Molecules to Medicine with mTOR: Translating Critical Pathways into Novel Therapeutic Strategies

policy-makers.

Preface

Kenneth Maiese, Editor

Throughout the world, the age of the population is increasing at a significant rate. In developed countries over the course of the last half-century, the number of individuals that are over the age of 65 has approximately doubled. This increase in life expectancy and age has been accompanied by a 1% decrease in the age-adjusted death rate over the years 2000 through 2011. Developing nations also share in the increased life expectancy of the world’s population. In these countries, the number of elderly people will rise from 5% of the population to approximately 10% of the population over future decades.

This positive news for the increased lifespan of individuals in developed nations as well as developing countries is somewhat buffered by the parallel rise in non-communicable diseases (NCDs). Improvements in healthcare have fostered increased longevity of the global population. For example, of the five leading causes of death that are cardiac disease, cancer, chronic lower respiratory disease, stroke, and traumatic accidents, stroke is no longer ranked as the third leading cause of death. Multiple factors most likely have led to this lower rank for stroke that involve heightened public awareness for the rapid treatment of stroke, a reduction in tobacco consumption, and improved care for disorders involving diabetes mellitus (DM), hypertension, and low-density lipoprotein cholesterol. Active treatment with recombinant tissue plasminogen activator has also led to a reduction in mortality and morbidity in stroke patients. Yet, therapeutic treatments to resolve disability and death from stroke continue to remain limited for the majority of patients.

Overall, a rise in the prevalence of NCDs continues with the advancing age of the world’s population. According to World Health Organization (WHO) records, greater than 60% of the annual 57 million global deaths are due to NCDs. Furthermore, almost 80% of these NCDs occur in low- and middle-income countries. Disorders such as DM will continue to grow according to WHO estimates, such that DM will be the seventh leading cause of death by the year 2030. As of 2013, approximately 350 million individuals are estimated to suffer from DM and millions of individuals are estimated to be currently undiagnosed with DM. The costs of caring for individuals with DM are extremely high. In the United States (US) alone, the costs of care for individuals with DM in 2012 equaled 8915 US dollars per person and almost 17% of the country’s gross domestic product. Although early diagnosis of individuals with DM may be critical to prevent the progression of this disease, the presence of impaired glucose tolerance in the young raises additional concerns for the future development of DM. Obesity, now increasing in incidence, is another risk factor for the development of DM since it leads to cellular oxidative stress, insulin resistance, and lipid-induced impairment of pancreatic β cells.

DM can affect multiple systems of the body and can impact cardiovascular disease, neurodegenerative disorders, the musculoskeletal system, cancer, and immune function. Acute and chronic neurodegenerative disorders lead to disability and death in more than 30 million individuals worldwide. It is predicted that the number of individuals with dementia will continue to grow, doubling every 20 years, and reach almost 115 million individuals by 2050. Most of the increase is expected to occur in developing countries, with Alzheimer’s disease being considered a significant public health concern. In regards to costs, care for those suffering from dementia equals more than 600 billion US dollars annually. With chronic neurodegenerative disorders such as Parkinson’s disease, approximately 50,000 new cases are present in the US alone each year and this number is predicted to double by 2030. During this same period, other disorders such as epilepsy are predicted to affect over 50 million people and peripheral nerve disease is estimated to be present in almost 300 million individuals. With cancer, almost 50% of cancer survivors are 70 years or older, indicating the increased prevalence with advancing age. In contrast, 5% of individuals with cancer are younger than 40 years of age. The number of cancer survivors will continue to grow and reach at least 19 million by 2024 in the US alone. For cardiovascular disease, this disorder remains the leading cause of death and increases with aging, but can easily affect all ages of the world’s population. Elevated cholesterol and hypertension are significant risk factors for cardiovascular disease and contribute to approximately 13% of all deaths.

In light of the limited courses of action to treat many NCDs that currently have only symptomatic or a handful of therapeutic options, development of novel therapeutic strategies to address the growing prevalence of NCDs with the accompanying advancing age of the global population is considered to be highly warranted to fill this void. The mechanistic target of rapamycin (mTOR) is one such strategic pathway that has gained high visibility as a critical target for multiple disease entities. mTOR is also known as the mammalian target of rapamycin and the FK506-binding protein 12-rapamycin complex-associated protein 1. mTOR is a 289-kDa serine-threonine protein kinase, present throughout the body, and oversees multiple functions that involve gene transcription, protein formation, oxidative stress, stem cell development, cell metabolism, cell survival, cell senescence, immunity, aging, tissue regeneration and repair, cancer, and pathways of programmed cell death that include autophagy and apoptosis.

Molecules to Medicine with mTOR: Translating Critical Pathways into Novel Therapeutic Strategies is a unique resource that employs a translational medicine approach to present novel work and new insights for the role of mTOR during normal physiology, in disease states, and for the development and refinement of therapeutic strategies applicable to multiple systems of the body. Prominent internationally recognized experts explore the complexity of mTOR signaling and the critical need for therapeutic targeting of this pathway, but maintain a clear and insightful presentation of clinical and basic research perspectives for a broad audience that encompasses healthcare providers, scientists, drug developers, and students.

Molecules to Medicine with mTOR: Translating Critical Pathways into Novel Therapeutic Strategies begins with the presentation of mTOR in cellular development, proliferation, and survival in Section I. Topics of cellular biology and new therapeutic strategies with mTOR that involve the maintenance of proliferation of stem cells, conceptus development, implantation and placentation, myogenesis, cartilage homeostasis, and cell survival during xenobiotic cell injury are presented. Section II extends these topics with the discussion of mTOR in the nervous system and the role of mTOR in congenital disease. In Section III, higher cognitive function and aging are explored with the examination of the role of mTOR in memory formation as well as cognitive impairment, aging processes of the brain, depression, addiction, and behavior modification. The cardiovascular system is discussed in Section IV, highlighting the role of mTOR in nutrient sensing, new vessel growth, cardiac ischemic disease, and the maintenance of cardiac function during metabolic disorders. In Section V, the vital role of mTOR in adaptive and innate immune function, organ transplantation, inflammatory pathways in the nervous system, and glial function are presented. Section VI further expands the analysis of mTOR in the endocrine system and provides scrutiny of the influence of mTOR in endocrine disorders, metabolic disease, diabetogenesis, and integrated pathways that involve both renal and nervous system impairment. The last segment of the book, Section VII, provides a vital perspective for the role of mTOR as a proliferative agent to foster tumor cell growth in a variety of settings and to affect cancer cell metabolic pathways. Molecules to Medicine with mTOR: Translating Critical Pathways into Novel Therapeutic Strategies provides an innovative examination of the pivotal function mTOR holds in the human body and highlights the compelling need to critically consider mTOR signaling pathways in the translation of cellular biology to effective and safe clinical treatments for the growing prevalence of multiple disorders that mirror the increasing age and lifespan of the global population.

