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Mitochondrial-nuclear interactions and lifespan control in fungi
Experimental Gerontology 34 (1999) 901–909
Mini-review
Mitochondrial-nuclear interactions and lifespan control in fungi Heinz D. Osiewacz*, Erik Kimpel Botanisches Institut, Molekulare Entwicklungsbiologie und Biotechnologie, Johann Wolfgang Goethe–Universita¨t, Marie–Curie–Str. 9, D-60439 Frankfurt am Main, Germany Received 12 July 1999; received in revised form 25 August 1999; accepted 26 August 1999
Abstract In fungi, mitochondrial–nuclear interactions are part of a complex molecular network involved in the control of aging processes. The generation of reactive oxygen species at the mitochondrial respiratory chain plays a major role in this network. Mitochondrial DNA instabilities, which are under the control of nuclear genes, affect the generation of reactive oxygen species and modulate the rate of aging. As mitochondria become dysfunctional, they transduce signals to the nucleus and induce the expression of a set of nuclear genes, a process termed retrograde regulation. Molecular data are emerging which suggest that retrograde regulation is involved in lifespan control. © 1999 Elsevier Science Inc. All rights reserved.
1. Introduction Aging is a fundamental process found in almost all biological systems. It may be defined as a time-dependent loss of function and an exponential increase in mortality rate. Today, the underlying mechanisms are still not understood in detail in any system. However, efforts to unravel these mechanisms in particular in humans are currently reinforced by various research programs. These initiatives, in particular in the area of biomedical aging research, are not only of academic interest but also of social significance. In the near future, in a “graying society,” a major challenge is the development of efficient therapies and prophylaxis strategies directed against the various age-related diseases and disabilities (e.g., Alzheimer’s disease, cancer, cardiovascular diseases) that severely impair human healthspan and thus the quality of life in older age. To reach these important goals, experimental gerontology is performed on different levels using various systems. In addition to mammalian species, a number of experimental * Corresponding author. Tel.: ⫹49-69-798-29264; fax: ⫹49-69-798-29363. E-mail address: [email protected] (H.D. Osiewacz) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 6 3 - 7
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models are investigated for different reasons. At least some of these systems (e.g., yeast, filamentous fungi, Caenorhabditis elegans) are much simpler in their organization and are better accessible to experimental manipulations than higher systems. In addition, in these models lifespan is much shorter than in humans or any mammalian species. In this mini-review, we focus on experimental aging research in a group of lower model systems: the fungi. Only a few species have been investigated in the past but some have now been analyzed in great detail. At the molecular level, fungi were the first systems in which it became clear that the mitochondrion and age-related changes in the mitochondrial DNA (mtDNA) are part of the process leading to degenerations at the organismal level. Because mitochondria are semiautonomous organelles, which is that only a very limited number of components are encoded by the mtDNA, nuclear-encoded gene products are essential for the function of these organelles. In this paper, we will emphasize mitochondrial-nuclear interactions involved in the control of lifespan in fungal aging models.
2. The biology of mitochondria Mitochondria are eukaryotic organelles involved in energy transduction, the production of adenosine triphosphate (ATP) from energy–rich biomolecules (e.g., glucose). These processes are essential for survival of most eukaryotes (obligate aerobes). Only some species, like the yeast Saccharomyces cerevisiae (facultative aerobes), can produce enough ATP by glycolysis, a pathway occurring in the cytoplasm that is independent of functional mitochondria. The mitochondrial matrix space, in which different metabolic pathways take place (e.g., the tricarbocylic acid cycle: TCA cycle), is surrounded by two biomembranes. The inner membrane contains the respiratory chain, a highly organized system of usually four membrane-bound protein complexes that are involved in electron transport. During this process, protons derived from energy–rich biomolecules metabolized in the cytoplasm or in the mitochondrial matrix are transported across the inner membrane into the intermembrane space. This process generates an electrochemical gradient (⌬⌿) that is the driving force by which ATP is synthesized at another membraneinserted protein complex, the ATP-synthase (complex V). Only very few of the individual proteins of the respiratory chain (e.g., 