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Ganoderma lucidum stimulates autophagy-dependent longevity pathways

The medicinal fungus Ganoderma lucidum is used as a dietary supplement and health tonic, but whether it affects longevity remains unclear. We show here that a water extract of G. lucidum mycelium extends lifespan of the nematode Caenorhabditis elegans. The G. lucidum extract reduces the level of fibrillarin (FIB-1), a nucleolar protein that correlates inversely with longevity in various organisms. Furthermore, G. lucidum treatment increases expression of the autophagosomal protein marker LGG-1, and lifespan extension is abrogated in mutant C. elegans strains that lack atg-18, daf-16, or sir-2.1, indicating that autophagy and stress resistance pathways are required to extend lifespan. In cultured human cells, G. lucidum increases concentrations of the LGG-1 ortholog LC3 and reduces levels of phosphorylated mTOR, a known inhibitor of autophagy. Notably, low molecular weight compounds (<10 kDa) isolated from the G. lucidum water extract prolong lifespan of C. elegans and the same compounds induce autophagy in human cells. These results suggest that G. lucidum can increase longevity by inducing autophagy and stress resistance.

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Aging is a malleable process that can be modulated by genes, diet and lifestyle [1]. For instance, rare mutations and single nucleotide polymorphisms (SNPs) in pathways involving forkhead box O3A (FOXO3A) and insulin-like growth factor-1 (IGF-1) receptor are associated with extended lifespan in centenarians [2, 3]. Caloric restriction (CR) also extends lifespan in various species including yeasts, nematodes, fruit flies, rodents and monkeys [46]. Given that animals fed CR diets usually consume their daily allocated food rapidly and in a single serving, the effects of CR may in reality be due to long periods of food abstinence (>16 hrs/day; i.e., intermittent fasting) [5, 7]. Similarly, exercise improves health markers and is widely believed to improve the healthspan and lifespan in humans [8]. Many plant and fungal compounds found in the diet, such as polyphenols, terpenoids and alkaloids also extend lifespan in model organisms and produce health benefits in humans [912]. Anti-aging interventions improve health and longevity by activating stress resistance pathways in the host via hormesis, which posits that low amounts of intermittent stress can improve cellular functions and produce health benefits, while higher levels of stress are detrimental [11, 1315]. The organism adapts in response to mild stress by reducing the amount of energy and resources allocated to growth and reproduction, and instead uses the limited resources to improve cell maintenance and survival [16]. Accordingly, CR, intermittent fasting, exercise and phytochemicals activate cellular pathways that induce autophagy, DNA repair, mitochondrial biogenesis and expression of antioxidant and detoxifying enzymes, which together improve cellular and organ functions [4, 7, 17]. Notably, autophagy plays a critical role in the anti-aging interventions identified to date by degrading damaged proteins and organelles in a process that is analogous to cellular recycling [18]. Given that the anti-aging lifestyle interventions may be difficult to implement on a daily basis, considerable interest has been devoted to identifying molecules that induce autophagy and promote longevity (i.e., CR mimetics). Many candidate compounds in this category have been isolated or derived from natural sources such as aspirin, metformin, rapamycin, glucosamine, polyphenols, and spermidine [10, 19]. Since CR mimetics such as aspirin and rapamycin can produce unwanted side effects, identification of new and safe CR mimetics is needed. In this context, Caenorhabditis elegans represents a useful model for identifying CR mimetics from natural health products due to its short lifespan, ease of manipulation, and tractable genetics [20], even though many precautions need to be considered [21]. Ganoderma lucidum (GL), also known as lingzhi or reishi, is a fungus with a long history of use in Asia as a tonic to improve health and vitality [22]. Recent research shows that GL produces various health benefits in animal models, including anti-inflammatory, anti-diabetic and anti-cancer effects [23, 24]. While screening for bioactive compounds derived from medicinal fungi, we observed that high molecular weight polysaccharides isolated from GL mycelia reduced obesity, inflammation, insulin resistance and fatty liver disease in mice fed with a high-fat diet [25]. Similarly, another study showed that polysaccharides isolated from GL extended the lifespan of C. elegans through a process dependent on daf-16 [26], the ortholog of the human FOXO transcription factors involved in stress resistance. Finally, another report demonstrated that a water extract of GL prolonged the lifespan of C. elegans by producing antioxidant effects and modulating germline signaling [27]. Nonetheless, the mechanisms whereby GL compounds may affect aging in animal models and human cells remain poorly understood. In the present study, we showed that a water extract of GL and a sub-fraction containing polysaccharides, oligosaccharides and other low-molecular-weight compounds promote longevity in nematodes by inducing autophagy. Notably, GL and the same sub-fraction also induce autophagy in human cells, indicating that the effects produced by the fungus may be conserved across different species.