Section I

mTOR in Cellular Development, Proliferation, and Survival

Outline

Chapter 1 Novel Stem Cell Strategies with mTOR

Chapter 2 mTOR

Chapter 3 mTORC1 in the Control of Myogenesis and Adult Skeletal Muscle Mass

Chapter 4 mTOR

Chapter 5 The Role of mTOR, Autophagy, Apoptosis, and Oxidative Stress During Toxic Metal Injury

Chapter 1

Novel Stem Cell Strategies with mTOR

Kenneth Maiese,    Cellular and Molecular Signaling, Newark, NJ, USA

Abstract

The incidence of noncommunicable diseases is increasing at an alarming rate that parallels the growing life expectancy of the global population. Stem cell therapeutic strategies hold exciting prospects for the treatment of numerous illnesses that can involve metabolic disorders, neurodegenerative disease, cardiac and vascular disease, and cancer. Intimately coupled to the development of novel treatment regimens with stem cells is the mechanistic target of rapamycin (mTOR), 289-kDa serine/threonine protein kinase that oversees multiple cellular processes ranging from cellular growth to cellular senescence. As an essential component of mTOR complex 1 and mTOR complex 2, mTOR governs stem cell development, maintenance, proliferation, and differentiation. mTOR can regulate pathways of apoptosis and autophagy either as a primary determinant or through trophic factor-mediated pathways. These pathways of programmed cell death are critical since they can lead to stem cell proliferation as well as to stem cell demise. Equally important for the biological and clinical outcomes driven by mTOR are the tightly linked pathways of Wnt signaling, Wnt1 inducible signaling pathway protein 1, silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae), AMP activated protein kinase, and forkhead transcription factors. mTOR holds compelling prospects to direct and focus new stem cell therapies for a broad array of disorders, but a number of challenges remain to gain further insight into the potential for varied clinical outcomes that can be fostered by mTOR.

Keywords

Cancer; cardiovascular; diabetes; forkhead; mTOR; neurodegeneration; stem cells; SIRT1; WISP; Wnt

1.1 Life Expectancy and the Significance of Global Noncommunicable Diseases

In developed nations, the age of the population has significantly risen. Life expectancy is approaching almost 80 years for all individuals and has been marked by a 1% decrease in the age-adjusted death rate from the years 2000 through 2011 [1]. The number of individuals over the age of 65 also has doubled during the prior 50 years [2]. If one examines large developing nations, such as China and India, it is expected that the proportion of elderly individuals will increase from the present levels of approximately 5% to almost 10% over the next several decades. Improvements in treatment resources and preventive care have contributed to the increased lifespan of the global population.

However, accompanied with the increase in life expectancy has been a rise in chronic disorders and noncommunicable diseases (NCDs; Table 1.1). Through data obtained by the World Health Organization, greater than 60% of the 57 million annual global deaths result from NCDs. Approximately 80% of these NCDs occur in low- and middle-income countries [3]. NCDs affect approximately 30% of the population under 60 in low- and middle-income countries. In high-income countries, 13% of the population under 60 is affected.

Table 1.1

Global Noncommunicable Diseases

The five leading causes of death are cardiac disease, cancer, chronic lower respiratory disease, stroke, and traumatic accidents [4]. Elevated cholesterol and hypertension are significant risk factors for cardiovascular disease. For example, hypertension contributes to approximately 13% of all deaths [5].

Disorders such as elevated cholesterol and hypertension can also lead to an increase in acute neurodegenerative disorders such as stroke, the fourth leading cause of death (Table 1.1) [6,7]. Other NCDs, such as diabetes mellitus (DM), can also contribute to acute neurodegenerative diseases that include cerebrovascular disease [8,9]. Stroke leads to multiple complications that affect both the livelihood and minimal daily function of an individual [10,11]. As a leading cause of death [4], stroke affects approximately 15 million individuals every year. In the United States, approximately 800,000 strokes occur per year at an annual cost of 75 billion US dollars [6]. It is of interest to note that stroke is no longer ranked as the third leading cause of death. Factors related to improved management of hypertension and low-density lipoprotein cholesterol disorders, reduction in tobacco consumption, heightened public education and awareness for the need for rapid treatment of cerebrovascular disorders, and more focused management on metabolic disorders such as DM may have contributed to this lower ranking for stroke [6,7]. In addition, treatment with recombinant tissue plasminogen activator has led to a reduction in mortality and morbidity in patients presenting with stroke [12,13]. Yet, overall therapeutic strategies for patients presenting with stroke remain limited for the majority of patients. Treatment with recombinant tissue plasminogen activator is applicable to only a subgroup of patients that requires a narrow therapeutic window.

Together, acute and chronic neurodegenerative disorders lead to disability and death in more than 30 million individuals worldwide annually (Table 1.1) [14]. Acute neurodegenerative disorders can place a severe burden on the world’s population [6,15]. Acute traumatic brain injury (TBI) leads to severe neurological disability [16,17]. A twofold detrimental effect on the clinical outcome of patients can occur with TBI. TBI can result in acute injury to the nervous system as well as subsequent chronic impairment [18–20]. In the United States, approximately 50,000 individuals die every year as a result of TBI and more than 100,000 individuals must live with chronic disability [21]. During severe trauma, approximately 50% of individuals eventually die.

With the aging population, a rise in the incidence of chronic neurodegenerative disorders is also expected to ensue [22,23]. One such example is Alzheimer’s disease (AD). The familial cases of AD consist of less than 2% of all presentations [24] and usually occur prior to age 55 [25]. Familial cases of AD occur as an autosomal dominant form of a mutated amyloid precursor protein (APP) gene as well as mutations in the presenilin 1 or 2 genes [26]. Familial AD is present in approximately 200 families worldwide and results from variable single-gene mutations on chromosomes 1, 14, and 21. Mutations on chromosome 1 lead to altered presenilin 2, mutations on chromosome 14 result in altered presenilin 1, and mutations on chromosome 21 lead to altered APP. In contrast, 10% of the global population over the age of 65 is affected with sporadic AD [27]. At present, more than 5 million individuals are diagnosed with AD and 3.5 million are under treatment at an annual cost of almost 4 billion US dollars. It is estimated that the number of those suffering from AD will increase significantly and rise to more than 30 million individuals over the next 15–20 years [14,27,28]. If one considers other chronic progressive neurodegenerative disorders such as Parkinson’s disease (PD) [29,30], almost 50,000 new cases present in the United States alone each year. Approximately 1–4% of individuals over age 60 suffer from PD globally and this number of affected individuals may double by the year 2030. By the year 2030, epilepsy is predicted to affect over 50 million people, neuropathies are estimated to afflict almost 300 million individuals, and neurological injuries may change the lives of 243 million individuals [31]. However, similar to acute neurodegenerative disorders, the availability of treatments that can prevent the initiation or block the progression of chronic neurodegenerative disorders is limited.

