the melted mutant, defective in dTOR signaling, displays reduction in body size due to decreased fat accumulation [131]. Reduction of TOR activity in Drosophila also results in reduced glucose and lipid levels, maintenance of insulin sensitivity, decreased heart failure rate and increased lifespan [49]. 9. TOR and neurodegeneration DR has been suggested to have a neuroprotective effect and to decrease age-related neuronal loss [132,133]. The underlying mechanisms for this neuroprotective effect of DR are poorly understood. DR is implicated in regulating adult neuronal stem cells, increasing neurogenesis in young rats, [134] and reducing decline of neurogenesis in older rats through a cell survival promoting effect [135]. Neurological disorders such as Huntington's, and Alzheimer's diseases as well as normal brain function might benefit from pharmacological modulation of mTOR signaling. mTOR signaling has been shown to play a role in protection against polyglutamine toxicity, which is associated with several human diseases, including Huntington's disease. Rapamycin treatment reduces polyglutamine toxicity in fly and mouse models via a mechanism that involves reduction of mTOR activity [136] and induction of an autophagy response that might be involved in clearance of protein aggregates [137]. It is speculated that this autophagy response induced by rapamycin treatment may help remove protein aggregates in other neurodegenerative disorders, such as β-amyloid accumulation in Alzheimer's patients. mTOR also plays a role in regulation of translation of tau mRNA and degradation of tau [138], a protein thought to contribute to

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the neurofibrillary tangles found in the brains of Alzheimer's patients. In Drosophila models, rapamycin treatment decreases tau levels, reducing its toxicity [139]. In response to oxidative stress, mTOR signaling is inhibited, inducing expression of stress response genes, such as YDR533C in yeast [140]. Autosomal recessive mutation of the human homolog of YDR533C, DJ-1, is associated with early onset Parkinson's disease, which has led to the postulation that inhibition of mTOR signaling in Parkinson's patients might provide a neuroprotective effect by induction of DJ-1 expression [141]. In addition, mTOR is a major downstream effector of the tuberous sclerosis complex (TSC), suggesting that pharmacologic modulation of mTOR signaling might benefit neuropsychiatric disorders, such as epilepsy, mental retardation and autism, all of which have been linked to TSC mutations [142]. Evidence demonstrating that mTOR is involved in synaptic protein synthesis links the mTOR signaling pathway with normal brain function as well as learning and memory. Synthesis and induction of long-term potentiation and long-term facilitation proteins, important for the long-term synaptic plasticity involved in learning and memory processes, are inhibited by rapamycin treatment [143]. mTOR and components critical for translation are enriched at postsynaptic junctions [144], allowing for rapid synthesis of proteins from pretranscribed mRNAs upon stimulus of a synapse to induce long-term changes in synaptic function and structure. 10. Conclusions While rapamycin may protect from and in some cases be part of a treatment regimen for age-related diseases, there are potential adverse consequences in otherwise healthy patients. Foremost among these may be its activity as an immunosuppressant. As such, it has been approved for clinical use following kidney transplant as a means to inhibit organ rejection [145]. As with many cancer trials, rapamycin is primarily used in combination therapy with other immunosuppressants such as cyclosporine. The role of TOR signaling in modulating immunity and the immunosuppressive mechanism of rapamycin is not well understood. A growing body of research indicates that mTOR regulates activation and proliferation of T cells while attenuating the inflammatory response [146,147]. How long-term low dose rapamycin administration would affect the immune system in healthy individuals remains unclear. Concerns in this regard are sufficient to make rapamycin a questionable candidate for administration to healthy individuals to promote healthspan. A better understanding of TOR signaling and immune system function may aid in understanding how modulation of mTOR activity affects age-related diseases and lifespan. It is worth noting that reduced immune function may be associated with dietary restriction (DR) in humans [148]. Other side effects of rapamycin treatment regimens have also been reported, although the drug is considered to be generally well-tolerated. What is apparent is that reduced TOR signaling is sufficient to increase lifespan in invertebrate organisms and, if brought about in the right manner, can protect against at least a subset of age-related diseases in mammals (Fig. 1). These findings make further mechanistic studies relating TOR signaling to aging an imperative. It will be particularly important to determine which downstream effects of mTOR activation accelerate aging and in what tissues these actions occur. Likewise, a better understanding of the relationship between TOR signaling, insulin/IGF-1 signaling and other signaling pathways involved in responding to DR will be required. Importantly, the field awaits the findings from the NIA Intervention Testing Program rapamycin study on mouse longevity [66,67]. Will lifespan be extended by inhibition of TOR? If so, this would mark the first pathway shown to modulate aging in four commonly used model aging systems: yeast, worms, flies, and mice. As such, examination of the already clinically relevant TOR inhibitors for effects across a spectrum of age-associated diseases in people would seem to be warranted.


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