Results G. lucidum reduces FIB-1 protein levels in nematodes Earlier work showed that rRNA synthesis and nucleolar size are inversely correlated with lifespan in C. elegans, fruit flies, mice, and humans [28]. Accordingly, the abundance of the fibrillarin protein FIB-1—a nucleolar rRNA 2’-O-methyltransferase involved in processing of pre-rRNA—negatively correlates with lifespan and can thus be used as a marker to study longevity [28]. We used a transgenic nematode strain (SJL1) described earlier [20, 29] which expresses a FIB-1 construct coupled with green fluorescent protein (GFP; Supplementary Figure 1A, 1B) to identify natural products that affect aging. Under fluorescence microscopy, we observed that GL reduced FIB-1::GFP intensity in a dose-dependent manner, compared with water control (Figure 1A). GL also reduced FIB-1 protein levels in wild-type N2 nematodes as assessed by Western blotting (Figure 1B, 1C). As a positive control, rapamycin also reduced FIB-1 protein levels in these assays (Figure 1A1C), consistent with the anti-aging effects produced by this compound in various organisms [18]. G. lucidum extends lifespan in C. elegans To determine whether G. lucidum can influence longevity, we measured the effects of GL on the lifespan of C. elegans. In these experiments, nematodes were fed with UV-killed Escherichia coli to prevent the possibility that GL may affect bacterial growth and energy intake. Consistent with the results shown above for FIB-1, GL used at 2 mg/plate extended median lifespan of C. elegans from 13.0 ± 2.8 to 18.5 ± 6.3 days compared to control water, representing a 45% extension (Figure 1D and Supplementary Table 1). In this case, the low GL concentration (2 mg/plate) was more effective than the high concentration (20 mg/plate) (Figure 1D and Supplementary Table 1), consistent with a hormetic dose response [11, 15]. Notably, the lifespan extension produced by GL (2 mg/plate) was more pronounced than the extension induced by rapamycin, which extended median lifespan to 16.0 ± 2.8 days (Figure 1D and Supplementary Table 1). GL also prolonged maximum lifespan from 18.8 ± 1.6 to 28.9 ± 2.3 days (Figure 1D and Supplementary Table 1, 20 mg/plate). Of note, treatment with GL did not affect pharyngeal pumping (Figure 1E, up to a concentration of 10 mg/plate), indicating that the extract did not extend lifespan by reducing food intake. In control experiments, both GL and rapamycin increased the number of nematodes that moved out of the bacterial lawn containing the tested substance (Supplementary Figure 1C). In addition, nematodes fed GL were larger than controls (Figure 1F), while their triglyceride content was reduced (Supplementary Figure 1D), producing changes similar to rapamycin (Figure 1F and Supplementary Figure 1D). G. lucidum extends lifespan by inducing autophagy Given that CR mimetics can usually activate autophagy [19], we examined the possibility that GL may extend lifespan by inducing this cellular process. We tested the effects of GL in mutant nematodes that lack atg-18, which encodes a protein required for autophagy [18]. While GL extended lifespan in wild-type N2 nematodes (Figure 2A and Supplementary Table 1), no lifespan extension was observed in nematodes lacking atg-18 (Figure 2B and Supplementary Table 1), indicating that autophagy is required for lifespan extension. Furthermore, GL did not extend lifespan in mutant worms lacking either daf-16 or sir-2.1 (Figure 2C, 2D, and Supplementary Table 1). Moreover, GL increased expression of the protein LGG-1 (Figure 2E2H), the ortholog of mammalian light chain 3 (LC3), which is involved in autophagy [18]. That is, transgenic DA2123 nematodes expressing GFP::LGG-1 showed higher numbers of LGG-1+ puncta following GL treatment compared to water-treated controls in which fluorescence was diffuse (Figure 2E, 2F). In these experiments, a hormetic dose response involving peak stimulation of autophagy at low concentrations (0.2 mg/plate) and lower levels of activation at high concentrations (20 mg/plate) was also observed (Figure 2F). Western blotting assays also revealed that GL treatment increased GFP::LGG-1 levels in transgenic DA2123 nematodes in a manner like rapamycin (Figure 2G, 2H). These results indicate that G. lucidum prolongs lifespan by activating autophagy and stress resistance pathways in nematodes. G. lucidum induces autophagy in human cells We tested whether GL induced autophagy in the human hepatoma Huh7 cell line, which is routinely used to assess this cellular process. Cell viability