DM also is a significant NCD for the world’s population (Table 1.1) [32]. The incidence of DM is increasing throughout the world and approximately 350 million individuals currently have DM [33–37]. Another 8 million individuals are believed to suffer from metabolic disorders and are currently undiagnosed [38–40]. The costs to care for individuals with DM are high. Approximately 9000 US dollars are spent in the United States for each individual with DM per year and overall care for patients with DM consumes 17% of the gross domestic product [41]. Early diagnosis and proper care of individuals with DM can impact care for this disease by modulating epigenetic changes in age-related genes involved with DM and other degenerative disorders [42–46]. Impaired glucose tolerance in the young raises additional concerns for the risk of developing DM [34]. Obesity is another risk factor for DM [33–37]. Obesity and excess body fat can precipitate pancreatic β-cell injury [47], cellular inflammation [8], impaired growth factor release [48–51], changes in protein tyrosine phosphatase signaling [37,52], oxidative stress [35,53], and insulin resistance [8,32,54,55].

DM can involve any system of the body [8,9]. DM leads to vascular disease resulting in endothelial cell senescence [56], injury to endothelial cells [51,57–62], cardiovascular disease [63–71], platelet dysfunction [72,73], atherosclerosis [11,74–76], loss of endothelial progenitor cells [52,77–81], dysfunctional maintenance and mobilization of endothelial progenitor cells [79,80], and impaired angiogenesis [62,69,82]. DM also can have negative effects on the immune system [33,70,83–88], renal function [89–93], hepatic metabolism [35,54,94–98], and musculoskeletal integrity [53,74,99–101]. DM affects all components of the central and peripheral nervous systems. DM can lead to retinal disease [39,102–104], peripheral nerve disorders [95,105], memory loss [106–108], psychiatric disorders [109,110], stroke [22,46,72,76,111,112], dementia such as AD [50,106,113,114], and impairment of neuronal longevity [50].

Interestingly, tight glucose control does not always result in the resolution of complications from DM [34,115]. Use of diet control treatments may be effective to prevent hyperglycemia events, but can potentially decrease organ mass through processes that involve autophagy [97]. These observations point to the need for additional strategies for the treatment of DM and its complications.

1.2 mTOR Structure and Signaling

It is clear that NCDs are becoming a growing concern for the global population given the concurrent rise in longevity and life expectancy. Increased lifespan is accompanied by a greater exposure to several NCDs that involve multiple systems of the body as well as processes closely tied to cellular metabolism. Given the need for novel treatments to effectively treat NCDs that currently have limited therapeutic options, the mechanistic target of rapamycin (mTOR) is increasingly being recognized as a critical target for drug discovery development against NCDs that involve cardiovascular disease, neurological disorders, metabolic disease, and cancer.

mTOR is also known as the mammalian target of rapamycin and the FK-506-binding protein 12-rapamycin complex-associated protein 1 (Table 1.2). It is a 289-kDa serine/threonine protein kinase that is encoded by a single gene, FRAP1 [116,117]. Initially, the target of rapamycin (TOR) was identified through the use of rapamycin-resistant TOR mutants in Saccharomyces cerevisiae with the genes TOR1 and TOR2 that yield two protein isoforms in yeast known as Tor1 and Tor2 [118]. The carboxyterminal domain of mTOR has four domains for catalytic activity that include FAT, FKBP12-rapamycin-binding domain (FRB), the catalytic PI3/PI4-related kinase domain, and FATC [116]. The FAT domain, which consists of FKBP12-rapamycin-associated protein (FRAP), ataxia-telangiectasia (ATM), and the transactivation/transformation domain-associated protein domain, resides adjacent to the FRB that allows association between mTOR and FKBP12 when bound to rapamycin. The PI3/PI4-related kinase domain and the small FATC domain follow these two domains. The N-terminal portion of mTOR contains at least a 20 HEAT (Huntingtin, Elongation factor 3, a subunit of Protein phosphatase-2A, and TOR1) repeat. This area promotes binding with the regulatory proteins Raptor (regulatory-associated protein of mTOR) and Rictor (rapamycin-insensitive companion of mTOR) [119]. Post-translational phosphorylation of mTOR can occur through a number of processes. The C-terminal domain with sequence homology to the catalytic domain of the phosphoinositide 3-kinase (PI 3-K) family [120] contains several phosphorylation sites that regulate mTOR. Within the HEAT domain, serine¹²⁶¹ can be phosphorylated in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) by insulin signaling through the PI 3-K pathway to increase mTOR activity [120]. Phosphorylation of serine¹²⁶¹ also results in mTOR serine²⁴⁸¹ autophosphorylation [120].

Table 1.2

mTOR Structure and Signaling

Akt, protein kinase B; AMPK, AMP activated protein kinase; eIF4E-4EBP1, eukaryotic initiation factor 4E-binding protein 1; FAT domain, FKBP12-rapamycin-associated protein (FRAP), ataxia-telangiectasia (ATM), and the transactivation/transformation domain-associated protein domain; HEAT, Huntingtin, Elongation factor 3, a subunit of protein phosphatase-2A, and TOR1; mTOR, mechanistic target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; PI 3-K, phosphoinositide 3-kinase; p70S6K, p70 ribosomal S6 kinase; TOR, target of rapamycin.

mTOR is a central component of the protein complexes mTORC1 and mTORC2 (Figure 1.1) [27,29,121,122]. Rapamycin is a macrolide antibiotic derived from Streptomyces hygroscopicus that inhibits both TOR activity and mTOR activity [123]. mTORC1 is more sensitive to the inhibitory effects of rapamycin than mTORC2 [124]. Rapamycin blocks mTORC1 activity by binding to immunophilin FK-506-binding protein 12 (FKBP12) that attaches to FRB at the C-terminal of mTOR to prevent the phosphorylation of mTOR [125]. Chronic administration of rapamycin can inhibit mTORC2 activity that may occur from the disruption of the assembly of mTORC2 [124].

