Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here, we show that OA benefits Caenorhabditis elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet-dependent enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal an unexpected mechanism of FA signal transduction, as well as a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate some benefits of the OA-rich Mediterranean diet.
Lipid droplet
Endoplasmic-reticulum-associated protein degradation
To explore the characteristics of cell apoptosis and proliferation of corpus cavernosum smooth muscle (CCSM) cells in diabetic rats.From a SD rat model of diabetes induced by a single dose of streptozotocin, CCSM cells were isolated for primary culture and identified using immunocytochemical assays for SMα-actin. The proliferation of CCSM cells was evaluated by WST-1 assay, and flow cytometry was used to detect the cells apoptosis. Real-time fluorescence quantitative RT-PCR (qRT-PCR) was used to analyze the relative expression of proliferation cell nucleus antigen (PCNA) and caspase-3 mRNA.The proliferation rate of the primarily cultured CCSM cells from diabetic rats was significantly decreased and the apoptosis rate significantly increased compared with those of the cells from the control rats. The expression of PCNA mRNA was significantly lowered while caspase-3 mRNA significantly increased in the corpus cavernosum of the diabetic rats (P<0.001).In rats with persisted hyperglycemia, a higher apoptosis rate and a lower proliferation rate both contribute to the reduction of CCSM cells.
Article7 October 2021Open Access Source DataTransparent process Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling Juliane C Campos Juliane C Campos Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil These authors contributed equally to this work Search for more papers by this author Ziyun Wu Ziyun Wu orcid.org/0000-0001-8239-1890 Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Department of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China These authors contributed equally to this work Search for more papers by this author Paige D Rudich Paige D Rudich orcid.org/0000-0001-6979-9701 Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Sonja K Soo Sonja K Soo Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Meeta Mistry Meeta Mistry Bioinformatics Core, Harvard School of Public Health, Harvard Medical School, Boston, MA, USA Search for more papers by this author Julio CB Ferreira Julio CB Ferreira orcid.org/0000-0003-2694-239X Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil Search for more papers by this author T Keith Blackwell Corresponding Author T Keith Blackwell [email protected] Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jeremy M Van Raamsdonk Corresponding Author Jeremy M Van Raamsdonk [email protected] orcid.org/0000-0001-8376-9605 Department of Genetics, Harvard Medical School, Boston, MA, USA Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Juliane C Campos Juliane C Campos Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil These authors contributed equally to this work Search for more papers by this author Ziyun Wu Ziyun Wu orcid.org/0000-0001-8239-1890 Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Department of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China These authors contributed equally to this work Search for more papers by this author Paige D Rudich Paige D Rudich orcid.org/0000-0001-6979-9701 Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Sonja K Soo Sonja K Soo Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Meeta Mistry Meeta Mistry Bioinformatics Core, Harvard School of Public Health, Harvard Medical School, Boston, MA, USA Search for more papers by this author Julio CB Ferreira Julio CB Ferreira orcid.org/0000-0003-2694-239X Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil Search for more papers by this author T Keith Blackwell Corresponding Author T Keith Blackwell [email protected] Research Division, Joslin Diabetes Center, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jeremy M Van Raamsdonk Corresponding Author Jeremy M Van Raamsdonk [email protected] orcid.org/0000-0001-8376-9605 Department of Genetics, Harvard Medical School, Boston, MA, USA Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Author Information Juliane C Campos1,2,3,4, Ziyun Wu1,2,3,5, Paige D Rudich6,7, Sonja K Soo6,7, Meeta Mistry8, Julio CB Ferreira4, T Keith Blackwell *,1,2,3 and Jeremy M Van Raamsdonk *,2,6,7,9 1Research Division, Joslin Diabetes Center, Boston, MA, USA 2Department of Genetics, Harvard Medical School, Boston, MA, USA 3Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA 4Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil 5Department of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 6Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada 7Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada 8Bioinformatics Core, Harvard School of Public Health, Harvard Medical School, Boston, MA, USA 9Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada *Corresponding authors. Tel: +1 617 309 2760; E-mail: [email protected] *Corresponding authors. Tel: +1 514 934 1934 ext. 76157; E-mail: [email protected] EMBO Reports (2021)22:e52964https://doi.org/10.15252/embr.202152964 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity in model organisms. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain results in upregulation of genes involved in innate immunity, which is driven by the mitochondrial unfolded protein response (mitoUPR) but also dependent on the canonical p38-mediated innate immune signaling pathway. Both of these pathways are required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitoUPR act in concert on the same innate immunity genes to promote pathogen resistance and longevity and that input from the mitochondria can extend longevity by signaling through these pathways. This indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan. SYNOPSIS Mild impairment of mitochondrial function results in enhanced innate immunity and lifespan extension. Both phenotypes are driven by activation of the mitochondrial unfolded protein response but are also dependent on p38-mediated innate immune signaling. Mild impairment of mitochondrial function enhances innate immunity in a p38-dependent manner through activation of ATFS-1. The mitochondrial unfolded protein response and p38-mediated innate immune signaling pathway act in concert to promote resistance to bacterial pathogens and extend lifespan. The p38-mediated innate immune signaling pathway plays a key role in determining longevity. Different levels of p38-mediated innate immune signaling are optimal for different contexts of lifespan extension. Introduction While aging was long believed to be a stochastic process of damage accumulation, research during the past three decades has demonstrated that lifespan can be strongly influenced by genetics. Single-gene mutations have been shown to extend longevity in model organisms, including yeast, worms, flies, and mice. Importantly, genes and interventions that increase lifespan tend to be conserved across species. For example, decreasing insulin-IGF1 signaling, which was first shown to increase lifespan in the worm C. elegans (Klass, 1977; Friedman & Johnson, 1988; Kenyon et al, 1993), has subsequently been shown to extend longevity in flies (Clancy et al, 2001) and mice (Holzenberger et al, 2003), and to be associated with longevity in humans (Flachsbart et al, 2017). This suggests that studying the aging process in model organisms can provide insights that are relevant to human aging. The first single gene mutation that was shown to extend lifespan was identified in C. elegans (Klass, 1977; Friedman & Johnson, 1988), and since then this organism has been extensively used to find additional genetic pathways associated with lifespan extension and to elucidate the underlying mechanisms. Among the earliest genes that were shown to influence longevity were genes involved in mitochondrial function. Mutations in the clk-1, nuo-6, and isp-1 genes affect different components of the mitochondrial electron transport chain, and all lead to increased lifespan (Wong et al, 1995; Lakowski & Hekimi, 1996; Feng et al, 2001; Yang & Hekimi, 2010b). In the case of nuo-6 and isp-1, which encode subunits of complex I and complex III, respectively, a complete loss-of-function mutation would be lethal, while point mutations that result in a mild impairment of mitochondrial function extend longevity. Similarly, decreasing the expression of genes involved in mitochondrial function with RNA interference also increases lifespan (Dillin et al, 2002; Lee et al, 2003). Importantly, mutations that affect mitochondrial function have also been shown to increase lifespan in other species, including flies (Copeland et al, 2009) and mice (Liu et al, 2005; Dell'agnello et al, 2007). While initially it was believed that the mechanism by which mild impairment of mitochondrial function increased lifespan was through a decrease in the production of reactive oxygen species (ROS) and the resulting oxidative damage (Feng et al, 2001), more recent studies show that mutations affecting mitochondrial function actually increase the levels of ROS (Yang & Hekimi, 2010a). The increase in ROS is required for the long lifespan of these mutants, as treatment with antioxidants