assays showed that GL did not affect the viability of Huh7 liver cells at doses of 0.5 to 2% (Supplementary Figure 2A). For IMR-90 lung fibroblasts, a non-cancerous cell line often used in aging-related studies, GL did not affect viability at doses of 0.5 and 1%, but cytotoxic effects were observed at 2% (Supplementary Figure 2C). Western blot analysis performed on Huh7 cells showed that GL decreased the level of phosphorylated mTOR (p-mTOR; Figure 3A, 3B), which acts as a repressor of autophagy [18]. Similarly, GL at a dose of 2% slightly reduced the phosphorylation of insulin receptor substrate-1 (p-IRS-1) (Figure 3A, 3B), an upstream activator of mTOR [18]. Furthermore, GL reduced phosphorylated GSK-3β (p-GSK-3β), but increased phosphorylation of Akt on serine 473 (p-Akt; Figure 3A, 3B), possibly due to a negative feedback mechanism, as observed earlier in various cancer cell lines treated with mTOR inhibitors such as everolimus [30] and rapamycin [31]. Similar results were obtained for p-mTOR and p-IRS-1 in IMR-90 fibroblasts, although no statistically significant changes were observed for p-Akt and p-GSK-3β in these cells (Figure 3C, 3D). Treatment of Huh7 cells with GL increased conversion of LC3B-I to LC3B-II (Figure 4A), which is used as a marker to monitor autophagy activation. As expected, treatment with the autophagy inhibitor, 3-methyladenine, reduced GL-induced LC3B-II protein accumulation (Figure 4A). On the other hand, GL did not affect the phosphorylation of other autophagy-related proteins, such as Beclin-1 and p38/p44/42 mitogen-activated protein kinases (MAPKs; Supplementary Figure 3A, 3B). Using Huh7 cells that express monomeric red fluorescent protein (RFP)-LC3 and GFP-LC3 [32], we observed that GL induced the formation of autophagosomes (consisting of RFP+ GFP+ puncta) and autolysosomes (RFP+ GFP– puncta, as GFP-LC3 fluorescence is quenched in autolysosomes) (Figure 4B, 4C; see also Supplementary Figure 3C, 3D). Similar results were obtained in IMR-90 lung fibroblasts stained with dyes that label autolysosomes and autophagosomes (Supplementary Figure 4A, 4B). Notably, GL also increased the number of autolysosomes as revealed by transmission electron microscopy (TEM), while the number of autophagosomes was not significantly affected in this case (Figure 4D, 4E). These observations indicate that GL can induce autophagy in cultured human cells. Effects of G. lucidum sub-fractions in C. elegans and human cells We used ultrafiltration to prepare sub-fractions containing polysaccharides, oligosaccharides and small compounds from GL (Supplementary Tables 25), and tested the ability of these sub-fractions to modulate FIB-1::GFP protein levels in transgenic SJL1 C. elegans. Sub-fraction 10K-1 containing high molecular weight compounds and polysaccharides (>10 kDa) did not affect FIB-1::GFP protein levels in the treated nematodes, whereas sub-fraction 10K-2 containing polysaccharides, oligosaccharides and other compounds of low molecular weight (<10 kDa) significantly reduced FIB-1::GFP protein levels, producing effects similar to that of rapamycin (Figure 5A). In lifespan assays, sub-fraction 10K-2 (2 mg/plate) extended median lifespan in nematodes, whereas 10K-1 did not produce statistically significant results (Figure 5B and Supplementary Table 1). Consistent with these results, sub-fraction 10K-2 increased levels of GFP::LGG-1 in transgenic DA2123 nematodes, while sub-fraction 10K-1 had no effect (Figure 5C). In human cells, sub-fractions 10K-1 and 10K-2 did not have a negative effect on cell viability (Supplementary Figure 2B, 2D). Sub-fraction 10K-2 reduced levels of p-mTOR and p-IRS-1 in Huh7 cells, while p-Akt and p-GSK-3β were unaffected (Figure 6A, 6B). In IMR-90 fibroblasts, sub-fraction 10K-2 reduced p-mTOR, p-IRS-1, p-Akt, and p-GSK-3β levels (Figure 6C, 6D). Notably, sub-fraction 10K-2 induced autophagy in human cells, as revealed by the increased number of autophagosomes (RFP+ GFP+ puncta) and autolysosomes (RFP+ GFP– puncta; Figure 7A, 7B; see also Supplementary Figure 3E, 3F). These results were confirmed in IMR-90 lung fibroblasts stained with dyes that label autolysosomes and autophagosomes (Supplementary Figure 4A, 4B). Similarly, 10K-2 increased autolysosomes in Huh7 cells based on TEM analysis (Figure 7C, 7D). Sub-fraction 10K-1 also induced autophagy in some of these experiments (Figure 7A7D, and Supplementary Figure 3E, 3F), but mTOR signaling was not affected in this case (Figure 6A6D).

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