Figure 1.1 mTOR oversees stem cell development, pluripotency, differentiation, maintenance, and programmed cell death in clinical disease. Multiple disease entities that involve neurodegenerative disorders, cardiovascular disease, DM, and cancer can be the result of oxidative-stress-mediated cell injury. Through oxidative stress and the generation of ROS, the pathways of mTOR and PI 3-K, protein kinase B (Akt), AMPK, and the TSC1/TSC2 complex become significant determinants of stem cell survival. mTOR is a vital component of mTORC1 and mTORC2. mTORC1 consists of Raptor, the proline-rich Akt substrate 40 kDa (PRAS40), Deptor, and mLST8/GβL. mTORC2 is composed of Rictor, mLST8, Deptor, the mSIN1, and the protein observed with Rictor-1 (Protor-1). Intimately tied to these pathways are Wnt, WISP1, SIRT1, forkhead transcription factors, and growth factors. Ultimately, mTOR signaling governs stem cell apoptosis, autophagy, maintenance, cell senescence, development, pluripotency, and differentiation.

mTORC1 consists of Raptor, the proline-rich Akt substrate 40 kDa (PRAS40), Deptor (DEP domain-containing mTOR interacting protein), and mLST8/GβL (mammalian lethal with Sec13 protein 8, abbreviated as mLST8 or G protein beta subunit-like (GβL)) (Table 1.2) [116]. Phosphorylation of Raptor through several pathways that can include the protein Ras homolog enriched in brain (Rheb) activates mTORC1 and allows it to bind to its complex constituents [126]. Rheb phosphorylates Raptor residue serine⁸⁶³ and other residues that include serine⁸⁵⁹, serine⁸⁵⁵, serine⁸⁷⁷, serine⁶⁹⁶, and threonine⁷⁰⁶. mTORC1 activity is limited if serine⁸⁶³ remains unphosphorylated [127]. Once mTOR is active, mTOR also can modulate Raptor activity that can be blocked by rapamycin [127]. PRAS40 inhibits mTOR activity and can prevent the binding of mTORC1 to Raptor [128]. Phosphorylation of PRAS40 by protein kinase B (Akt) frees PRAS40 from Raptor and allows PRAS40 to be sequestered by the cytoplasmic docking protein 14-3-3 to activate mTORC1 [128–132]. Deptor inhibits mTORC1 activity by binding to the FAT domain of mTOR. Without Deptor, the activity of Akt, mTORC1, and mTORC2 are enhanced [133]. In contrast to PRAS40 and Deptor, mLST8 promotes mTOR kinase activity through p70 ribosomal S6 kinase (p70S6K) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) that bind to Raptor [134]. PRAS40 can block mTORC1 activity by preventing the association of p70S6K and 4EBP1 with Raptor [14,128]. In addition, mLST8 promotes mTOR kinase activity and is known to control insulin signaling through the transcription factor FoxO3 [135], be necessary for Akt and protein kinase C-α (PKCα) phosphorylation [135], and is required for the association between Rictor and mTOR [135].

mTOR signaling is closely associated with the pathways of PI 3-K, Akt, and AMP activated protein kinase (AMPK) (Table 1.2) [64,136]. Akt regulates the activity of the hamartin (tuberous sclerosis 1)/tuberin (tuberous sclerosis 2) (TSC1/TSC2) complex, an inhibitor of mTORC1 [137–140]. Although several regulatory phosphorylation sites are known to exist for TSC1, control of the TSC1/TSC2 complex is primarily mediated though the phosphorylation of TSC2 by Akt, extracellular signal-regulated kinases, activating protein p90 ribosomal S6 kinase 1, AMPK, and glycogen synthase kinase-3β. TSC2 functions as a GTPase-activating protein (GAP) converting a small G protein Rheb (Rheb-GTP) to the inactive GDP-bound form (Rheb-GDP). Once Rheb-GTP is active, Rheb-GTP associates with Raptor to regulate the binding of 4EBP1 to mTORC1 and increase mTORC1 activity [141]. Akt phosphorylates TSC2 on multiple sites that leads to the destabilization of TSC2 and disruption of its interaction with TSC1. Phosphorylation of TSC2 at serine⁹³⁹, serine⁹⁸¹, and threonine¹⁴⁶² results in the binding of TSC2 to protein 14-3-3, disruption of the TSC1/TSC2 complex, and subsequent activation of Rheb and mTORC1 [142]. During some paradigms of cellular protection, a limited activity of TSC2, as well as AMPK [143], appears necessary since complete knockdown of TSC2 can prevent cellular protection [144].

AMPK also controls the activity of the TSC1/TSC2 complex and blocks mTORC1 function. AMPK phosphorylates TSC2 to increase GAP activity to turn Rheb-GTP into Rheb-GDP and thus inhibits the activity of mTORC1 [145]. AMPK also controls TSC1/TSC2 activity through RTP801 (REDD1/product of the Ddit4 gene) [146]. AMPK activity can increase REDD1 expression during hypoxia to suppress mTORC1 activity by releasing TSC2 from its inhibitory binding to protein 14-3-3 [146]. In addition, AMPK modulates the sirtuin silent mating type information regulation 2 homolog 1 (S. cerevisiae) (SIRT1) activity that can be vital for stem cell survival and proliferation [22]. AMPK increases the cellular NAD+/NADH ratio that leads to deacetylation of the SIRT1 targets peroxisome proliferator-activated receptor-gamma coactivator 1 and the forkhead transcription factors FoxO1 [68] and FoxO3a [147]. AMPK increases nicotinamide phosphoribosyltransferase activity that catalyzes the conversion of nicotinamide to nicotinamide mononucleotide [85], increases nicotinamide adenine dinucleotide (NAD+) levels, decreases levels of the SIRT1 inhibitor nicotinamide, and promotes SIRT1 transcription [9,148,149]. SIRT1 expression in combination with AMPK activation promotes the induction of autophagy that can protect endothelial cells exposed to oxidized low-density lipoproteins [150]. SIRT1 may function similar to AMPK as an inhibitor of mTOR to block its activity [35].

In regards to mTORC2, mTORC2 consists of Rictor, mLST8, Deptor, the mammalian stress-activated protein kinase interacting protein (mSIN1), and the protein observed with Rictor-1 (Protor-1) [6,119,151–154]. Rictor [155] and mSIN1 [156] can activate Akt at serine⁴⁷³ and facilitate threonine³⁰⁸ phosphorylation by phosphoinositide-dependent kinase 1 to promote cell survival [14,151,156]. The kinase domain of mTOR has been shown to phosphorylate mSIN1, preventing the lysosomal degradation of mSIN1 [157]. Protor-1 is a Rictor-binding subunit of mTORC2 and can activate serum and glucocorticoid induced protein kinase 1 (SGK1), since loss of Protor-1 reduces the hydrophobic motif phosphorylation of SGK1 and the substrate N-Myc down-regulated gene 1 in the kidney [158]. mTORC2 also phosphorylates and activates SGK1, a member of the protein kinase A/protein kinase G/protein kinase C family of protein kinases [159]. mTORC2 controls cytoskeleton remodeling through PKCα and oversees cell migration through the Rac guanine nucleotide exchange factors P-Rex1 and P-Rex2 and through Rho signaling [160]. In contrast to the inhibition of mTORC1, mTORC2 is activated by the TSC1/TSC2 complex through the N-terminal region of TSC2 and the C-terminal region of Rictor [161]. Absence of the TSC1/TSC2 complex leads to the loss of mTORC2 kinase activity in vitro [161].

