Rapamycin For Longevity: series 4 part 1

Rapamycin For Longevity: series 4 part 1

Abstract:

In 2009, rapamycin was reported to increase the lifespan of mice when implemented later in life (see last article by Harrison et al). This study resulted in changes to how researchers viewed aging. Harrison et al’s study provided the first evidence that a pharmacological agent could have an impact on aging when administered later in life, i.e., an intervention that did not have to be implemented early in life before the negative impact of aging. Over the past decade, there has been an explosion in the number of reports studying the effect of rapamycin on various diseases, physiological functions, and biochemical processes in mice and other models. In this review, Selvarani et al will focus on those areas in which there is strong evidence for rapamycin’s effect on aging and age-related diseases in mice, e.g., lifespan, cardiac disease/function, central nervous system, immune system, and cell senescence. They advocate that it is time that preclinical studies be focused on taking rapamycin to the clinic, e.g., as a potential treatment for Alzheimer’s disease.

Introduction:

Rapamycin, also known by the trade names of sirolimus, everolimus, or rapamune to name a few, is a macrocyclic lactone produced by Streptomyces hygroscopicus, which was isolated from soil samples collected from Easter Island by Georges Nogrady in the late 1960s [1]. Scientists at Ayerst Pharmaceuticals in Canada discovered that Streptomyces hygroscopicus produced a compound that would kill fungi, which they named rapamycin after the native name of Easter Island, Rapa Nui. The initial interest in rapamycin focused on its antifungal properties. When it was found that rapamycin inhibited the growth of eukaryotic cells, research on rapamycin turned to rapamycin’s supposed immunosuppressive and anticancer properties. Rapamycin treatment was approved by the FDA in 1994 to prevent organ rejection in liver transplant patients. In addition to being used as an anti-rejection drug, rapamycin and its rapalogs are being used today to prevent restenosis after coronary angioplasty, and they are being tested in many clinical trials as antitumor agents, e.g., FDA approved the use of rapamycin in treatment of pancreatic cancer patients in 2011.

Research in the late 1980s turned to identifying the mechanism by which rapamycin blocks the growth of eukaryotic cells. Heitman et al [2] discovered the protein, target of rapamycin (TOR), in yeast that was responsible for rapamycin’s ability to inhibit growth. Three groups in 1994 independently identified the mammalian counterpart, mTOR [3–5]. TOR, itself a  serine/threonine kinase, was found to be a master-regulator in the response of eukaryotic cells to nutrients, growth factors, and cellular energy status, and this is now known as the TOR pathway or mTOR signaling pathway. Harrison et al [6] in 2009 reported that rapamycin increased the lifespan of both male and female heterogeneous mice with data garnered at three separate testing sites. This study was a major discovery in the field of gerontology. Please note, gerontology is the study of the physical and cellular changes of aging. In any case, Harrison et al’s study is important because its results are the first viable evidence produced that the lifespan of a mammal can be significantly increased by a pharmacological agent. The journal, Science, selected this study as one of the major scientific break-throughs in 2009 (Science 326, 1598–1607), the first discovery in aging to be selected by Science as a break-through. Over the past decade, there has been an explosion in the number of reports studying the effect of rapamycin on aging and age-related diseases, and there have been several reviews describing various aspects of rapamycin on aging [7–11]. In this article, Selvarani et al discuss, compile, and review the data that's been collected over the past decade on the effect of rapamycin on lifespan and age-related diseases.

