Maximum lifespans differ up to 100-fold between extant mammalian species, ranging from 211 years in the bowhead whale to 2.1 years in the forest shrew (Myosorex varius) [24]. Recent studies have shown that extremely long-lived species, such as African elephants, bowhead whales, brandt's bats and naked mole rats, have evolved their unique mechanisms of lifespan extension. However, the shared mechanisms underlying mammalian longevity are complex and still not fully understood. In this study, we performed molecular evolutionary analyses of 72 genes involved in mTOR network across 48 mammals, and identified the potential mechanisms underlying extending lifespan in mammals. Our results revealed 12 positively selected genes (TSC2, TSC1, RAPTOR, PIK3CA, PDPK1, ATP6V1C2, WDR24, DEPDC5, NPRL3, LAMTOR2, IGF1R and PRKCB) and four convergent amino acid changes involved in four genes (SESN2, ATP6V1H, IRS1 and TTI1) were unique to long-lived species. Evolutionary rates of five genes (MLST8, LAMTOR4, EIF4E2, AKT2, and TSC2) were significantly related to the MLS. Combined, a total of 20 genes (with one overlapped gene, TSC2) were identified to have significant evolution signs in the long-lived mammals, which may help explain the role of mTOR pathway in the evolution of mammalian lifespan extension.
Enhanced autophagy to resist aging-related diseases in long-lived mammals
One hallmark of aging is the accumulation of various of damaged organelles, misfolded protein or DNA mutations, as well as the incidence of chronic diseases such as neurodegeneration rises with age [16]. Autophagy is a conserved physiological process that relies on lysosomes to degrade intracellular macromolecules (such as excess or defective proteins and organelles), which is essential for the organism to maintain its homeostasis [17]. It was shown that enhanced autophagy was associated with extended lifespan in model organisms (such as worm and mouse) [18], and increasing evidences indicated that seveal genes (including LAMTOR4, LAMTOR2 and NPRL3) involved in mTOR network regulated autophagy and neurodegenerative diseases [25, 26].
In this study, we found that four positively selected genes (PRKCB, WDR24, NPRL3 and LAMTOR2) and one longevity-associated genes (LAMTOR4) in long-lived species were involved in the autophagy response and aging-related diseases. LAMTOR (also known as Ragulator) is a scaffold protein complex that regulates mTOR signaling in response to amino acids, thus influencing cell growth and proliferation. LAMTOR2, a subunit of LAMTOR, is involved in autophagy and functions as a selective autophagy regulator by mediating autophagosome–lysosome fusion [27]. Similarly, NPRL2 and NPRL3 were also involved in regulation of autophagy as deletions of both homologous genes resulted in significant defects in nitrogen-starvation-induced autophagy in yeast [28]. Researches from fruit flies and zebrafish have demonstrated that WDR24 plays a role in regulating autophagy initiation [29]. For instance, WDR24 knockdown results in developmental defects, at least in part, due to dysregulated autophagy, which could be partially restored by WDR24 overexpression [30]. By contrast, overexpression of PRKCB leads to an inhibition of autophagy [31]. Moreover, of five longevity-associated genes identified in our study, LAMTOR4 was revealed associated with aging diseases. LAMTOR4 mutants reduced the number of phagocytic cells — microglia, leading to weakened phagocytosis and reduced the clearance of Aβ protein in Alzheimer disease [32]. There is increasing evidence that autophagy is an effective neuroprotective mechanism and malfunctioning autophagy frequently leads to neurodegenerative diseases, such as Parkinson's disease or Alzheimer's disease [33]. Collectively, enhanced autophagy to resist aging-related diseases may be an important mechanism of life extension in long-lived mammals.
Lifespan extension through modulating cancer and aging genes
Previous studies have shown that several extremely long-lived species have evolved unique cancer resistance mechanisms to extend their lifespan. For example, cell contact inhibition induced by high-molecular-mass hyaluronan were responsible for the naked mole rat’s cancer resistance whereas the interferon-mediated ‘concerted cell death’ were believed to prevent bland mole rat’s tumor transformation [34]. In our study, eight of 20 genes with significant evolution signals were identified in long-lived species were known as cancer genes according to OncoKB database [35], including TSC1, TSC2, PIK3CA, RAPTOR, IGF1R, PDPK1, PRKCB and AKT2. For example, TSC2 (tuberous sclerosis complex 2) was a tumor suppression gene that was reported that specific mutations of this gene led to the development of renal and extra-renal tumors in mice [36]. Moreover, TSC1 and TSC2 form tumor suppressor complex to control cell growth and prevent disease [37]. In our study, both TSC1 and TSC2 genes were subject to positive selection special for long-lived species, and TSC2 evolutionary rate was significantly related to MLS. Another long-lived mammals-specific positive selection gene—RAPTOR (Regulatory associated protein of mTOR complex 1) was responsible for the induction of mTORC1 during tumorigenesis and progression, such as RAPTOR overexpression in colorectal cancer tissues and cell lines [38]. Notably, of the eight cancer-related genes, six (TSC1, TSC2, PIK3CA, IGF1R, PDPK1 and AKT2) are known senescence related genes according to GenAge database in human/model organisms [24], and genetic manipulation of these genes can directly lead to shortening or extending lifespan of model organisms. For instance, knockout of daf-2 (homologous to IGF1R in mammals) in nematodes (Caenorhabditis elegans), leads to doubled lifespan extension [39]. Overexpression of Tsc1 and Tsc2 in Drosophila extended mean lifespan by 14% and 12%, respectively [40]. As observed above, a number of genes related to cancer and aging were examined to have significant evolution signals in long-lived species, suggesting that mammals might increase lifespan by regulation cancer and aging related genes.
Convergent autophagy in long-lived species
Convergent autophagy in long-lived species explains the possible mechanism that contributing to extended lifespan. Our results exhibited that there were four convergent amino acid mutations at four genes (ATP6V1H, TTI1, IRS1, SESN2) in deeply distant long-lived species: the naked mole rat, blind mole rat, manatee, bats (Brandt's bat, Large-eared bat and Big brown bat) and primates (Human, Gorilla, Chimpanzee, Pygmy chimpanzee). Of them, three convergent amino acid sites of three genes located in key function domains (SESN2, ATP6V1H and IRS1). ATP6V1H was proven to participate in the formation of phagosomes and a recent comparative transcriptomes analysis showed that ATP6V1H was highly expressed in long-lived bats [8]. SESN2, known as stress-inducible protein, may function in the regulation of cell growth, survival and autophagy [41], overexpression of SESN2 can promote autophagy in neuroblastoma cells [42]. TTI1 is a highly conserved regulator of DNA damage response, which can maintain the stability of genome and therefore extend lifespan [43]. Two convergent genes in long-lived species in our study are strongly associated with autophagy, suggesting convergent autophagy might be another mechanism to extend lifespan in long-lived mammals. Of course, more experiments are needed to explore if the convergent site of autophagy gene contribute to longevity in the future.