13 in Podospora anserina) are encoded by the mtDNA found in several molecules in each mitochondrion. These are proteins of complexes I, III, IV, and V. Complex II and all of the other proteins of the TCA cycle are nuclear-encoded and are imported from the cytoplasm. Highly organized translocases located in both membranes (translocase of the outer membrane: TOM; translocase of the inner membrane: TIM) control the import and targeting of cytoplasmic proteins to the different mitochondrial locations: the outer and the inner membrane, the intermembrane space, and the matrix. The individual components of these complexes are all nuclear-encoded. The same holds true for other import and export complexes, like the porin and the adenine nucleotide transporter (ANT). Apart from the few proteins of the respiratory chain encoded by the mtDNA, in some organisms, additional open reading frames are encoded by the mtDNA. In most cases, the products of these ORFs are unknown, but in a few, specific functions have been attributed to them (see below). As a consequence of separation of the genetic information encoding the different components of functional mitochondria, well-coordinated expression and selective transport of components is essential for mitochondrial biogenesis. Control of nuclear-mito-
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chondrial interactions are particularly important in growing cells, tissues, or organisms in which mitochondria are propagating by division. In addition, it is critical that the mtDNA itself is replicated with high fidelity. All enzymes needed for mtDNA expression (transcription, translation) and all enzymes involved in mtDNA replication are nuclearencoded. In contrast, all RNA’s of the mitochondrial protein biosynthesis apparatus are encoded by the mtDNA. Given the essential function of mitochondria in energy transduction it is not surprising that dysfunctional mitochondria lead to severe impairments and specific degenerative phenotypes. For more than 20 years now, mitochondria and specific genetic instabilities in mitochondria have been put forward to explain aging processes observed in the filamentous fungus P. anserina and, shortly later, in the related ascomycetes Neurospora crassa and N. intermedia (for review see Osiewacz, 1995, 1997). After a brief summary of these early data, we will concentrate on some more recent investigations and will draw a picture explaining fungal senescence as the result of mitochondrial dysfunction and processes in which mitochondrial-nuclear interactions are involved.
3. Age-related mitochondrial dysfunction The first clear evidence that mitochondria, more specifically mtDNA instabilities, play a major role in aging and in the control of lifespan was derived from genetic data and from a subsequent molecular analysis of the filamentous fungus P. anserina. In contrast to most fungi, all wild-type strains of P. anserina senesce after prolonged vegetative propagation. The senescent phenotype is characterized by a reduction of the linear growth rate, a reduced formation of aerial hyphae, and finally by cellular death (Rizet, 1953; Marcou, 1961). Lifespan varies between a few days and about four months depending on the geographical race (Esser and Tudzynski, 1980). The aging process is under the control of genetic factors, both nuclear and extranuclear traits, and depends on environmental conditions. A comparative analysis of the mtDNA of different wild strains revealed that in senescent cultures, a specific DNA species accumulates. This DNA, termed plDNA (plasmid-like DNA), is a covalently closed circular molecule and is derived from the first intron (pl-intron) of cytochrome c oxidase subunit I gene (COI) (Stahl et al., 1978; Cummings et al., 1979; Ku¨ck et al., 1981; Osiewacz and Esser, 1984; Cummings et al., 1985). The pl-intron is a mobile genetic element. It is able to transpose into different positions of the mtDNA (Sellem et al., 1993) leading to the formation of repeated sequences. Importantly, transposition of the intron seems to be dependent on the activity of a reverse transcriptase, a protein encoded by the open reading frame of the pl-intron (Osiewacz and Esser, 1984; Michel and Lang, 1985; Steinhilber et al., 1986; Fassbender et al., 1994). The sequence duplications of the pl-intron are thought to be the targets of homologous recombination that give rise to both, the formation and accumulation of defective mtDNA’s and the plDNA molecules during aging of P. anserina wild-type cultures (Ku¨ck et al., 1985; Osiewacz, 1992). Depending on whether or not the resulting mtDNA subcircles contain an origin of replication, they are retained or become lost. As a result, the mtDNA of senescent cultures of wild-type strains of P. anserina harbor large deletions, spanning essential coding sequences; consequently the cultures die at the hyphal tips due to severe energy deficits. Interestingly, in N. crassa and N. intermedia different strains show a senescent phenotype that was also found to be related to mitochondrial dysfunction due to age-