Effect of rapamycin on the lifespan of mice

The first data suggesting that rapamycin might affect longevity came from studies with invertebrates, think species that lack a spine, like Caenorhabditis elegans. In 2003, Vellai et al [12] showed that a TOR mutation seemingly increased the lifespan of C. elegans and other groups showed that certain mutations in TOR increased the lifespan of yeast [13] and Drosophila[14]. Subsequently, it was found that rapamycin increased lifespan in yeast [15]. Based on this data, David Sharp (University of Texas Health Science Center at San Antonio) proposed that the NIA Intervention Testing Program tests the effect of feeding rapamycin to mice. The study was initiated in 2006, and in 2009, Harrison et al [6] reported the first data showing that feeding rapamycin (14 ppm or ~ 2.24 mg/kg based on average food consumption of mice) increased the lifespan of both male and female mice. Not only was this the first report to show that lifespan could be increased pharmacologically in both male and female mice, but more importantly, the increase in lifespan was observed when rapamycin was given to mice late in life (19 months). Until Harrison et al’s study, it was generally believed that initiating any intervention late in life would have minimal impact on longevity, i.e., treatment would need to be initiated early in life before major age-related decrements occurred and then the treatment  intervention would have to be maintained the rest of life. Interestingly, the increase in lifespan by rapamycin was similar when implemented at 4 months [16], 9 months [17], or 19 months [6] of age, seemingly irrespective of age. Since the initial report in 2009, there have been fourteen additional studies showing that rapamycin increased the lifespans of male and female mice. As shown in Table 1, the effect of rapamycin on lifespan is robust because it has been replicated in many different laboratories with different strains of laboratory mice, ranging from inbred strains (e.g., C57BL/6 and 129) to the UM-HET3 mice (a heterogeneous strain of mice generated by a 4-way cross) and with different rapamycin dosing regimens. Only the study by Bitto et al [20] reported that rapamycin had no effect on lifespan. At a high dose (8 mg/kg/day) given i.p., rapamycin had no effect on the lifespan of female mice; however, this dose and route of rapamycin increased the lifespan of male rats 61%. On the other hand, 126 ppm of rapamycin administration via dietary supplements increased the lifespan of both female and male mice 39% and 45%, respectively.

To date, there currently are no reports that show that rapamycin has a negative effect on the lifespan of normal, healthy, laboratory strains of mice.

Three additional points of interest with respect to rapamycin’s longevity effect can be seen from Table1. First, rapamycin is effective over a wide dose range. Even at high doses, it does not have a negative effect on lifespan. Second, rapamycin increases the lifespan of both male and female mice, which is wholly unique to rapamycin because all the other anti-aging interventions identified by the NIA Intervention Testing Program are sex specific, i.e., they significantly increased lifespan in one sex but have little or no effect on the other sex [18]. However, as the data in Table 1. show, the increase in lifespan is greater in female mice than male mice in those studies that have compared the effect of rapamycin on lifespan in both males and females. However, this difference becomes minimized at high doses of rapamycin [19, 20]. Thus, it appears that female mice tend to be more sensitive to the life-extending actions of rapamycin. Third, the study by Bitto et al [20] showed that only 3 months of a high dose of rapamycin (126 ppm) late in life was able to increase lifespan dramatically, again pointing to the late life benefits of rapamycin as well as showing that rapamycin need not be continuously administered to mice for a beneficial effect.Table 2. lists the studies showing that rapamycin significantly increased the lifespan of genetic mouse models that mimic various diseases found in humans. Most of these studies used mouse models of accelerated cancer, and as would be expected, these studies show an increase in lifespan as well as reduction in the progression of the neoplastic tumors specific to each model. These studies are described in more detail below. In addition, five studies have used mouse models of other disease phenotypes. As a side note, when denoting mice strains such as “Lmna−/−” the “Lmna” denotes the specific gene modified and the “−/−'' is used to describe the specific genotype; with “+” denoting a working copy of the allele and “-” denoting a non-working copy of the allele. Because mice are diploids they have two alleles per gene, hence the mice with two non-workings copies of the Lmna gene, a complete gene knockout, are denoted as “−/−”. Furthermore “+/-” denotes heterozygosity, “+/+” denotes functional homozygosity, and “-/-'' denotes a non-functional homozygosity due to either a non-functional but present gene or a complete absence of the gene itself. Continuing on, using a mouse model (Lmna−/−) that mimics Hutchinson-Gilford progeria, a disease that is characterized by rapid and premature aging. Ramos et al [21] showed that rapamycin increased lifespan over 50% and improved cardiac and skeletal muscle function in the Lmna−/− mice. Khapre et al [22] studied the effect of rapamycin on the lifespan ofBmal1−/− mice because knocking out Bmal1, a transcription factor that is key to the circadian clock,  increased mTORC1 activity and reduced lifespan and disrupted circadian rhythm. They showed that rapamycin increased the lifespan of the Bmal1−/− mice leading Khapre et al [22] to propose that the regulation of mTORC1 activity by Bmal1 is key to the circadian clock. Two groups have studied the effect of rapamycin on mouse models with mutations leading to mitochondrial dysfunction which leads to a variety of debilitating mitochondrial diseases due to mitochondrial function being so integral for any given normal healthy life. Siegmund et al [23] studied TK2KI/KI mice, which have a nuclear mutation in the mitochondrial nucleotide salvage enzyme thymidine kinase resulting in reduced replication of mtDNA and an increase in mtDNA instability. Rapamycin dramatically increased the lifespan of the TK2KI/KI without having any detectable improvement in mitochondrial dysfunction. The authors concluded that rapamycin enhanced longevity in the TK2KI/KI mice through alternative energy reserves and/or triggering indirect signaling events. Johnson et al [7] initially showed that rapamycin attenuated mitochondrial disease symptoms and progression in Ndufs4−/− mice, which lack a subunit in mitochondria complex I and is a mouse model of Leigh syndrome. Leigh syndrome is characterized by the onset usually occurring in the first year of life and is marked by a rapid decline in cellular function as well as seizures, altered states of consciousness, dementia,  and ventilatory failure. Johnson et al [24] subsequently showed that rapamycin increased the lifespan of the Ndufs4−/−mice, especially at very high doses of rapamycin, which were 28-fold higher than the dose of rapamycin initially showed to increase the lifespan of mice by Harrison et al [6]. Reifsnyder et al [25] studied a mouse model of type 2 diabetes, the BKS-Leprdb mouse. They found that rapamycin doubled the lifespan of female mice but had no detectable effect on the lifespan of male mice. Rapamycin improved both kidney and cardiac functions in the female BKS-Leprdb mice.

