- Email: [email protected]
Walk the (Germ) Line
Cell Metabolism
Previews LepRb function is needed to demonstrate that the region is critical for the effects of endogenous leptin on food intake. Nonetheless, the LHA LepRb neurons are clearly positioned to influence the VTA dopamine centers. The LHA receives extensive projections from the shell of the nucleus accumbens (Kelley et al., 2005), and like OX neurons, these new LepRb-expressing neurons may be influenced by GABAergic nucleus accumbens projections forming a parallel LHA-VTAnucleus accumbens-LHA circuit. A challenge that remains is understanding what this distributed response to leptin means. Presumably, it allows for effective and tightly regulated behavioral responses to changes in metabolic state. However, even in the hypothalamus, we have yet to determine how the neuronal response to leptin changes in the satiated versus restricted state, for example. Ultimately, analysis of brainregion neuronal activity during feeding is needed to generate a more complete picture of what happens and how meta-
bolic hormones influence the component neural circuits. Practical experimental issues compel us to consider and study brain regions as independent units. This is a conundrum of the modern neuroscience research effort: we spend great time and effort to develop mouse lines and viral vectors to manipulate genes in specific regions in an effort to understand an integrated system. In this context, it is important to note that single-region manipulations (e.g., arcuate nucleus, LHA, or VTA) may or may not reflect what happens in the normal state. However, the hope is that we understand the complete system once we have defined the parts and determined how they respond to metabolic signals. This innovative work from Myers and colleagues has identified and characterized a set of interesting neurons in a key brain center. Their emphasis on defining downstream neural circuits also orients us toward the next challenge: how the brain integrates and interprets metabolic signals to result in behavioral responses.
REFERENCES Fulton, S., Woodside, B., and Shizgal, P. (2000). Science 287, 125–128. Fulton, S., Pissios, P., Manchon, R.P., Stiles, L., Frank, L., Pothos, E.N., Maratos-Flier, E., and Flier, J.S. (2006). Neuron 51, 811–822. Grill, H.J., Schwartz, M.W., Kaplan, J.M., Foxhall, J.S., Breininger, J., and Baskin, D.G. (2002). Endocrinology 143, 239–246. Hommel, J.D., Trinko, R., Sears, R.M., Georgescu, D., Liu, Z.W., Gao, X.B., Thurmon, J.J., Marinelli, M., and DiLeone, R.J. (2006). Neuron 51, 801–810. Kelley, A.E., Baldo, B.A., Pratt, W.E., and Will, M.J. (2005). Physiol. Behav. 86, 773–795. Leinninger, G.M., Jo, Y.-H., Leshan, R.L., Louis, G.W., Yang, H., Barrera, J.G., Wilson, H., Opland, D.M., Faouzi, M.A., Gong, Y., et al. (2009). Cell Metab. 10, this issue, 89–98. Olds, J., and Milner, P. (1954). J. Comp. Physiol. Psychol. 47, 419–427. Palmiter, R.D. (2007). Trends Neurosci. 30, 375–381. Salamone, J.D., Correa, M., Mingote, S.M., and Weber, S.M. (2005). Curr. Opin. Pharmacol. 5, 34–41. Stellar, E. (1954). Psychol. Rev. 61, 5–22. Wise, R.A. (2006). Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1149–1158.