can decrease their lifespan (Yang & Hekimi, 2010a; Van Raamsdonk & Hekimi, 2012). While multiple factors contributing to the long lifespan of these mitochondrial mutants have been identified (Walter et al, 2011; Baruah et al, 2014; Yee et al, 2014; Munkacsy et al, 2016; Senchuk et al, 2018; Wu et al, 2018), the precise mechanisms of lifespan extension remain incompletely understood. In our previous work, we have shown that two stress-responsive transcription factors, DAF-16/FOXO3 and ATFS-1/ATF5, are required for the long lifespan of nuo-6 and isp-1 worms (Senchuk et al, 2018; Wu et al, 2018). DAF-16/FOXO3 is a FOXO transcription factor that is directly regulated by phosphorylation in response to insulin-IGF1 signaling, a growth factor signaling pathway that begins with the insulin-IGF1 receptor DAF-2 (Kenyon, 2010). While DAF-16 normally resides in the cytoplasm, when signaling through the insulin-IGF1 pathway is reduced DAF-16 accumulates in nuclei. DAF-16 also translocates to the nucleus in response to various stresses. In the nucleus, DAF-16 upregulates genes involved in stress response and metabolism (Murphy et al, 2003; Tepper et al, 2013). DAF-16 has been shown to be required in multiple different contexts of lifespan extension (Kenyon et al, 1993; Apfeld & Kenyon, 1999; Berman & Kenyon, 2006; Syntichaki et al, 2007; Senchuk et al, 2018). Activating transcription factor associated with stress 1 (ATFS-1/ATF5) is the transcription factor that mediates the mitochondrial unfolded protein response (mitoUPR) (Jovaisaite et al, 2014), a stress response pathway that responds to mitochondrial stress in order to restore mitochondrial function. ATFS-1 contains a nuclear localization signal (NLS) and a mitochondrial targeting sequence (MTS). Under normal conditions, ATFS-1 is targeted to the mitochondria where it is imported and degraded. Under conditions of mitochondrial stress, mitochondrial import of ATFS-1 is prevented, resulting in accumulation of ATFS-1 in the cytoplasm, where the NLS translocates ATFS-1 into the nucleus in order to restore mitochondrial homeostasis through alterations in expression of genes involved in protein folding and metabolism (Nargund et al, 2012). While DAF-16 and ATFS-1 can both contribute to defense against bacterial pathogens (Pellegrino et al, 2014; Dues et al, 2019), the primary innate immune signaling pathway that responds to bacterial pathogen stress is a mitogen-activated protein kinase (MAPK) signaling pathway, which has been found to be conserved from invertebrates to mammals (Hoffmann et al, 1999; Kimbrell & Beutler, 2001; Irazoqui et al, 2010). In this pathway, NSY-1/ASK1 (MAPK kinase kinase) signals to SEK-1/MKK3/MKK6 (MAPK kinase), which signals to PMK-1/p38 (MAPK) (Kim et al, 2002; Kim & Ewbank, 2018) (Appendix Fig S1). Downstream of this pathway, the transcription factor ATF-7/ATF2/ATF-7/CREB5 acts to modulate the expression of genes involved in innate immunity (Shivers et al, 2010; Fletcher et al, 2019a). While ATF-7 normally acts as a repressor of gene function, when it is phosphorylated by PMK-1, ATF-7 functions as an activator of p38/ATF-7-regulated immunity gene expression (Appendix Fig S1). In this work, we show that the long-lived mitochondrial mutants, nuo-6 and isp-1, exhibit an upregulation of genes involved in innate immunity that is driven by the activation of the mitoUPR, but also dependent on the p38-mediated innate immune signaling pathway, leading to an increased resistance to bacterial pathogens. We find that the p38-mediated innate immune signaling pathway is required for the long lifespan of nuo-6 and isp-1 mutants. Finally, we demonstrate that the activation of the mitoUPR is sufficient to upregulate innate immunity genes and is also required for their upregulation in nuo-6 mutants. Overall, this work demonstrates the importance of the mitoUPR in upregulating innate immunity in response to signals from the mitochondria and delineates a clear role of innate immune signaling pathways in determining lifespan. Results Long-lived mitochondrial mutants exhibit broad upregulation of genes involved in innate immunity that is dependent on p38-mediated innate immune signaling While mild impairment of mitochondrial function has been shown to extend longevity, the underlying mechanisms are yet to be fully elucidated. When mitochondrial function is impaired, mitochondria are able to communicate with the nucleus to alter nuclear gene expression. To obtain a comprehensive, unbiased view of the transcriptional changes that result from impairment of mitochondrial function, we used RNA sequencing (RNA-seq) to examine gene expression in two long-lived mitochondrial mutants, nuo-6 and isp-1. After