1.3 mTOR and Oxidative Cell Stress

mTOR has a critical role in cell signaling pathways and regulates multiple cellular processes that include cellular proliferation, cellular senescence, metabolism, gene transcription, cellular survival, and cellular death (Figure 1.1). Targeting mTOR and its signaling pathways that control stem cell maintenance may prove essential for the development of new strategies for NCDs. Stem cells have the potential for treating multiple disorders. As a result, the ability of mTOR to oversee processes of cell injury and cell death becomes a vital consideration in the development of stem-cell-mediated therapies.

Oxidative stress can markedly impact cellular survival and longevity [11,53,162–167]. During oxidative stress, reactive oxygen species (ROS) are generated that include nitrogen-based free radical species such as nitric oxide and peroxynitrite as well as superoxide free radicals, hydrogen peroxide, and singlet oxygen [163]. Mitochondria also lead to the generation of ROS. Mitochondria produce adenosine triphosphate (ATP) through the oxidation of glucose, pyruvate, and NAD+ that are present in the cytosol. NAD+ and flavin adenine dinucleotide (FAD) are reduced to NADH and FADH2 in the tricarboxylic acid cycle. This redox energy from NADH and FADH2 is transferred to oxygen through the electron transport chain. Protons are then transferred from complexes I, III, and IV in the inner membrane to the intermembrane space with a subsequent proton gradient that is formed across the inner membrane. Complex V (ATP synthase) accumulates the energy from this gradient to produce ATP from adenosine diphosphate and inorganic phosphate (Pi).

As a result of this aerobic production for ATP, ROS are generated. At reduced levels, production of ROS may be important for tolerance against hypoxia and ischemia. Moderate levels of ROS also may be required for the regulation of inflammatory cell activation [168]. However, increased levels of ROS can lead to cellular injury and result in mitochondrial and other organelle injury, DNA damage, protein misfolding, and neuronal synaptic dysfunction [17,22,164,169,170]. Protective pathways serve to insulate against damage from ROS and involve vitamins B, C, D, and K [76,85,171–173], glutathione peroxidase [173,174], and superoxide dismutase [163,169,175–182].

1.4 mTOR, Stem Cell Survival, Apoptosis, and Autophagy

Closely associated with oxidative stress cell injury are the pathways of programmed cell death that involve apoptosis and autophagy (Figure 1.1) [183–186]. Apoptosis has an early phase that involves the loss of plasma membrane lipid asymmetry and a later phase that leads to genomic DNA degradation [187–189]. The loss of asymmetry of membrane phosphatidylserine (PS) distribution is an early component of apoptosis that can be reversible [32,81,190]. However, if not reversed, cell death ensues and genomic DNA degradation prevails [187,188,191–195]. During the later phase of apoptotic cell injury, cellular DNA is destroyed, which is usually not a reversible process [39,179,196,197]. Externalization of PS residues and the onset of genomic DNA degradation are the result of a series of activation of nucleases and proteases that occur during apoptosis [148,198]. The early phase of apoptosis with membrane PS externalization activates inflammatory cells to engulf and remove injured cells [191,193,199,200]. Inhibition of membrane PS externalization is necessary to prevent the loss of functional cells that are temporarily injured and expressing membrane PS residues.

Under most circumstances, activation of mTOR and its related pathways of PI 3-K and Akt can prevent apoptotic cell death in stem cells. Loss of mTOR activity, such as with rapamycin, leads to endothelial progenitor cell apoptotic death. These detrimental effects during mTOR diminished activity may be related to inhibition of growth factor signaling [201]. Growth factors, such as erythropoietin (EPO) [49,202], are protective against apoptosis through mTOR activity against multiple insults. These include sepsis-associated encephalopathy [203], oxidative stress [129], cerebral microglial injury [204], and beta-amyloid (Aβ) toxicity [205]. EPO relies upon mTOR for cell development, differentiation, and survival [11,35]. EPO requires mTOR for the differentiation of neural precursor cells to achieve a neuronal phenotype [206] and for the protection of retinal progenitor cells from oxidative stress [207]. EPO controls mTOR and its downstream signaling pathways that involve PRAS40 to increase neuronal survival during oxygen-glucose deprivation [129]. EPO, through mTOR and Wnt signaling, maintains microglial survival during oxidative stress [204]. EPO can block Aβ toxicity through Wnt signaling and mTOR pathways and prevent caspase activation and apoptosis [205]. During hypoxia-reoxygenation stress, EPO increases mTOR activity to protect hippocampus-derived neuronal cells [208]. In the mTOR pathway, AMPK may also affect the biological function of EPO. The ability of EPO to oversee neuroinflammation may be linked to AMPK activity [209]. EPO may require a specific level of AMPK activity to alleviate detrimental effects of oxidative stress [210]. In addition, the concentration and activity of EPO may influence the protective actions of mTOR and signaling pathways associated with AMPK. High concentrations of EPO may promote cellular damage and lessen the activity of mTOR [211].

Other growth factors in addition to EPO also rely upon mTOR to maintain stem cell integrity. In experimental models with mice, the growth factors epidermal growth factor (EGF) and fibroblast growth factor (FGF) are protective of stem cells and neurons [212–216]. EGF and FGF use mTOR to maintain the proliferation of neural stem and progenitor cells [217]. EGF requires PI 3-K, Akt, and mTOR pathways to block cell injury during metabolic stress [218] and to prevent memory impairment [219]. Brain-derived neurotrophic factor (BDNF) uses mTOR activation for memory consolidation [220]. Yet, under some conditions, growth factor protection that is dependent upon autophagy may require blockade of mTOR activity. Cortical neurons are protected by BDNF through the induction of autophagy and the inhibition of mTOR during oxygen deprivation [221].

Autophagy is another pathway of programmed cell death that is dependent upon mTOR signaling to influence stem cell survival. Autophagy is a process that recycles components in the cell cytoplasm to remove nonfunctional organelles for disposal and tissue remodeling [15,185,222,223]. Autophagy has classifications that include microautophagy, chaperone-mediated autophagy, and macroautophagy [32]. Macroautophagy is the primary category of autophagy. This process consists of the sequestration of cytoplasmic proteins and organelles into autophagosomes that combine with lysosomes for degradation and recycling [32,66,80,223,224]. Microautophagy results in the invagination of lysosomal membranes for the sequestration and digestion of cytoplasmic components. Chaperone-mediated autophagy employs cytosolic chaperones for the transport of cytoplasmic components across lysosomal membranes [14].

