| 其他摘要 | Anti-aging mechanisms have been a hot issue in the frontier of life science.In studies of terrestrial model organisms in different evolutionary status, genetic factors and environmental factors have been found to be determinants of longevity.The insulin / insulin-like growth factor-1 (Insulin/IGF-1signaling pathway, IIS) pathway is the most conserved aging pathway throughout evolution. Mutations affecting the functionality of core genes in this pathway can effectively extend organismal lifespan. However, extending lifespan through genetic mutations or mutagenesis often comes at the cost of compromising other health performances in the organisms. Therefore, it is crucial to study the natural mechanisms regulating lifespan extension without compromising health, which may provide new avenues for exploring the determinants of animal lifespan. The bay scallop Argopecten irradians and the Peruvian scallop A. purpuratus evolved from a common ancestor. In the process of long-term adaptation to different environments, genetic mutations in their respective genomes led to different life cycles. The lifespan of A. irradians was less than 14 months, while the lifespan of A. purpuratus live up to 7-10 years, making them ideal models for studying the mechanisms that determine lifespan under natural conditions. However, there are few reports on the mechanism of lifespan determination in marine invertebrates. Thus, this study aims to systematically characterize the structure, expression, and protein activity of four core genes (IGF2, IGF1R, PETN, and FoxO) of the IIS/FoxO pathway in both scallops, and to clarify the mechanism of core genes of IIS/FoxO pathway in nutrition sensing, genome stability and lifespan determination The main findings in this study are:
1. The genetic structures and spatiotemporal expression patterns of the core genes IGF2, IGF1R, PETN, and FoxO in A. irradians and A. purpuratus were systematically characterized, clarifying the large generic variation of core genes between the two scallops. Gene cloning revealed that the core genes of the IIS/FoxO pathway in two scallops are single-copy genes. Sequence homology alignment revealed numerous of cytosine (C) to thymine (T) transition mutations in AiIGF2 compared to ApIGF2, leading to the change of ptotential codon and increased hydrophobicity of the basic amino acids, which may contribute to the accumulation of functional altered peptides, resulting in a short-lived phenotype of A. irradians. Twenty-one SNPs and one specific glycosylation site were identified in the extracellular ligand-binding domain of AiIGF1R and ApIGF1R. Two amino acid variations were found near the 21st Akt phosphorylation site in AiFoxO and ApFoxO proteins, suggesting that this variation may affect the extent of Akt-mediated phosphorylation of FoxO proteins. Specifically, the 76th Thr-specific phosphorylation site of AiFoxO protein coincides with the DNA-binding domain, potentially influencing FoxO transcriptional activity. Amino acid variations occurred in the core region adjacent to the "HCxxGxxR" motif in AiPTEN and ApPTEN proteins, which may lead to differential phosphatase activity of PTEN. Additionally, the expression levels of IGF2 and IGF1R in the mantle and gonads of two scallops increased with age, while the expression levels of FoxO and PTEN decreased with age, suggesting that the mantle and gonads are representative tissues of scallop aging.
2. The effects of different genetic variations in IGF2, IGF1R, PETN, and FoxO on protein activity were analyzed in A. irradians and A. purpuratus. Along with organismal growth and development, the interaction intensity between IGF1R and IGF2 increased gradually, and the interaction intensity between IGF1R protein and ligand IGF2 was higher in short-lived A. irradians than that in long-lived A. purpuratus. The kinase activity and phosphorylation levels of IGF1R in two scallops showed a trend of initial increase followed by decrease with age, but the kinase activity and phosphorylation levels of IGF1R during the aging period remained higher than those during the juvenile period, indicating that IGF1R kinase actively responds to signals generated during scallop development to regulate growth, development, and survival. Moreover, the phosphatase activity of PTEN in A. purpuratus was consistently higher than that in A. irradians, and the phosphatase activity of PTEN in aging scallops was very low, indicating that PTEN inactivation mediates cellular aging. In A. irradians, the Akt phosphorylation level of FoxO protein increased with age, and compared to A. purpuratus, A. irradians exhibited higher phosphorylation of FoxO, suggesting that scallops require FoxO with lower phosphorylation levels to activate the expression of downstream longevity-related target genes, thereby extending organismal lifespan.