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related modifications in the mtDNA. However, in these cases it was not the accumulation of molecules with deletions that accumulate during senescence. In these cases, the modifications were due to the integration of normally autonomous elements into mtDNA (Bertrand et al., 1985; Akins et al., 1986; Chan et al., 1991; Court et al., 1991). The integration of these elements into essential sequences leads to a disruption of the corresponding genetic information. During aging of cultures, the impaired mtDNA molecules become suppressive by an unknown mechanism leading to mitochondrial dysfunction and death of the corresponding cultures. Most intriguingly, in the cases described, deficits in energy transduction are correlated with mtDNA instabilities and senescence. In P. anserina, it was even believed that mtDNA reorganizations that were found in the different senescing wild-strains are a prerequisite of aging. Evidence was derived from the analyses of different wild-type strains and from certain immortal strains in which the mtDNA was found to be stabilized (Tudzynski et al., 1982; Schulte et al., 1988). However, recently, the characterization of additional long-lived mutants of P. anserina revealed that these strains still senesce despite the fact that the severe wild-type specific mtDNA reorganizations and specifically the amplification of the pl-intron do not occur. The long-lived mutant grisea is such a mutant of P. anserina (Prillinger and Esser, 1977). The inheritance of this long-lived phenotype (56% increased lifespan) is according to the Mendelian rules. The strain is further characterized by a reduced pigmentation of the ascospore (gray instead of black), a reduced growth rate and an impairment in the formation of female gametangia. Interestingly, the phenotype can be reverted by additional copper in the growth medium (Marbach et al., 1994). By using a complementation approach, the wild-type copy of Grisea was cloned and subsequently the mutant copy was selected from a mutant gene bank by hybridization with the cloned wild-type sequence of Grisea. The characterization of the two alleles revealed that the mutant is a splicing deficiency mutant and that GRISEA is an ortholog of MAC1, a yeast transcription factor involved in the control of cellular copper homeostasis (Osiewacz and Nuber, 1996; Borghouts and Osiewacz, 1998). Most surprisingly, it turned out that the age-related amplification of the pl-intron, which was thought to be a prerequisite for aging, does not occur in senescent cultures of the mutant (Borghouts et al., 1997). The same holds true for strains in which the nuclear genes Su12 and As6 are mutated, two genes coding for the cytosolic ribosomal proteins S7 and S19, respectively (Silar et al., 1997). In two further mutants, mutants that encode two components of the TOM complex, the amplification of the pl-intron was also not observed. However, in senescent cultures of these strains specific deleted mtDNA molecules accumulated (Jamet–Vierny et al., 1997). And finally, a mutant of P. anserina in which the pl-intron is exactly deleted from the mtDNA and thus is not available for amplification, does still senesce (Begel et al., 1999). From these data it is now clear that, although mtDNA reorganizations do have an important impact on the lifespan of the above mentioned filamentous fungi, the specific amplification of the pl-intron is not the prerequisite for senescence in P. anserina cultures but rather a modulator of the rate of aging. It is intriguing that plDNA has been found to be involved in aging of all analyzed wild-type strains. It thus may play a specific role in wild-type aging and may be related to the specific environmental niche, that is herbivorous dung, to which P. anserina has adapted. Mutant strains like grisea that are viable under laboratory conditions would not survive under natural conditions. Although these mutants do senesce, parts of the natural aging mechanism may be missing. These mutants are of
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special relevance because, in them, different pathways of the molecular aging machinery are visible, which in wild-type are overlaid by other mechanisms. But what are the basic mechanisms of aging that lead to senescence even if the wild-type specific gross mtDNA reorganizations do not occur? From the available data it seems most likely that in P. anserina also the generation of reactive oxygen species (ROS) and the age-dependent increase of oxidative stress is part of the basic molecular aging mechanism. ROS formation is not restricted to the wild-type but also occurs in nuclear mutant strains, like the long-lived mutant grisea, although, due to a stabilization of the mtDNA, ROS