Although the overwhelming majority of studies on the effect of rapamycin on longevity in mice have shown a significant increase in lifespan, there are five studies that have reported either no effect or reduced lifespan when treated with rapamycin. Two studies using transgenic mouse models of amyotrophic lateral sclerosis (G93A and H46R/H48Q) reported no increase in lifespan when given rapamycin [26, 27]. Sataranatarajan et al [28] reported that 14 ppm, a dose of rapamycin that increases the lifespan of C57BL/6 mice, reduced the lifespan of the obese and diabetic C57BL/KsJleprdb/db mice, 13% in males and 15% in females. The reduced lifespan of the db/db mice by rapamycin was associated with an increase in suppurative inflammation, which was the primary cause of death in the db/db mice. Ferrara-Romeo et al [29] reported that 42 ppm rapamycin reduced the lifespan of telomerase-deficient mice (G2-Terc−/−) 16% compared with over a 50% increase in the lifespan of the G2-Terc+/+ mice. Fang et al [30] found that rapamycin reduced the lifespan of growth hormone receptor knockout (GHR-KO) mice (15% for males and 5% for females) even though the same dose of rapamycin increased the lifespan of the wild-type, control mice. The reduced lifespan of the GHR-KO mice was associated with impaired glucose and lipid homeostasis and increased inflammation. In essence, the overwhelming amount of evidence produced by these studies support the theory of rapamycin’s lifespan extending effects.