The proteins TOR and mTOR are directly tied with genes that control autophagy [27,29,225]. At least 33 autophagic related genes (Atg) have been identified in yeast that can affect multiple disorders including DM, vascular disease, cancer, and neurodegenerative disorders [10,185,222,223,226–230]. Of these autophagic related genes, Atg1 and Atg13 (also known as Apg13) are associated with PI 3-K, Akt, and the TOR pathways. Dephosphorylation of Atg13 that can occur during starvation leads to the activation of Atg1 and the induction of autophagy. Phosphorylation of Atg13 through a TOR-dependent pathway releases Atg13 from Atg1 and reduces Atg1 activity. In mammals, the homologs of Atg1 are UNC-51 like kinase 1 (ULK1) and ULK2 [29]. Mammalian Atg13 binds to ULK1, ULK2, and FIP200 (focal adhesion kinase family interacting protein of 200 kDa) to activate ULKs, foster phosphorylation of FIP200 by ULKs, and lead to the activation of autophagy [231]. mTOR activity blocks the induction of autophagy by phosphorylating Atg13 and ULKs to inhibit the ULK–Atg13–FIP200 complex.

Under some conditions, autophagy serves as a vital component to promote stem cell survival that involves both SIRT1 and mTOR pathways. In embryonic stem cells, SIRT1 is protective [232] and appears to have an inverse relationship with mTOR [22]. SIRT1 can depress mTOR-mediated pathways as well as promote autophagy to preserve the integrity of human embryonic stem cells exposed to oxidative stress [233]. SIRT1 can foster inhibition of mTOR signaling to promote neuronal growth [234]. SIRT1 activity is necessary to promote autophagy to maintain proteostasis, produce energy during nutrient deprivation, and maintain muscle stem cell activation [235]. In endothelial cells exposed to oxidized low-density lipoproteins that can lead to atherosclerosis, SIRT1 up-regulation in combination with AMPK activity leads to autophagy that is necessary for cellular protection [150].

However, activation of autophagy during mTOR inhibition can also lead to stem cell demise in some circumstances. Autophagy may foster cellular senescence [236]. Endothelial progenitor cells that regenerate vascular endothelium become dysfunctional with the activation of autophagy during exposure to elevated glucose [80].

Wnt signaling pathways with mTOR can also modulate programmed cell death to play a role in stem cell survival. Wnt proteins are cysteine-rich glycosylated proteins that regulate stem cell proliferation and tumor cell growth [237–243]. During the activation of autophagy, breast cancer stem cells have been shown to succumb to apoptosis and the inhibition of Wnt signaling [228]. In addition, growth factors, such as EPO, may require Wnt signaling for the preservation of mesenchymal stem cells [244] and to limit proapoptotic forkhead transcription factor activity [245,246].

A downstream target in the Wnt pathway is Wnt1 inducible signaling pathway protein 1 (WISP1), which is also known as CCN4 [9]. WISP1 is a member of the six secreted extracellular matrix-associated CCN family of proteins that are involved in cellular survival and stem cell proliferation [247]. WISP1 is present in the brain, heart, kidney, lung, pancreas, placenta, epithelium, ovaries, small intestine, and spleen [247]. WISP1 is closely tied to mTOR and can significantly influence stem cell survival and proliferation. WISP1 can control induced pluripotent stem cell reprogramming [248,249]. WISP1 is also one of several genes that are overexpressed during pancreatic regeneration [250] and can support vascular regeneration during saphenous vein crush injury [251]. However, WISP1 oversees cellular senescence [252] and does not appear to foster excessive cellular proliferation under circumstances involving aging vascular cells [253]. WISP1 can also be differentially regulated in stem cells. WISP1 expression is increased during stem cell migration [254] and is repressed during hepatic differentiation in adipose-derived stem cells [238]. WISP1 also relies upon pathways of mTOR to prevent injury to cells [6,29]. WISP1 activates mTOR, inhibits PRAS40 [131], and limits TSC2 activity [144] to increase microglial cell survival during oxidative stress and Aβ toxicity. WISP1 also controls the post-translational phosphorylation of AMPK [35,64,136,255]. WISP1 regulates AMPK activation by differentially decreasing phosphorylation of TSC2 at serine¹³⁸⁷, a target of AMPK, and increasing phosphorylation of TSC2 at threone¹⁴⁶², a target of Akt [144]. This enables WISP1 to provide a minimal but not excessive level of TSC2 and AMPK activity to promote cellular survival during toxic insults [144].

1.5 mTOR, Stem Cell Proliferation, and Stem Cell Differentiation

mTOR can oversee the proliferation of stem cells in multiple systems of the body (Figure 1.1) [29]. Embryonic stem cell proliferation is decreased during the deletion of the C-terminal six amino acids of mTOR that leads to inhibition of kinase activity [256]. Deletion of the mTOR gene results in limited trophoblast growth, faulty implantation, and the inability to establish embryonic stem cells [257]. mTOR can also control undifferentiated stem cell growth in human embryonic stem cells as well as lead to activation of cell differentiation. Under some conditions, mTOR activity leads to mesenchymal stem cell senescence [258]. Loss of mTOR activity can result in cell pluripotency, cell proliferation, and inhibition of mesoderm and endoderm activities in embryonic stem cells [259]. In contrast, activation of mTOR and its downstream signal transduction pathways can lead to cell differentiation. Increased mTOR and p70S6K activity results in embryonic stem cell differentiation [260].

1.6 mTOR, Stem Cells, and Metabolic Disease

mTOR pathways are critical components for insulin signaling (Figure 1.1) [64]. mTOR inhibition, such as with rapamycin administration, results in reduced β-cell function and mass, insulin resistance, decreased insulin secretion, and DM [261]. Blockade of mTOR activity with rapamycin also reduces food intake and prevents fat-diet-induced obesity in mice. However, rapamycin administration can impair glucose uptake through the blockade of insulin-generated Akt activation and alteration in the translocation of glucose transporters to the plasma membrane as has been demonstrated in skeletal muscle [99]. Furthermore, loss of p70S6K activity results in hypoinsulinemia, insulin insensitivity to glucose secretion, glucose intolerance, and decreased pancreatic β-cell size [262]. Activation of the mTOR pathways p70S6K and 4EBP1 in pancreatic β-cells in mice leads to improved insulin secretion and resistance to β-cell streptozotocin toxicity and obesity [263].