generation at the respiratory chain seems to be reduced. Normally, and this is also thought to be the case in higher systems including mammals, the mitochondrial respiratory chain does not exclusively transfer four electrons to molecular oxygen at complex IV but, if disturbed for different reasons, single electrons “leak” to molecular oxygen (for review see Wallace, 1999). This leads to the generation of the superoxide anion that can be converted to hydrogen peroxide (H2O2). In the presence of reduced transition metals, like Fe(II) and Cu(I), the highly reactive hydroxyl radical is formed (Fig. 1a). It is this molecule that is able to very efficiently damage almost every cellular component (e.g., membranes, proteins, DNA) leading to molecular damage and subsequent cellular dysfunction (Fig. 1a). Apart from being involved in damaging processes, ROS are thought to serve as molecular signals. In particular in plants, the expression of an alternative terminal oxidase, the cyanide resistant and salicyl hydroxamic acid (SHAM) sensitive oxidase (AOX), is thought to be induced by ROS such as superoxide, hydrogen peroxide, or a hydroxyl radical (for review see: Vanlerberghe and McIntosh, 1997). The AOX is an enzyme encoded by a nuclear gene. It has been demonstrated to occur in plants, some yeasts (not Saccharomyces cerevisiae), and some protists. In plants, the AOX is known to contribute to a defense system by preventing over-reduction that leads to the production of ROS (Wagner and Moore, 1997; Sluse and Jarmuszkiewicz, 1998). Interestingly, although not proven experimentally, such a reduced production of ROS as the result of the induction of an AOX seems to be responsible for lifespan extension in certain long-lived mutants of P. anserina. In one immortal mutant, ex1, it has been demonstrated that the activity of the AOX is strongly increased in comparison to the wild-type strain (Schulte et al., 1988). Importantly, the induction of the Aox gene is correlated with a deletion of part of the mitochondrial COI gene giving rise to a block in respiratory chain at complex IV. Due to the induction of the AOX the electron transport pathway branches most likely at the ubiquione pool (Fig. 1b). The idea that the reduced production of ROS in long-lived mutant ex1 is responsible for lifespan extension is supported by the observation that mutant ex1 senesces if oxidative stress is increased by the addition of millimolar amounts of Fe2⫹ salts to the medium (Frese and Stahl, 1992). The induction of nuclear genes as a result of mitochondrial dysfunction, as just described for the immortal mutant ex1 of P. anserina, has also been observed to occur in Saccharomyces cerevisiae where it was termed retrograde regulation (Liao and Butow, 1993). Interestingly, very recently, retrograde regulation has been correlated with lifespan control in S. cerevisiae (Kirchman et al., 1999). Until now, in this species, mitochondria were not considered to be of significance with respect to lifespan control. In part, this was due to the ability of yeast, as a facultative aerobe, to survive in the absence of functional mitochondria. In a recent study different strains with impaired mitochondrial functions were found to display a long-lived phenotype. Interestingly, in these strains Cit2, a marker gene of retrograde regulation was up-regulated. It seems that a signal, the nature of which
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Fig. 1. (a) Oxidative phosphorylation (OXPHOS) at the inner mitochondrial membrane. Electrons enter the respiratory chain at complex I and II and are transported to complex IV. The four electrons are transferred to molecular oxygen resulting in the formation of water. At the same time, protons are transported at complexes I, III, and IV across the membrane into the intermembrane space. At complex V protons are transported in the opposite direction leading to the condensation of ADP and phosphate (Pi) to ATP. If the respiratory chain is affected for different reasons, single electrons may be transferred to molecular oxygen giving rise to the superoxide anion. MnSOD may convert this ROS to hydrogen peroxide that may be reduced to water by glutathione peroxidase (GPx). In the presence of reduced iron or copper, the aggressive hydoxyl radical may be formed that is able to damaged other cellular molecules. (2b) In mitochondria of higher plants, some fungi, and some protists, mitochondrial dysfunction, which normally would lead to an increased formation of ROS, induce the expression of an alternative oxidase (AOX). The alternative electron transport pathway branches at the ubiquinone pool. The AOX, like complex IV, transfers four electrons to one molecule oxygen leading to the formation of water. It seems that the activity of the AOX reduces the formation of mitochondrial ROS significantly. This protein is considered to be part of the defense systems against oxidative stress.