determining which genes were differentially expressed compared with wild-type worms, we identified groups of genes that showed enrichment. Among the genes that showed enrichment were genes involved in innate immunity. These genes encode proteins that function to inhibit the growth and survival of pathogenic bacteria and to repair or remove damage to the worm (Troemel et al, 2006; Chikka et al, 2016; Fletcher et al, 2019a). Accordingly, we decided to investigate the role of innate immunity in the long lifespan of these long-lived mitochondrial mutants. To determine the extent to which genes involved in innate immunity are upregulated in the long-lived mitochondrial mutants, nuo-6 and isp-1, we first examined eight genes, which others have used to monitor innate immune activity (Shivers et al, 2010; Pellegrino et al, 2014; Block et al, 2015; Chikka et al, 2016; Jeong et al, 2017; Wu et al, 2019). These genes included T24B8.5/sysm-1, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8, and C32H11.4. All of these genes are upregulated in response to exposure to the bacterial pathogen Pseudomonas aeruginosa strain PA14, and five of eight have been shown to be direct targets of the p38-mediated innate immune signaling pathway in a ChIP-seq analysis of ATF-7 (Fletcher et al, 2019a). In examining the expression of these genes in our RNA-seq data, we found that all eight genes were significantly upregulated in both nuo-6 and isp-1 worms (Fig 1A). Figure 1. Innate immunity genes are upregulated in long-lived mitochondrial mutants Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild type of six biological replicates per strain. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1, and ATF-7. Gene expression changes were examined by quantitative RT–PCR of three biological replicates per strain. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Differentially expressed genes in long-lived mitochondrial mutants show significant overlap with genetic targets of the p38-mediated innate immune signaling pathway. Significantly modulated genes in long-lived mitochondrial mutants nuo-6 and isp-1 (six biological replicates per strain) were compared to genes that are modulated in response to exposure to the bacterial pathogen P. aeruginosa strain PA14 in a PMK-1- and ATF-7-dependent manner as identified by Fletcher et al (2019a). There is a highly significant degree of overlap between genes upregulated by activation of the p38-mediated innate immune pathway and genes upregulated in nuo-6 and isp-1 mutants. Similarly, there is a highly significantly degree of overlap between genes downregulated by activation of the p38-mediated innate immune pathway and genes downregulated in nuo-6 and isp-1 mutants. The P-values indicate the significance of the difference between the observed number of overlapping genes between the two gene sets and the expected number of overlapping genes if the genes were picked at random. Data information: Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test in panels A and B. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test in panel C. Download figure Download PowerPoint To determine whether the upregulation of innate immunity genes in the long-lived mitochondrial mutants requires the p38-mediated innate immune signaling pathway (NSY-1 → SEK-1 → PMK-1 → ATF-7), we generated double mutants of nuo-6 and isp-1 with all of these genes before measuring gene expression with quantitative RT–PCR (qPCR). We used loss-of-function mutants for nsy-1, sek-1, and pmk-1. Since ATF-7 normally acts as a repressor, we used the qd22 gain-of-function mutation for this gene. This mutation prevents phosphorylation of ATF-7 by PMK-1, thereby making the mutant ATF-7 a constitutive repressor (Shivers et al, 2010) (Appendix Fig S1). As in the RNA-seq data, the results from the qPCR experiments showed that innate immune genes are upregulated in nuo-6 and isp-1 worms. Importantly, we found that in both nuo-6 and isp-1 worms that disruption of genes involved in the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7(gof)) prevented the upregulation of innate immunity genes (Figs 1B and EV1). Click here to expand this figure. Figure EV1. Upregulation of innate immunity genes in long-lived mitochondrial mutants requires the p38-mediated innate immune signaling pathway A–C. Mutation of genes involved in the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7(gof)) decreases the expression of genes involved in innate immunity in wild-type (A), nuo-6 (B), and isp-1 (C) worms. Gene expression was determined by quantitative real-time RT–PCR on three biological replicates of pre-fertile young adult worms. Data information: Error bars indicate