Walk the (Germ) Line D. Leanne Jones1,* 1Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2009.07.008
Germ cells possess the unique ability to transmit genetic information from generation to generation. In a recent paper, Curran et al. (2009) explore the possibility that some features of germ cells, including enhanced genomic stability, can be acquired by the soma as a mechanism to increase longevity. Germ cells are highly specialized cells that form gametes and the only cells within an organism that contribute genes to offspring. Since the genetic information contained within germ cells is passed from generation to generation, the germline is often referred to as immortal. Germline stem cells (GSCs) are responsible for the continual production of germ cells and mature gametes throughout life; however, GSCs are lost in older animals, and those GSCs that do remain display decreases or arrest in cell cycle progression, potential hallmarks of germline aging
(Cheng et al., 2008; reviewed in Jones, 2007). Remarkably, however, serial transplantation experiments, designed to assay the ability of murine spermatogonial stem cells (SSCs) to repeatedly reconstitute spermatogenesis in recipient mice devoid of endogenous germ cells, demonstrated that mammalian SSCs harbor tremendous regenerative potential. SSCs continued to self-renew and generate colonies of germ cells upwards of 1000 days, well beyond the life span of wild-type laboratory strains of mice (Ryu et al., 2006). Recent data have re-
78 Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc.
vealed strategies that germ cells utilize to protect the genetic information contained within them (Das et al., 2008; Malone et al., 2009; Robert et al., 2005; reviewed in O’Donnell and Boeke, 2007), but are these mechanisms sufficient to insure vigilant protection of the germline indefinitely, and can germline-protection mechanisms be exploited by the soma to enhance longevity? A recent study provides insight into these questions (Curran et al., 2009). Noting that RNAi in Caenorhabditis elegans (C. elegans) is deployed as a
Cell Metabolism
Previews protective stress response and that RNAi functions in the germline remarkably efficiently, the authors speculated that the enhanced RNAi observed in somatic tissues in various mutant backgrounds could be associated with a transformation of somatic tissues to a more germline-like state. To test this hypothesis, Curran et al. first demonstrated that worms that are long-lived due to mutations in the insulinlike signaling pathway (daf-2/insulin/IGF1-like receptor [InR], age-1/PI3 kinase) show ectopic expression of a germline reporter in somatic tissues, including the intestine and hypodermis. In addition, quantitative RT-PCR analysis confirmed that germline-specific genes pie-1 and pgl-1–3 were upregulated in insulin-like signaling mutants. To verify that this increase was due to expression in the soma and not increased expression in the germline, the authors demonstrated that pie-1 and pgl-1–3 levels were increased in animals that were mutant for daf-2/InR and germline deficient due to a mutation in glp-4, a gene required for germline formation. DAF-16/FOXO is the key downstream transcriptional effector of the insulin-like signaling pathway. Upon activation of the insulin-like signaling pathway, DAF-16/ FOXO is phosphorylated and retained within the cytoplasm. However, when insulin-like signaling is low, DAF-16/ FOXO is active and enters the nucleus to activate expression of key target genes. The upregulation of germline genes in somatic tissues was abolished in shortlived daf-16/FOXO; daf-2/InR double mutant animals, indicating that somatic expression of germline genes occurs in a DAF-16/FOXO-dependent manner. Consistent with a new role for DAF-16/ FOXO in the expression of germline genes, Curran et al. demonstrated that DAF-16/ FOXO binds directly to sequences in the pie-1 promoter using both electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). If somatic tissues have assumed germline characteristics in long-lived insulinlike signaling mutants, Curran et al. speculated that these somatic cells might have acquired an enhanced ability to protect against genotoxic stress and damage. To test this hypothesis, they used RNAi to deplete genes known to be involved in protecting the genome in wild-type and daf-2/ InR mutant backgrounds. In wild-type