Effect of rapamycin on cancer in mouse models

Rapamycin is predicted to reduce the progression of cancer because it has been shown to inhibit cell growth and proliferation. In addition, mTOR is frequently over-activated in cancer, and mTORC1 has often been observed to be deregulated in a wide variety of human cancers [31]. Data generated in the 3 years after the discovery that rapamycin increased lifespan in 2009 by Harrison et al showed that rapamycin and rapalogs (e.g Sirolimus, everolimus, temsirolimus, ridaforolimus, etc.) reduced the efficacy of various cancers induced in mice. A few of these studies are summarized in Table 3. and show that mTOR inhibitors have an antineoplastic effect on a broad range of cancers. Note, antineoplastics are a class of drugs and compounds used to treat cancer, such as chemotherapy. For example, Rivera et al [32] studied the effect of ridaforolimus on the growth of various human tumor xenografts, grafts of tissue from a donor of a different species, in mice. They showed that the administration of ridaforolimus inhibited the growth of prostate (PC-3), colon (HCT-116), breast (MCF7), lung (A549), and pancreas (PANC-1) cancer cells. These results help solidify rapamycin’s proposed anti-cancer abilities.

The data in Table 2. shows the effect of rapamycin and its analogs on the survival of various mouse models with genetically engineered mutations in genes involved in cancer. Particularly striking are the three studies with APCMin/+ (ApcD716) mice, which are a mouse model of human colorectal cancer. Most human colorectal cancers have somatic mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, and APCMin/+ mice develop multiple intestinal neoplasia. The APCMin/+ mice are relatively short lived, living a maximum of ~ 200 days compared with 800 to 900 days for normal laboratory mice. Three groups [33–35] showed that treating APCMin/+ mice with rapamycin or everolimus reduced intestinal neoplasia with regards to polyp number and size in the APCMin/+ mice. In addition, these studies showed that rapamycin or everolimus dramatically increased the lifespan of the APCMin/+ mice. For example, Hasty et al [35] found that a high level of rapamycin (42 ppm) resulted in a lifespan longer than that observed in normal laboratory mice, over a fourfold increase in lifespan of the APCMin/+ mice.

Three groups have studied the effect of rapamycin on mice with deletions in the p53 gene, a transcription factor with broad biological functions, including as a tumor suppressor in humans [36]. Komarova et al [37] and Christy et al [38] found that rapamycin treatment resulted in a modest, but significant increase in the lifespan of p53+/− mice. Komarova et al [37] reported that rapamycin reduced the incidence of tumors in the p53+/− mice; however, Christy et al [38] did not observe any significant changes in tumor incidence in p53+/− mice treated with rapamycin. Comas et al [39] reported that rapamycin increased the lifespan of p53−/− mice; however, Christy et al [38] did not observe a significant increase in the lifespan of their studies  p53−/− mice.

Two reports have described the effect of rapamycin on transgenic mice overexpressing Her-2/neu. These mice are classified as transgenic because foreign genetic material has been artificially introduced. Additionally, HER2 is a member of the human epidermal growth factor receptor family and amplification/overexpression of this oncogene, a class of genes that have the capacity to cause cancer, has been unsurprisingly shown to play a role in certain types of breast cancer. Rapamycin treatment resulted in a modest, but significant increase in the lifespan of Her-2/neu transgenic mice [40, 41] and dramatically delayed the incidence of tumors in the Her-2/neu transgenic mice. Hernando et al [42] reported that everolimus dramatically increased the lifespan of Ptet−/− mice, a model of leiomyosarcomas, a cancer of smooth muscle. The Ptet−/− mice develop widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, and everolimus significantly reduced the growth rate of these tumors. Livi et al [43] studied the effect of rapamycin on Rb1+/−mice. The retinoblastoma gene (Rb1) was the first tumor suppressor gene identified in humans and prevents excessive cell growth by inhibiting cell cycle progression. Rapamycin increased the lifespan of the Rb1+/− mice and reduced the incidence of thyroid C cell carcinomas as well as delaying the appearance and reducing the size of pituitary tumors. Hurez et al [44] studied the effect of rapamycin on immunocompromised, cancer prone Rag2−/−, and IFN-γ−/− mice. Cancer immune surveillance is reduced in these two mouse models and rapamycin increased the lifespan of both Rag2−/− and IFN-γ−/− mice; however, no data were presented on the effect of rapamycin on the actual incidence of tumors in these mice.