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is not clear at present, is transmitted from impaired mitochondria leading to the induction of a set of nuclear genes. Among these, genes affecting lifespan seem to be induced. Currently, the nature of these genes is unclear. A gene encoding a SHAM sensitive alternative oxidase cannot be induced because in S. cerevisiae such an enzyme does not occur. In conclusion, investigations of about the last twenty years revealed a clear mitochondrial basis of aging in fungi. Mitochondria are very complex organelles. Their biogenesis is dependent on various well– ordered processes (e.g., coordinate expression of nuclear and mitochondrial genes, targeting of gene products, assembly of multienzyme complexes). Any defect in any process may lead to severe dysfunctions and may be responsible for the various degenerative syndromes (including aging) occurring in nature. Importantly, a mitochondrial etiology of aging is not restricted to filamentous fungi but also holds true for yeast and other species including humans (for review see: Osiewacz and Hermanns, 1992; Wallace, 1999). Although the molecular mechanisms involved in the control of aging may differ in their details from system to system it is clear that mitochondrial oxidative stress plays a major role in lifespan control in various systems. It is also clear that, in the future, experimental aging research performed with different species, including simple model systems, will be relevant to elucidating the molecular details of nuclear-mitochondrial interactions, ROS generating systems, and efficient cellular defense systems.
Acknowledgments We wish to thank Dr. M. Silliker (De Paul University, Chicago, USA) for carefully reading of the manuscript and the Deutsche Forschungsgemeinschaft (Bonn, Germany) for the continuous support of the experimental work.
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Mini-review
Mitochondrial-nuclear interactions and lifespan control in fungi Heinz D. Osiewacz*, Erik Kimpel Botanisches Institut, Molekulare Entwicklungsbiologie und Biotechnologie, Johann Wolfgang Goethe–Universita¨t, Marie–Curie–Str. 9, D-60439 Frankfurt am Main, Germany Received 12 July 1999; received in revised form 25 August 1999; accepted 26 August 1999
Abstract In fungi, mitochondrial–nuclear interactions are part of a complex molecular network involved in the control of aging processes. The generation of reactive oxygen species at the mitochondrial respiratory chain plays a major role in this network. Mitochondrial DNA instabilities, which are under the control of nuclear genes, affect the generation of reactive oxygen species and modulate the rate of aging. As mitochondria become dysfunctional, they transduce signals to the nucleus and induce the expression of a set of nuclear genes, a process termed retrograde regulation. Molecular data are emerging which suggest that retrograde regulation is involved in lifespan control. © 1999 Elsevier Science Inc. All rights reserved.
1. Introduction Aging is a fundamental process found in almost all biological systems. It may be defined as a time-dependent loss of function and an exponential increase in mortality rate. Today, the underlying mechanisms are still not understood in detail in any system. However, efforts to unravel these mechanisms in particular in humans are currently reinforced by various research programs. These initiatives, in particular in the area of biomedical aging research, are not only of academic interest but also of social significance. In the near future, in a “graying society,” a major challenge is the development of efficient therapies and prophylaxis strategies directed against the various age-related diseases and disabilities (e.g., Alzheimer’s disease, cancer, cardiovascular diseases) that severely impair human healthspan and thus the quality of life in older age. To reach these important goals, experimental gerontology is performed on different levels using various systems. In addition to mammalian species, a number of experimental * Corresponding author. Tel.: ⫹49-69-798-29264; fax: ⫹49-69-798-29363. E-mail address: [email protected] (H.D. Osiewacz) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 6 3 - 7
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models are investigated for different reasons. At least some of these systems (e.g., yeast, filamentous fungi, Caenorhabditis elegans) are much simpler in their organization and are better accessible to experimental manipulations than higher systems. In addition, in these models lifespan is much shorter than in humans or any mammalian species. In this mini-review, we focus on experimental aging research in a group of lower model systems: the fungi. Only a few species have been investigated in the past but some have now been analyzed in great detail. At the molecular level, fungi were the first systems in which it became clear that the mitochondrion and age-related changes in the mitochondrial DNA (mtDNA) are part of the process leading to degenerations at the organismal level. Because mitochondria are semiautonomous organelles, which is that only a very limited number of components are encoded by the mtDNA, nuclear-encoded gene products are essential for the function of these organelles. In this paper, we will emphasize mitochondrial-nuclear interactions involved in the control of lifespan in fungal aging models.