SEM. All differences from control are significant P < 0.05. Statistical significance was assessed for each gene independently using a one-way ANOVA with Dunnett's multiple comparisons test. Download figure Download PowerPoint To confirm these results using an alternative approach, we crossed a fluorescent reporter strain for one of the innate immunity genes (T24B8.5/sysm-1) (Shivers et al, 2010) to nuo-6 and isp-1 mutants and examined the effect of knocking down sek-1 through RNA interference (RNAi). Again, we found that T24B8.5/sysm-1 is upregulated in nuo-6 and isp-1 worms and that this upregulation is dependent on SEK-1 (Fig 1C). Combined, these results demonstrate that innate immunity genes are upregulated in the long-lived mitochondrial mutants, nuo-6 and isp-1, and that this upregulation is dependent on the p38-mediated innate immune signaling pathway. To further examine the expression of innate immunity genes in nuo-6 and isp-1 mutants, we compared the differentially expressed genes in these mutants to a more comprehensive and unbiased list of genes involved in innate immunity. A recent study defined the changes in gene expression that result from exposure to the bacterial pathogen Pseudomonas aeruginosa strain PA14 and determined which of these changes in gene expression are dependent on PMK-1 and ATF-7 (Fletcher et al, 2019a). In total, they reported 300 genes that were upregulated by exposure to PA14 in a PMK-1- and ATF-7-dependent manner, and 230 genes that were downregulated by exposure to PA14 in a PMK-1- and ATF-7-dependent manner. We compared these lists of PA14-modulated, PMK-1-dependent, ATF-7-dependent genes to genes that we found to be significantly upregulated or downregulated in nuo-6 and isp-1 mutants. We found that of the genes that are upregulated by PA14 exposure in a PMK-1- and ATF-7-dependent manner, 38% (P = 1.5 × 10−35) and 30% (P = 3.7 × 10−27) are also upregulated in nuo-6 and isp-1 mutants, respectively (Fig 1D; P-values indicate the significance of the difference between the observed number of overlapping genes between the two gene sets and the expected number of overlapping genes if the genes were picked at random). There is a high degree of overlap between genes upregulated in nuo-6 and isp-1 mutants (71%), and this includes the genes involved in innate immunity (Dataset EV1; 71 overlapping genes of 89/115 innate immunity genes upregulated in nuo-6 or isp-1, respectively). In contrast, there was no significant overlap of the same genes upregulated by PA14 exposure with genes downregulated in nuo-6 and isp-1 mutants. In examining the list of genes that are downregulated by exposure to PA14 in a PMK-1- and ATF-7-dependent manner, we found that a significant number of these genes are also downregulated in nuo-6 mutants (20%; P = 8.4 × 10−7) and isp-1 mutants (20%; P = 7.5 × 10−5). To more comprehensively compare the PA14-modulated, PMK-1-dependent, ATF-7-dependent gene expression changes to gene expression changes in nuo-6 and isp-1 mutants, we generated heat maps comparing the expression of PA14-modulated, PMK-1-dependent, ATF-7-dependent genes between wild-type worms and nuo-6 or isp-1 mutants. Among the genes that are upregulated by PA14 exposure in a PMK-1- and ATF-7-dependent manner, many of these genes are upregulated in nuo-6 and isp-1 mutants compared with wild-type worms, while a small subset show decreased expression (Appendix Figs S2 and S3). Among the genes that are downregulated by PA14 exposure in a PMK-1- and ATF-7-dependent manner, many of these genes are downregulated in nuo-6 and isp-1 mutants compared with wild-type worms, while a number of these genes also show increased expression (Appendix Figs S4 and S5). Overall, these results indicate that a mild impairment of mitochondrial function caused by mutations in nuo-6 or isp-1 leads to a broad upregulation of genes involved in innate immunity that is dependent on the p38-mediated innate immune signaling pathway. At the same time, a smaller proportion of these genes show decreased expression suggesting that the correct balance of innate immune gene expression may be required to optimize stress resistance and lifespan. Long-lived mitochondrial mutants show increased resistance to bacterial pathogens, which requires p38-mediated innate immune signaling Based on our observation that the long-lived mitochondrial mutants, nuo-6 and isp-1, have increased expression of many genes involved in innate immunity, we next sought to determine whether this increase in their expression resulted in enhanced resistance to bacterial pathogens. To test pathogen resistance, we exposed worms to PA14 in a slow kill assay where worms die from the ingestion and internal