Effect of rapamycin on cardiac function and disease in mice

The first indication that rapamycin might be important for the heart was the discovery that coronary stents coated with rapamycin prevented restenosis and stent thrombosis compared with non-coated or other drug-eluting stents [45], which led to FDA approval of rapamycin-coated coronary stents in 2003. Subsequently, two other mTOR inhibiting rapalogs, everolimus and zotarolimus, are currently used to prevent restenosis and thrombosis in patients who require coronary stents [46].

The effect of rapamycin and rapalogs on the cardiovascular system initially was not clear, especially in humans. In clinical studies with transplant patients, rapalogs induced a negative plasma cardiovascular risk profile, e.g., an increase in LDL cholesterol and triglyceride concentrations in plasma [47]. Rapamycin also has been reported to have deleterious effects on endothelial function which is the ability of a blood vessel to constrict and dilate. These deleterious effects on endothelial function were seen in laboratory animals and in human coronary arteries from sirolimus-eluting stents [48, 49]. Rapamycin also has been reported to accelerate cell-senescence within endothelial progenitor cells [50]; however, as described below, most of the recent studies indicate that rapamycin reduces cellular senescence. Overall, these early studies are in conflict with the large number of studies in mice listed in Table 4. that have studied the effect of rapamycin on atherosclerosis in mice.

Table 4. lists the studies that have examined the effect of rapamycin or everolimus on various aspects of heart disease and heart function in mice. Four groups have studied the effect of rapamycin or everolimus on the occurrence of atherosclerotic lesions in the aortic arch of either ApoE−/− or LDLR−/− mice fed a high-fat diet to induce atherosclerotic plaque formation which as plaque builds up the arterial walls lose flexibility by becoming thick and stiff. All four studies showed that rapamycin reduced aortic atheromas, and Jahrling et al [51] found that this was paralleled by an improvement in cerebral blood flow and vascular density in LDLR−/− mice fed a high-fat diet. Because treating transplant patients with rapalogs has been shown to increase blood levels of cholesterol and triglycerides, the four groups also measured blood levels of cholesterol in their mouse models of atherosclerosis. Three found that rapamycin treatment had no effect on blood levels of cholesterol or triglycerides groups [51–53]. Mueller et al [54] reported that the blood levels of LDL and VLDL cholesterol were slightly higher in the everolimus-treated mice but observed no change in triglycerides. It is interesting to note that Ross et al [55] observed no effect of rapamycin (1.0 mg/kg/day) treatment on blood triglyceride levels in the non-human primate, marmoset.

A large number of studies have evaluated the effect of rapamycin on cardiomyopathy and hypertrophy induced by physical, pharmacological, or genetic engineering in mice and rats. All nine studies show that rapamycin prevents or attenuates cardiomyopathy or hypertrophy in both mice and rats. Two studies examined the effect everolimus in rats or rapamycin in mice on myocardial infarction [56, 57]. Rapamycin improved cardiac function, reduced infarct size in rats, and reduced hypertrophy and fibrosis in mice.

The three studies on the effect of heart function in old mice are the most relevant to this review. The studies by Simon Melov’s group at the Buck Institute [58] and Peter Rabinovitch’s group at the University of Washington [59] showed that rapamycin treatment for 2.5 to 3 months attenuated cardiac dysfunction and reduced cardiac hypertrophy seen with age. In other words, short-term rapamycin treatment was able to reverse cardiac dysfunction and hypertrophy that occurred in the old mice. The ability of rapamycin to improve cardiac function is not limited to mice. Urfer et al [60] showed that giving rapamycin (0.1 mg/kg, 3 times/week) for 10 weeks to middle-aged companion dogs improved both systolic and diastolic cardiac function. Recently, Rabinovitch’s group showed that the improvement in diastolic function after 2 months of rapamycin treatment of old mice persisted for 2 additional months after rapamycin treatment was discontinued, demonstrating that rapamycin can have lasting effects on cardiac function even after it is discontinued [113].