worms, loss of such genes led to reduced viability and increased DNA damage, as assayed by expression of a normally out-offrame lacZ-GFP reporter transgene. In contrast, long-lived daf-2/InR mutants showed lower levels of developmental arrest and increased viability, relative to wild-type, as well as significantly less expression of the lacZ-GFP ‘‘strand slippage’’ reporter. These data suggest that somatic cells in long-lived daf-2/InR mutant worms have engaged pathways that regulate genomic surveillance, rendering them more resistant to DNA damage. In order to determine how well life span extension correlates with somatic expression of germline genes, Curran et al. screened a collection of long-lived mutant worms for somatic expression of PGL-1 protein. Inactivation of two components of the cytosolic chaperonin complex, cct-4 and cct-6, led to increased longevity, with concomitant misexpression of PGL-1 in the soma (Curran and Ruvkun, 2007). The cytosolic chaperonin complex negatively regulates the expression of the transcription factor skn-1/Nrf in the intestine, which activates the expression of stress response target genes (Kahn et al., 2008). Consistent with this role for skn-1/Nrf 1, the highest levels of PGL-1 were observed in the intestine of worms with reduced cct-4 and cct-6. Interestingly, skn-1/Nrf mediates life span extension in insulin-like signaling mutants independently of daf-16/FOXO (Tullet et al., 2008), indicating that multiple transcriptional effectors integral to this longevity-promoting pathway not only activate stress resistance, but also impinge on expression of germline genes. Although the authors do demonstrate that loss of genes such as pie-1 or pgl-1 reduced the life span extension observed upon loss of daf-2/InR, no data are provided to indicate that somatic misexpression of germline genes is sufficient to extend life span or protect the soma from genotoxic stress. Furthermore, not all long-lived worms, including those that are either dietary restricted (eat-2) or impaired for mitochondrial function (clk-1), display somatic expression of pie-1 and pgl-1. In addition, it is not clear whether the soma acquires germline qualities during the long-lived dauer stages. It is possible that various subsets of germline genes are expressed under different conditions, and an analysis of all genes that are upregulated in glp4, daf-2 double
mutants should provide additional candidate genes that might confer germline-like qualities onto or engage additional stressresistance/genome-protecting pathways in the soma. However, it is clear that life span extension can occur via additional pathways. Expression of germline genes in tissues such as the intestine and hypodermis, which is the functional equivalent of the skin, is particularly interesting, as these are tissues that are maintained by somatic stem cells in many organisms. The ability of these tissues to turn over provides a mechanism to replace any damaged cells with new ones. Therefore, these provocative data demonstrating activation of germline genes in the soma of long-lived worms may represent an alternative strategy to maintain ‘‘healthy’’ tissues and organs in the absence of a resident stem cell population. Elucidation of additional mechanisms utilized by the germline to preserve and transmit genetic information from generation to generation may provide additional pathways that could be engaged in somatic cells to protect relatively short-lived cells, ultimately contributing to life span extension. REFERENCES Cheng, J., Tu¨rkel, N., Hemati, N., Fuller, M.T., Hunt, A.J., and Yamashita, Y.M. (2008). Nature 456, 599– 604. Curran, S.P., and Ruvkun, G. (2007). PLoS Genet 3, e56. Curran, S.P., Wu, X., Riedel, C.G., and Ruvkun, G. (2009). Nature 459, 1079–1084. Das, P.P., Bagijn, M.P., Goldstein, L.D., Woolford, J.R., Lehrbach, N.J., Sapetschnig, A., Buhecha, H.R., Gilchrist, M.J., Howe, K.L., Stark, R., et al. (2008). Mol. Cell 31, 79–90. Jones, D.L. (2007). Stem Cell Rev. 3, 192–200. Kahn, N.W., Rea, S.L., Moyle, S., Kell, A., and Johnson, T.E. (2008). Biochem. J. 409, 205–213. Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam, R., and Hannon, G.J. (2009). Cell 137, 522–535. O’Donnell, K.A., and Boeke, J.D. (2007). Cell 129, 37–44. Robert, V.J., Sijen, T., van Wolfswinkel, J., and Plasterk, R.H. (2005). Genes Dev. 19, 782–787. Ryu, B.Y., Orwig, K.E., Oatley, J.M., Avarbock, M.R., and Brinster, R.L. (2006). Stem Cells 24, 1505–1511. Tullet, J.M., Hertweck, M., An, J.H., Baker, J., Hwang, J.Y., Liu, S., Oliveira, R.P., Baumeister, R., and Blackwell, T.K. (2008). Cell 132, 1025– 1038.
Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc. 79
Previews LepRb function is needed to demonstrate that the region is critical for the effects of endogenous leptin on food intake. Nonetheless, the LHA LepRb neurons are clearly positioned to influence the VTA dopamine centers. The LHA receives extensive projections from the shell of the nucleus accumbens (Kelley et al., 2005), and like OX neurons, these new LepRb-expressing neurons may be influenced by GABAergic nucleus accumbens projections forming a parallel LHA-VTAnucleus accumbens-LHA circuit. A challenge that remains is understanding what this distributed response to leptin means. Presumably, it allows for effective and tightly regulated behavioral responses to changes in metabolic state. However, even in the hypothalamus, we have yet to determine how the neuronal response to leptin changes in the satiated versus restricted state, for example. Ultimately, analysis of brainregion neuronal activity during feeding is needed to generate a more complete picture of what happens and how meta-
bolic hormones influence the component neural circuits. Practical experimental issues compel us to consider and study brain regions as independent units. This is a conundrum of the modern neuroscience research effort: we spend great time and effort to develop mouse lines and viral vectors to manipulate genes in specific regions in an effort to understand an integrated system. In this context, it is important to note that single-region manipulations (e.g., arcuate nucleus, LHA, or VTA) may or may not reflect what happens in the normal state. However, the hope is that we understand the complete system once we have defined the parts and determined how they respond to metabolic signals. This innovative work from Myers and colleagues has identified and characterized a set of interesting neurons in a key brain center. Their emphasis on defining downstream neural circuits also orients us toward the next challenge: how the brain integrates and interprets metabolic signals to result in behavioral responses.
REFERENCES Fulton, S., Woodside, B., and Shizgal, P. (2000). Science 287, 125–128. Fulton, S., Pissios, P., Manchon, R.P., Stiles, L., Frank, L., Pothos, E.N., Maratos-Flier, E., and Flier, J.S. (2006). Neuron 51, 811–822. Grill, H.J., Schwartz, M.W., Kaplan, J.M., Foxhall, J.S., Breininger, J., and Baskin, D.G. (2002). Endocrinology 143, 239–246. Hommel, J.D., Trinko, R., Sears, R.M., Georgescu, D., Liu, Z.W., Gao, X.B., Thurmon, J.J., Marinelli, M., and DiLeone, R.J. (2006). Neuron 51, 801–810. Kelley, A.E., Baldo, B.A., Pratt, W.E., and Will, M.J. (2005). Physiol. Behav. 86, 773–795. Leinninger, G.M., Jo, Y.-H., Leshan, R.L., Louis, G.W., Yang, H., Barrera, J.G., Wilson, H., Opland, D.M., Faouzi, M.A., Gong, Y., et al. (2009). Cell Metab. 10, this issue, 89–98. Olds, J., and Milner, P. (1954). J. Comp. Physiol. Psychol. 47, 419–427. Palmiter, R.D. (2007). Trends Neurosci. 30, 375–381. Salamone, J.D., Correa, M., Mingote, S.M., and Weber, S.M. (2005). Curr. Opin. Pharmacol. 5, 34–41. Stellar, E. (1954). Psychol. Rev. 61, 5–22. Wise, R.A. (2006). Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1149–1158.
Walk the (Germ) Line D. Leanne Jones1,* 1Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2009.07.008
Germ cells possess the unique ability to transmit genetic information from generation to generation. In a recent paper, Curran et al. (2009) explore the possibility that some features of germ cells, including enhanced genomic stability, can be acquired by the soma as a mechanism to increase longevity. Germ cells are highly specialized cells that form gametes and the only cells within an organism that contribute genes to offspring. Since the genetic information contained within germ cells is passed from generation to generation, the germline is often referred to as immortal. Germline stem cells (GSCs) are responsible for the continual production of germ cells and mature gametes throughout life; however, GSCs are lost in older animals, and those GSCs that do remain display decreases or arrest in cell cycle progression, potential hallmarks of germline aging
(Cheng et al., 2008; reviewed in Jones, 2007). Remarkably, however, serial transplantation experiments, designed to assay the ability of murine spermatogonial stem cells (SSCs) to repeatedly reconstitute spermatogenesis in recipient mice devoid of endogenous germ cells, demonstrated that mammalian SSCs harbor tremendous regenerative potential. SSCs continued to self-renew and generate colonies of germ cells upwards of 1000 days, well beyond the life span of wild-type laboratory strains of mice (Ryu et al., 2006). Recent data have re-
78 Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc.