3. Combined with experiments in vivo and in vitro, the mechanisms of adaptive regulation of the IIS/FoxO pathway in A. irradians and A. purpuratus under nutritional sensing and ionizing radiation are clarified. After dietary restriction, the expression levels of core genes in the IIS/FoxO pathway changed consistently in both scallops. The expression levels of upstream signal sensors IGF2 and IGF1R were significantly downregulated, while the expression levels of downstream effectors FoxO and PTEN were significantly upregulated, with A. irradians showing a faster and greater response to dietary restriction. Dietary restriction led to a decrease in the phosphorylation level of stress response factor FoxO protein, an increase in nuclear localization, and activation of downstream antioxidant stress and autophagy-related target genes ULK2, ATG8, SOD, CAT, and GADD45, as well as a significant decrease in the cellular aging marker β-Gal activity, suggesting that the IIS/FoxO pathway participates in dietary restriction-mediated longevity regulation in A. irradians and A. purpuratus, with FoxO protein activity being essential for dietary restriction-mediated lifespan extension. After acute ionizing radiation, the survival rate of A. purpuratus was significantly higher than that of A. irradians. The expression levels of DNA damage repair-related genes ApFoxO, ApPTEN, ApGADD45 and ApP53 in A. purpuratus were significantly upregulated, while those in A. irradians were downregulated. However, the expression levels of autophagy-related genes AiULK2 and AiATG8 markedlky decreased, and pro-apoptotic gene AiBcl-2 expression signally increased in A. irradians, indicating that A. purpuratus have stronger damage resistance and repair capabilities during ionizing radiation, possibly by activating certain DNA damage repair mechanisms to maintain genomic integrity and delay organismal aging, while A. irradians may initiate programmed cell death due to their weaker damage repair mechanisms.
4. The expression of core genes IGF1R, PTEN and FoxO of IIS/FoxO pathway was silenced by RNAi technique, validating the cascade relationship of the genes in scallops and their roles in the scallop lifespan regulation. Meanwhile, the regulatory network of the IIS/FoxO pathway in scallop lifespan determination was comprehensively analyzed by GST pull-down combined with mass spectrometry and high throughput genomics. The silencing of ApPTEN resulted in a significant decrease in ApFoxO expression, indicating that PTEN positively regulates FoxO in scallops. After silencing the expression of FoxO and PTEN, the expression levels of downstream antioxidant target genes SOD and CAT were significantly decreased, the expression levels of stress resistance related genes GADD45 and GST and autophagy related genes ULK2 and ATG8 also showed the significant downward trend, and the activity of aging marker β-Gal was markedlly increased, indicating that FoxO and PTEN in scallops cooperated to regulate oxidative stress, thus positively regulating lifespan. After inhibition of IGF1R, the expression levels of secondary messenger Akt and PI3K in IIS/FoxO pathway were significantly downregulated, the expression of downstream effect factors PTEN and FoxO were observably increased, the activities of antioxidant enzymes SOD and CAT showed a significant increase, and the activity of aging marker β-Gal was markedly decreased, indicating that there was a hierarchical relationship between IGF1R, FoxO and PTEN, and silent expression of IGF1R could alleviate oxidative stress and delay the change of parameters related to aging in aging scallops. The mass spectrometric analysis of pull-down proteins showed that apoptosis inhibitor BIRC6 protein, glutathione peroxidase (GSHPx), DNA damage repair Rad50 protein and protease inhibitor SPINK1 were abundant. Transcriptome analysis showed that the expressions of Sirt, NADK2, NMNAT1 and NAMPT involved in nicotinic acid and nicotinamide metabolism, PANK2 involved in pantothenic acid and acetyl CoA metabolism and LIPA involved in lysosome degradation were significantly upregulated. At the same time, differentially expressed genes were enriched in multiple signaling pathways involved in longevity regulation, including AMPK signal pathway, FoxO signal pathway, PI3K/Akt signal pathway, NF-κB signal pathway, Sirt signal pathway, DNA damage response, p53 signal pathway and longevity regulation signal pathway. It is suggested that IIS/FoxO pathway can interconnect with the AMPK signaling pathway related to energy metabolism, the Sirt signaling pathway related to epigenetics, and the mTOR signaling pathway related to nutrient sensing to regulate amino acid, cAMP, and NAD+ levels in the organism, thereby controlling processes such as antioxidant stress, autophagy, DNA repair, immunity, mitochondrial homeostasis, and energy metabolism, jointly regulating scallop aging and longevity.
In summary, this study provides a thorough and detailed analysis of the mechanisms underlying the role of the IIS/FoxO pathway in the regulation of lifespan in A. irradians and A. purpuratus for the first time. It confirms the IIS/FoxO pathway may regulate the anti-oxidative stress, DNA damage repair, autophagy, and immunity, among other anti-aging processes involved in the aging of scallops. The completion of this work will advance the research on the molecular mechanisms underlying lifespan determination in marine bivalves, providing new insights for the study of lifespan determination mechanisms in other animals, including humans, and enhancing our understanding of aging and longevity mechanisms. Additionally, the completion of this work will accelerate the genetic breeding process of scallops, providing theoretical basis for the cultivation of long-lived hybrid scallops and improving product quality. |
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