Yet, activation of mTOR does not always lead to improved cellular metabolism and may require limited activity through the inhibition of this pathway. mTOR can function in a negative feedback loop and produce glucose intolerance by inhibiting the insulin receptor substrate 1 (IRS-1). mTOR signaling through the TSC1/TSC2 complex can inactivate IRS-1 and phosphorylate p70S6K to block IRS-1 activity [264]. With this pathway and mTOR activation, mTOR leads to inhibitory phosphorylation of IRS-1, impaired Akt signaling, and insulin resistance in studies with high-fat-fed obese rats [265]. Consumption of high-fat diets can also activate the renin–angiotensin–aldosterone system with increased circulating angiotensin II that activates mTOR and p70S6K to phosphorylate IRS-1 and foster insulin resistance [266].

Additional studies also suggest that limited activity of mTOR may be beneficial during DM. In studies that have examined caloric restriction in male mice, genes with the greatest statistical change after caloric restriction involved those associated with sirtuin activation and mTOR inhibition [267]. SIRT1 activation can prevent endothelial senescence during hyperglycemia [268], limit endothelial atherosclerotic lesions during elevated lipid states [269], and prevent endothelial cell apoptosis during experimental DM [193,270]. SIRT1 and mTOR signaling appear to have an inverse relationship in the control of cellular metabolism. Hepatic SIRT1 deficiency yields hepatic glucose overproduction, hyperglycemia, initiation of oxidative stress, and inhibition of the gene encoding Rictor that results in impaired mTORC2 and Akt signaling [271]. SIRT1 can function as a negative regulator of unfolded protein response signaling and inhibit mTOR in DM to attenuate hepatic steatosis, ameliorate insulin resistance, and restore glucose homeostasis [272]. During experimental DM models with high glucose exposure to mesangial cells, activation of SIRT1 with mTOR inhibition is required to promote mesangial cell proliferation [273]. SIRT1 is necessary for the protection of endothelial progenitor cells during high glucose exposure [274] and decreased SIRT1 levels have been observed in endothelial progenitor cells in patients with DM [78]. SIRT1 also has been shown to foster angiogenesis for vascular repair mechanisms during DM [275]. SIRT1 may provide progenitor cell protection through the maintenance of mitochondrial pathways. Autophagy and mitochondrial impairment occur in endothelial progenitor cells during exposure to elevated glucose exposure, suggesting a mechanism for dysfunction of endothelial progenitor cells during DM [80]. SIRT1 has been shown to prevent mitochondrial depolarization, cytochrome c release, and Bad, caspase 1, and caspase 3 activation [193,233,270,276].

1.7 mTOR, Stem Cells, and the Nervous System

In the nervous system, a loss of mTORC1 activity in neural stem cells results in reduced lineage expansion, blocked differentiation, and failed neuronal production (Figure 1.1) [277]. mTOR is necessary for insulin-induced neuronal differentiation in neuronal progenitor cells [278]. mTOR also governs the timing and control of neurogenesis. Inhibition of mTOR through the RTP801/REDD1 pathway delays neuronal differentiation. Over time, the expression of RTP801/REDD1 is altered with maturation. Levels of RTP801/REDD1 in newborn and mature neurons become diminished and mTOR activity is increased to promote the maturation of neurons [279]. Interestingly, with aging, the loss of mTOR activity may affect neural stem cell proliferation. mTOR signaling in the aged brain is reduced and is accompanied by a reduction in the proliferation of active neural stem cells [280]. mTOR activity is also necessary for the expression of the neuronal phenotype of postmortem neuronal precursors [206].

Growth factors that employ mTOR can also direct stem cell development. EPO promotes Wnt signaling to maintain the survival of mesenchymal stem cells [244]. EPO also requires mTOR to regulate bone homeostasis with osteoblastogenesis and osteoclastogenesis [281]. Differentiation of neural precursor cells that may be used for the treatment of neurodegenerative disorders is dependent upon both EPO and mTOR [206].

Importantly, the degree of mTOR activity may affect different populations of stem cells. Inhibition of mTOR activity can result in stem cell differentiation into astrocytic cells [217]. Combined Akt and mTORC1 inhibition can influence stem cell proliferation and has been shown to lead to reduced neuronal stem cell self-renewal and earlier neuronal and astroglial differentiation [282]. Non-neuronal neighboring cells may represent another factor to influence the growth of neuronal stem cells. Endothelial cells can foster mTOR activity and lead to the expansion of long-term glioblastoma stem-like cells [283].

1.8 mTOR, Stem Cells, and the Vascular System

Protein kinase signaling plays an important role in stem cell development in the cardiovascular system (Figure 1.1). In particular, mTOR is one of several components important for the proliferation of human embryonic stem-cell-derived cardiomyocytes [284]. mTOR can regulate the proliferation of hematopoietic stem cells and progenitor cells [285]. Loss of mTOR results in endothelial cell death [201]. Similar to neural stem cells that are dependent upon growth factors and mTOR for survival, failed endothelial progenitor cell development may be the result of decreased growth factor signaling and loss of mTOR activity [201]. Insulin-like growth factor relies upon mTOR and Akt for the development of cardiac progenitor cells [286]. mTOR may be required for endothelial progenitor cells to initiate angiogenesis that can subsequently offer neuroprotection during cerebral ischemia [287]. In some studies, mTOR activity has been demonstrated to have a dual role. mTOR not only leads to stem cell proliferation, but also promotes apoptotic cell death, suggesting that a defined degree of mTOR activity is necessary to maximize stem cell viability [288]. For the maintenance of hematopoietic stem cells with blocked differentiation, reduction in mTOR signaling with decreased phosphorylation of p70S6K is required [289].

1.9 mTOR, Stem Cells, and Tumorigenesis

Strongly associated with proliferative pathways, mTOR also may lead to detrimental effects and promote tumor cell growth through cancer stem cell proliferation. mTOR activity is associated with neurofibromatosis type 1, tuberous sclerosis, Lhermitte–Duclos disease, and glioblastoma multiforme [29]. As a result, the US Food and Drug Administration (FDA) has approved rapamycin (sirolimus) and several rapamycin derivative compounds, known as rapalogs, that inhibit mTOR for the treatment of subependymal giant cell astrocytoma associated with tuberous sclerosis, renal cancer, and neuroendocrine pancreatic tumors [116,139].