proliferation of the PA14 bacteria (Tan et al, 1999a, 1999b; Kirienko et al, 2014). We found that both nuo-6 and isp-1 worms exhibited significantly increased survival on PA14 bacteria compared with wild-type worms (Fig 2A). The increase in survival of nuo-6 and isp-1 worms did not result from reduced exposure to the pathogenic bacteria as their tendency to avoid PA14 was equivalent to wild-type worms (Appendix Fig S6). Figure 2. Long-lived mitochondrial mutants exhibit increased resistance to bacterial pathogens that is dependent on the presence of p38-mediated innate immune signaling pathway A–D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared with wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C), and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. A bar graph of all of these results can be found in Appendix Fig S7. Data information: ***P < 0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A are repeated in panels B, C, and D for direct comparison. Source data are available online for this figure. Source Data for Figure 2 [embr202152964-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint To determine the extent to which their enhanced resistance to bacterial pathogens is dependent on the p38-mediated innate immune signaling pathway, we next examined PA14 resistance in nuo-6 and isp-1 worms in which genes in this pathway were disrupted. In wild-type worms, mutations in nsy-1, sek-1, pmk-1, or atf-7(gof) significantly decrease the survival of worms exposed to PA14 (Fig 2B). Similarly, we found that disruptions of these innate immune signaling genes in nuo-6 (Fig 2C) and isp-1 (Fig 2D) worms also result in a significant decrease in survival on PA14 bacteria. In nuo-6 worms, survival was decreased back to wild type by mutations in nsy-1, pmk-1, or atf-7(gof), while a larger decrease in survival was observed with the sek-1 mutation. In isp-1 worms, mutations in nsy-1, sek-1, and atf-7(gof) all decreased survival to a greater extent than in nuo-6 mutants, and in each case, the survival of the double mutant was less than in wild-type worms (Appendix Fig S7). This suggests that isp-1 worms may be more reliant on the p38-mediated innate immune signaling pathway for their enhanced pathogen resistance than nuo-6 worms. F
Abstract Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here we show that OA benefits C. elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low, and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet (LD)-mediated enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome, and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal a surprising mechanism of FA signal transduction, and a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate benefits of the OA-rich Mediterranean diet.
Endoplasmic-reticulum-associated protein degradation
Abstract The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans , chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism and respiration, because can mediate the increased lifespan incurred by dietary restriction. We show that actually SKN-1B is not essential for dietary restriction longevity and instead, controls a variety of food-related behaviours. It acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-β), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.
Today, with the rapid development of the geographic information industry, automatic road extraction from satellite imagery is a basic requirement. Most existing methods have been designed based on binary segmentation. However, these methods do not consider the topological features of road networks, which include point, edge, and direction. In this study, a topology-based multi-task convolution network is designed, namely Bi-HRNet, which can effectively learn the key features of nodes and their directions. First, the proposed network learns the node heatmap of roads, and then the pixel coordinates are extracted from the node heatmap via non-maximum suppression (NMS). At the same time, the connectivity between nodes is predicted. To improve the integrity and accuracy of connectivity, we propose a bidirectional connectivity prediction strategy, which can learn the bidirectional categories instead of direction angles. The bidirectional categories are designed based on “top-to-bottom” and “bottom-to-top” strategies, which can improve the accuracy of the connectivity between nodes. To illustrate the effectiveness of the proposed Bi-HRNet, we compare our method with several methods on different datasets. The experiments show that our method achieves a state-of-the-art performance and significantly outperforms various previous methods.
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