2. The biology of mitochondria Mitochondria are eukaryotic organelles involved in energy transduction, the production of adenosine triphosphate (ATP) from energy–rich biomolecules (e.g., glucose). These processes are essential for survival of most eukaryotes (obligate aerobes). Only some species, like the yeast Saccharomyces cerevisiae (facultative aerobes), can produce enough ATP by glycolysis, a pathway occurring in the cytoplasm that is independent of functional mitochondria. The mitochondrial matrix space, in which different metabolic pathways take place (e.g., the tricarbocylic acid cycle: TCA cycle), is surrounded by two biomembranes. The inner membrane contains the respiratory chain, a highly organized system of usually four membrane-bound protein complexes that are involved in electron transport. During this process, protons derived from energy–rich biomolecules metabolized in the cytoplasm or in the mitochondrial matrix are transported across the inner membrane into the intermembrane space. This process generates an electrochemical gradient (⌬⌿) that is the driving force by which ATP is synthesized at another membraneinserted protein complex, the ATP-synthase (complex V). Only very few of the individual proteins of the respiratory chain (e.g., 13 in Podospora anserina) are encoded by the mtDNA found in several molecules in each mitochondrion. These are proteins of complexes I, III, IV, and V. Complex II and all of the other proteins of the TCA cycle are nuclear-encoded and are imported from the cytoplasm. Highly organized translocases located in both membranes (translocase of the outer membrane: TOM; translocase of the inner membrane: TIM) control the import and targeting of cytoplasmic proteins to the different mitochondrial locations: the outer and the inner membrane, the intermembrane space, and the matrix. The individual components of these complexes are all nuclear-encoded. The same holds true for other import and export complexes, like the porin and the adenine nucleotide transporter (ANT). Apart from the few proteins of the respiratory chain encoded by the mtDNA, in some organisms, additional open reading frames are encoded by the mtDNA. In most cases, the products of these ORFs are unknown, but in a few, specific functions have been attributed to them (see below). As a consequence of separation of the genetic information encoding the different components of functional mitochondria, well-coordinated expression and selective transport of components is essential for mitochondrial biogenesis. Control of nuclear-mito-
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chondrial interactions are particularly important in growing cells, tissues, or organisms in which mitochondria are propagating by division. In addition, it is critical that the mtDNA itself is replicated with high fidelity. All enzymes needed for mtDNA expression (transcription, translation) and all enzymes involved in mtDNA replication are nuclearencoded. In contrast, all RNA’s of the mitochondrial protein biosynthesis apparatus are encoded by the mtDNA. Given the essential function of mitochondria in energy transduction it is not surprising that dysfunctional mitochondria lead to severe impairments and specific degenerative phenotypes. For more than 20 years now, mitochondria and specific genetic instabilities in mitochondria have been put forward to explain aging processes observed in the filamentous fungus P. anserina and, shortly later, in the related ascomycetes Neurospora crassa and N. intermedia (for review see Osiewacz, 1995, 1997). After a brief summary of these early data, we will concentrate on some more recent investigations and will draw a picture explaining fungal senescence as the result of mitochondrial dysfunction and processes in which mitochondrial-nuclear interactions are involved.
3. Age-related mitochondrial dysfunction The first clear evidence that mitochondria, more specifically mtDNA instabilities, play a major role in aging and in the control of lifespan was derived from genetic data and from a subsequent molecular analysis of the filamentous fungus P. anserina. In contrast to most fungi, all wild-type strains of P. anserina senesce after prolonged vegetative propagation. The senescent phenotype is characterized by a reduction of the linear growth rate, a reduced formation of aerial hyphae, and finally by cellular death (Rizet, 1953; Marcou, 1961). Lifespan varies between a few days and about four months depending on the geographical race (Esser and Tudzynski, 1980). The aging process is under the control of genetic factors, both nuclear and extranuclear traits, and depends on environmental conditions. A comparative analysis of the mtDNA of different wild strains revealed that in senescent cultures, a specific DNA species accumulates. This DNA, termed plDNA (plasmid-like DNA), is a covalently closed circular molecule and is derived from the first intron (pl-intron) of cytochrome c oxidase subunit I gene (COI) (Stahl et al., 1978; Cummings et al., 1979; Ku¨ck et al., 1981; Osiewacz and Esser, 1984; Cummings et al., 1985). The pl-intron is a mobile genetic element. It is able to transpose into different positions of the mtDNA (Sellem et al., 1993) leading to the formation