In regards to stem cell proliferation that can foster tumor growth, neuronal activity that promotes high-grade glioma proliferation in neural precursor and oligodendroglial precursor cells has recently been linked to mTOR activity (Figure 1.1) [290,291]. Blockade of mTOR activity can prevent the conversion of astrocytoma cells to oligodendroglioma cells that foster the development of glioblastoma multiforme [292]. Inhibition of mTOR signaling may limit the population of cancer stem cells that can lead to disease recurrence and therapeutic resistance [293]. For example, activation of mTOR has been correlated with chemotherapy resistance of breast cancer cells with stem cell characteristics [294]. Furthermore, in nasopharyngeal carcinoma, cancer stem cell properties are reduced and invasion potential is restrained with the inhibition of mTOR signaling [295].

However, the role of mTOR during tumor growth is complex since mTOR signaling is linked to other proliferative pathways. An example of this involves the Wnt signaling pathway. During injury paradigms, Wnt signaling can be cytoprotective [239,241,296–298]. Activation of Wnt pathways can block inflammatory cell loss during neurodegenerative disorders [187,192,205,299], limit cerebral ischemia [300,301], protect cells during experimental DM [57,58,302], foster neurogenesis [303] and stem cell differentiation [304], and promote wound healing [305]. Growth factors also require Wnt to prevent cerebral endothelial cell injury [58], limit apoptosis during forkhead transcription factor activation [57,306], preserve mesenchymal stem cells [244], maintain immune cells in the nervous system [204], and block Aβ toxicity in cerebral microglia [205]. However, Wnt signaling may lead to glioma proliferation [307,308], metastatic disease [309–312], and malignant melanoma [313]. Prolonged exposure of growth factors such as EPO that rely upon Wnt signaling can have detrimental effects including the proliferation of cancer [314–316] and blood–brain barrier injury [317]. In addition, downstream pathways of Wnt, such as WISP1, can promote distant metastatic disease [318]. Variants of WISP1 are aggressive in promoting cell growth [319]. In contrast, nonvariant WISP1 expression can block tumor cell invasion, motility, and metastatic disease [320]. Under these scenarios of tumor cell growth with Wnt signaling, mTOR activity may represent a biological checkpoint that can limit excessive cell growth that is promoted by Wnt signaling. For example, mTOR activation maintains cell senescence and prevents tumor cell growth that is normally fostered by Wnt [321].

1.10 Future Considerations

The 289-kDa serine/threonine protein kinase mTOR oversees stem cell development, maintenance, proliferation, and differentiation in multiple systems of the body. In human embryonic stem cells, mTOR controls undifferentiated stem cell growth as well as the processes that lead to stem cell differentiation. During metabolic disorders such as DM, activation of mTOR may be necessary for proper pancreatic β-cell function and insulin sensitivity. However, a limited level of mTOR activity with SIRT1 activation is required to restore glucose homeostasis, foster mesangial cell proliferation, promote endothelial progenitor cell number, and allow for vascular repair mechanisms during DM. In the nervous system, mTOR activity is necessary for lineage expansion, cell differentiation, and neuronal cell production. During aging, mTOR activity is diminished and leads to a reduction in the proliferation of active neural stem cells. In the cardiovascular system, mTOR regulates proliferation of hematopoietic stem cells, cardiac progenitor cells, and endothelial progenitor cells that are necessary for angiogenesis. mTOR also plays a prominent role in tumorigenesis through cancer stem cell maintenance, proliferation, and invasion. The function of mTOR in cancer is complex, since at times mTOR can serve to block tumor cell growth.

In the development of stem cell strategies for NCDs, several considerations for mTOR need to be addressed to move forward. First, what are the pathways of programmed cell death that would be most conducive to promote stem cell maintenance and proliferation? Under a number of conditions, mTOR activation can prevent apoptotic cell death and may assist growth factor protection of stem cells through the blockade of apoptosis. However, some populations of stem cells, such as cortical neurons, use autophagic pathways for cell protection that inhibit mTOR. In combination with SIRT1 activation and inhibition of mTOR, autophagy can also preserve human embryonic stem cells exposed to oxidative stress and promote neuronal cell growth. However, the pathways of autophagy associated with mTOR are not straightforward. Activation of autophagy with mTOR inhibition can also result in stem cell demise. Autophagy may lead to cellular senescence and result in endothelial progenitor cell dysfunction. Further necessary analysis may involve the investigation of the role of pathways such as Wnt signaling and WISP1 that also employ mTOR signaling and can promote stem cell survival, but under some circumstances do not promote excessive cellular proliferation.

Another challenge for targeting mTOR involves the ability to direct stem cell differentiation. How can mTOR activity be finely controlled and balanced to oversee stem cell differentiation and maintain pluripotency? Absent or limited mTOR activity leads to cell pluripotency and cell proliferation. In contrast, activation of mTOR leads to stem cell differentiation, stem cell senescence, and potentially stem cell depletion.

Cellular metabolism for stem cell survival also comes into play with mTOR. mTOR activation can improve insulin secretion, reduce insulin resistance, and promote β-cell function and mass. Yet, mTOR activity as part of a feedback loop also has been shown to lead to inhibitory phosphorylation of IRS-1, impair Akt signaling, and promote insulin resistance in studies with high-fat diets. Furthermore, during mTOR inhibition in DM, SIRT1 can attenuate hepatic steatosis, ameliorate insulin resistance, restore glucose homeostasis, and promote mesangial cell proliferation. Control of mTOR activity and its function in feedback mechanisms must be considered during both physiological glucose homeostasis and during disorders of metabolism to effectively maintain stem cell survival and proliferation.

Similar considerations also must be addressed for mTOR and its impact on other biological systems and disorders as well. In the nervous system, mTOR activation increases lineage expansion, leads to cell differentiation, increases the neuronal cell population, and controls the timing of neurogenesis. However, mTOR activity in neighboring cells, such as endothelial cells, can also influence the neuronal population and lead to detrimental effects that result in the expansion of long-term glioblastoma stem-like cells. mTOR activation in the cardiovascular system is also required for endothelial progenitor cell and cardiac progenitor cell development. However, mTOR activity in the cardiovascular system can have a dual role that not only leads to cell proliferation, but also results in apoptotic cell death, implying that a defined degree of mTOR activity is necessary to maximize stem cell viability. As a proliferative agent, mTOR can also promote tumor cell growth through cancer stem cells. mTOR inhibition may be necessary to block cancer stem cell growth that can lead to disease recurrence and therapeutic resistance. However, mTOR holds a complicated role in tumor cell growth, since it is linked to other proliferative cell pathways necessitating careful governance of the cellular growth properties for mTOR. mTOR may function as a biological checkpoint by maintaining cell senescence and blocking tumor cell growth that is normally fostered through proliferative pathways such as Wnt signaling.

Acknowledgments

This research was supported by the following grants to Kenneth Maiese: American Diabetes Association, American Heart Association, NIH NIEHS, NIH NIA, NIH NINDS, and NIH ARRA.

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