vealed strategies that germ cells utilize to protect the genetic information contained within them (Das et al., 2008; Malone et al., 2009; Robert et al., 2005; reviewed in O’Donnell and Boeke, 2007), but are these mechanisms sufficient to insure vigilant protection of the germline indefinitely, and can germline-protection mechanisms be exploited by the soma to enhance longevity? A recent study provides insight into these questions (Curran et al., 2009). Noting that RNAi in Caenorhabditis elegans (C. elegans) is deployed as a
Cell Metabolism
Previews protective stress response and that RNAi functions in the germline remarkably efficiently, the authors speculated that the enhanced RNAi observed in somatic tissues in various mutant backgrounds could be associated with a transformation of somatic tissues to a more germline-like state. To test this hypothesis, Curran et al. first demonstrated that worms that are long-lived due to mutations in the insulinlike signaling pathway (daf-2/insulin/IGF1-like receptor [InR], age-1/PI3 kinase) show ectopic expression of a germline reporter in somatic tissues, including the intestine and hypodermis. In addition, quantitative RT-PCR analysis confirmed that germline-specific genes pie-1 and pgl-1–3 were upregulated in insulin-like signaling mutants. To verify that this increase was due to expression in the soma and not increased expression in the germline, the authors demonstrated that pie-1 and pgl-1–3 levels were increased in animals that were mutant for daf-2/InR and germline deficient due to a mutation in glp-4, a gene required for germline formation. DAF-16/FOXO is the key downstream transcriptional effector of the insulin-like signaling pathway. Upon activation of the insulin-like signaling pathway, DAF-16/ FOXO is phosphorylated and retained within the cytoplasm. However, when insulin-like signaling is low, DAF-16/ FOXO is active and enters the nucleus to activate expression of key target genes. The upregulation of germline genes in somatic tissues was abolished in shortlived daf-16/FOXO; daf-2/InR double mutant animals, indicating that somatic expression of germline genes occurs in a DAF-16/FOXO-dependent manner. Consistent with a new role for DAF-16/ FOXO in the expression of germline genes, Curran et al. demonstrated that DAF-16/ FOXO binds directly to sequences in the pie-1 promoter using both electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). If somatic tissues have assumed germline characteristics in long-lived insulinlike signaling mutants, Curran et al. speculated that these somatic cells might have acquired an enhanced ability to protect against genotoxic stress and damage. To test this hypothesis, they used RNAi to deplete genes known to be involved in protecting the genome in wild-type and daf-2/ InR mutant backgrounds. In wild-type
worms, loss of such genes led to reduced viability and increased DNA damage, as assayed by expression of a normally out-offrame lacZ-GFP reporter transgene. In contrast, long-lived daf-2/InR mutants showed lower levels of developmental arrest and increased viability, relative to wild-type, as well as significantly less expression of the lacZ-GFP ‘‘strand slippage’’ reporter. These data suggest that somatic cells in long-lived daf-2/InR mutant worms have engaged pathways that regulate genomic surveillance, rendering them more resistant to DNA damage. In order to determine how well life span extension correlates with somatic expression of germline genes, Curran et al. screened a collection of long-lived mutant worms for somatic expression of PGL-1 protein. Inactivation of two components of the cytosolic chaperonin complex, cct-4 and cct-6, led to increased longevity, with concomitant misexpression of PGL-1 in the soma (Curran and Ruvkun, 2007). The cytosolic chaperonin complex negatively regulates the expression of the transcription factor skn-1/Nrf in the intestine, which activates the expression of stress response target genes (Kahn et al., 2008). Consistent with this role for skn-1/Nrf 1, the highest levels of PGL-1 were observed in the intestine of worms with reduced cct-4 and cct-6. Interestingly, skn-1/Nrf mediates life span extension in insulin-like signaling mutants independently of daf-16/FOXO (Tullet et al., 2008), indicating that multiple transcriptional effectors integral to this longevity-promoting pathway not only activate stress resistance, but also impinge on expression of germline genes. Although the authors do demonstrate that loss of genes such as pie-1 or pgl-1 reduced the life span extension observed upon loss of daf-2/InR, no data are provided to indicate that somatic misexpression of germline genes is sufficient to extend life span or protect the soma from genotoxic stress. Furthermore, not all long-lived worms, including those that are either dietary restricted (eat-2) or impaired for mitochondrial function (clk-1), display somatic expression of pie-1 and pgl-1. In addition, it is not clear whether the soma acquires germline qualities during the long-lived dauer stages. It is possible that various subsets of germline genes are expressed under different conditions, and an analysis of all genes that are upregulated in glp4, daf-2 double
mutants should provide additional candidate genes that might confer germline-like qualities onto or engage additional stressresistance/genome-protecting pathways in the soma. However, it is clear that life span extension can occur via additional pathways. Expression of germline genes in tissues such as the intestine and hypodermis, which is the functional equivalent of the skin, is particularly interesting, as these are tissues that are maintained by somatic stem cells in many organisms. The ability of these tissues to turn over provides a mechanism to replace any damaged cells with new ones. Therefore, these provocative data demonstrating activation of germline genes in the soma of long-lived worms may represent an alternative strategy to maintain ‘‘healthy’’ tissues and organs in the absence of a resident stem cell population. Elucidation of additional mechanisms utilized by the germline to preserve and transmit genetic information from generation to generation may provide additional pathways that could be engaged in somatic cells to protect relatively short-lived cells, ultimately contributing to life span extension. REFERENCES Cheng, J., Tu¨rkel, N., Hemati, N., Fuller, M.T., Hunt, A.J., and Yamashita, Y.M. (2008). Nature 456, 599– 604. Curran, S.P., and Ruvkun, G. (2007). PLoS Genet 3, e56. Curran, S.P., Wu, X., Riedel, C.G., and Ruvkun, G. (2009). Nature 459, 1079–1084. Das, P.P., Bagijn, M.P., Goldstein, L.D., Woolford, J.R., Lehrbach, N.J., Sapetschnig, A., Buhecha, H.R., Gilchrist, M.J., Howe, K.L., Stark, R., et al. (2008). Mol. Cell 31, 79–90. Jones, D.L. (2007). Stem Cell Rev. 3, 192–200. Kahn, N.W., Rea, S.L., Moyle, S., Kell, A., and Johnson, T.E. (2008). Biochem. J. 409, 205–213. Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam, R., and Hannon, G.J. (2009). Cell 137, 522–535. O’Donnell, K.A., and Boeke, J.D. (2007). Cell 129, 37–44. Robert, V.J., Sijen, T., van Wolfswinkel, J., and Plasterk, R.H. (2005). Genes Dev. 19, 782–787. Ryu, B.Y., Orwig, K.E., Oatley, J.M., Avarbock, M.R., and Brinster, R.L. (2006). Stem Cells 24, 1505–1511. Tullet, J.M., Hertweck, M., An, J.H., Baker, J., Hwang, J.Y., Liu, S., Oliveira, R.P., Baumeister, R., and Blackwell, T.K. (2008). Cell 132, 1025– 1038.
Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc. 79