of repeated sequences. Importantly, transposition of the intron seems to be dependent on the activity of a reverse transcriptase, a protein encoded by the open reading frame of the pl-intron (Osiewacz and Esser, 1984; Michel and Lang, 1985; Steinhilber et al., 1986; Fassbender et al., 1994). The sequence duplications of the pl-intron are thought to be the targets of homologous recombination that give rise to both, the formation and accumulation of defective mtDNA’s and the plDNA molecules during aging of P. anserina wild-type cultures (Ku¨ck et al., 1985; Osiewacz, 1992). Depending on whether or not the resulting mtDNA subcircles contain an origin of replication, they are retained or become lost. As a result, the mtDNA of senescent cultures of wild-type strains of P. anserina harbor large deletions, spanning essential coding sequences; consequently the cultures die at the hyphal tips due to severe energy deficits. Interestingly, in N. crassa and N. intermedia different strains show a senescent phenotype that was also found to be related to mitochondrial dysfunction due to age-
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related modifications in the mtDNA. However, in these cases it was not the accumulation of molecules with deletions that accumulate during senescence. In these cases, the modifications were due to the integration of normally autonomous elements into mtDNA (Bertrand et al., 1985; Akins et al., 1986; Chan et al., 1991; Court et al., 1991). The integration of these elements into essential sequences leads to a disruption of the corresponding genetic information. During aging of cultures, the impaired mtDNA molecules become suppressive by an unknown mechanism leading to mitochondrial dysfunction and death of the corresponding cultures. Most intriguingly, in the cases described, deficits in energy transduction are correlated with mtDNA instabilities and senescence. In P. anserina, it was even believed that mtDNA reorganizations that were found in the different senescing wild-strains are a prerequisite of aging. Evidence was derived from the analyses of different wild-type strains and from certain immortal strains in which the mtDNA was found to be stabilized (Tudzynski et al., 1982; Schulte et al., 1988). However, recently, the characterization of additional long-lived mutants of P. anserina revealed that these strains still senesce despite the fact that the severe wild-type specific mtDNA reorganizations and specifically the amplification of the pl-intron do not occur. The long-lived mutant grisea is such a mutant of P. anserina (Prillinger and Esser, 1977). The inheritance of this long-lived phenotype (56% increased lifespan) is according to the Mendelian rules. The strain is further characterized by a reduced pigmentation of the ascospore (gray instead of black), a reduced growth rate and an impairment in the formation of female gametangia. Interestingly, the phenotype can be reverted by additional copper in the growth medium (Marbach et al., 1994). By using a complementation approach, the wild-type copy of Grisea was cloned and subsequently the mutant copy was selected from a mutant gene bank by hybridization with the cloned wild-type sequence of Grisea. The characterization of the two alleles revealed that the mutant is a splicing deficiency mutant and that GRISEA is an ortholog of MAC1, a yeast transcription factor involved in the control of cellular copper homeostasis (Osiewacz and Nuber, 1996; Borghouts and Osiewacz, 1998). Most surprisingly, it turned out that the age-related amplification of the pl-intron, which was thought to be a prerequisite for aging, does not occur in senescent cultures of the mutant (Borghouts et al., 1997). The same holds true for strains in which the nuclear genes Su12 and As6 are mutated, two genes coding for the cytosolic ribosomal proteins S7 and S19, respectively (Silar et al., 1997). In two further mutants, mutants that encode two components of the TOM complex, the amplification of the pl-intron was also not observed. However, in senescent cultures of these strains specific deleted mtDNA molecules accumulated (Jamet–Vierny et al., 1997). And finally, a mutant of P. anserina in which the pl-intron is exactly deleted from the mtDNA and thus is not available for amplification, does still senesce (Begel et al., 1999). From these data it is now clear that, although mtDNA reorganizations do have an important impact on the lifespan of the above mentioned filamentous fungi, the specific amplification of the pl-intron is not the prerequisite for senescence in P. anserina cultures but rather a modulator of the rate of aging. It is intriguing that plDNA has been found to be involved in aging of all analyzed wild-type strains. It thus may play a specific role in wild-type aging and may be related to the specific environmental niche, that is herbivorous dung, to which P. anserina has adapted. Mutant strains like grisea that are viable under laboratory conditions would not survive under natural conditions. Although these mutants do senesce, parts of the natural aging mechanism may be missing. These mutants are of
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special relevance because, in them, different pathways of the molecular aging machinery are visible, which in wild-type are overlaid by other mechanisms. But what are the basic mechanisms of aging that lead to senescence even if the wild-type specific gross mtDNA reorganizations do not occur? From the available data it seems most likely that in P. anserina also the generation of reactive oxygen species (ROS) and the age-dependent increase of oxidative stress is part of the basic molecular aging mechanism. ROS formation is not restricted to the wild-type but also occurs in nuclear mutant strains, like the long-lived mutant grisea, although, due to a stabilization of the mtDNA, ROS generation at the respiratory chain seems to be reduced. Normally, and this is also thought to be the case in higher systems including mammals, the mitochondrial respiratory chain does not exclusively transfer four electrons to molecular oxygen at complex IV but, if disturbed for different reasons, single electrons “leak” to molecular oxygen (for review see Wallace, 1999). This leads to the generation of the superoxide anion that can be converted to hydrogen peroxide (H2O2). In the presence of reduced transition metals, like Fe(II) and Cu(I), the highly reactive hydroxyl radical is formed (Fig. 1a). It is this molecule that is able to very efficiently damage almost every cellular component (e.g., membranes, proteins, DNA) leading to molecular damage and subsequent cellular dysfunction (Fig. 1a). Apart from being involved in damaging processes, ROS are thought to serve as molecular signals. In particular in plants, the expression of an alternative terminal oxidase, the cyanide resistant and salicyl hydroxamic acid (SHAM) sensitive oxidase (AOX), is thought to be induced by ROS such as superoxide, hydrogen peroxide, or a hydroxyl radical (for review see: Vanlerberghe and McIntosh, 1997). The AOX is an enzyme encoded by a nuclear gene. It has been demonstrated to occur in plants, some yeasts (not Saccharomyces cerevisiae), and some protists. In plants, the AOX is known to contribute to a defense system by preventing over-reduction that leads to the production of ROS (Wagner and Moore, 1997; Sluse and Jarmuszkiewicz, 1998). Interestingly, although not proven experimentally, such a reduced production of ROS as the result of the induction of an AOX seems to be responsible for lifespan extension in certain long-lived mutants of P. anserina. In one immortal mutant, ex1, it has been demonstrated that the activity of the AOX is strongly increased in comparison to the wild-type strain (Schulte et al., 1988). Importantly, the induction of the Aox gene is correlated with a deletion of part of the mitochondrial COI gene giving rise to a block in respiratory chain at complex IV. Due to the induction of the AOX the electron transport pathway branches most likely at the ubiquione pool (Fig. 1b). The idea that the reduced production of ROS in long-lived mutant ex1 is responsible for lifespan extension is supported by the observation that mutant ex1 senesces if oxidative stress is increased by the addition of millimolar amounts of Fe2⫹ salts to the medium (Frese and Stahl, 1992). The induction of nuclear genes as a result of mitochondrial dysfunction, as just described for the immortal mutant ex1 of P. anserina, has also been observed to occur in Saccharomyces cerevisiae where it was termed retrograde regulation (Liao and Butow, 1993). Interestingly, very recently, retrograde regulation has been correlated with lifespan control in S. cerevisiae (Kirchman et al., 1999). Until now, in this species, mitochondria were not considered to be of significance with respect to lifespan control. In part, this was due to the ability of yeast, as a facultative aerobe, to survive in the absence of functional mitochondria. In a recent study different strains with impaired mitochondrial functions were found to display a long-lived phenotype. Interestingly, in these strains Cit2, a marker gene of retrograde regulation was up-regulated. It seems that a signal, the nature of which
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Fig. 1. (a) Oxidative phosphorylation (OXPHOS) at the inner mitochondrial membrane. Electrons enter the respiratory chain at complex I and II and are transported to complex IV. The four electrons are transferred to molecular oxygen resulting in the formation of water. At the same time, protons are transported at complexes I, III, and IV across the membrane into the intermembrane space. At complex V protons are transported in the opposite direction leading to the condensation of ADP and phosphate (Pi) to ATP. If the respiratory chain is affected for different reasons, single electrons may be transferred to molecular oxygen giving rise to the superoxide anion. MnSOD may convert this ROS to hydrogen peroxide that may be reduced to water by glutathione peroxidase (GPx). In the presence of reduced iron or copper, the aggressive hydoxyl radical may be formed that is able to damaged other cellular molecules. (2b) In mitochondria of higher plants, some fungi, and some protists, mitochondrial dysfunction, which normally would lead to an increased formation of ROS, induce the expression of an alternative oxidase (AOX). The alternative electron transport pathway branches at the ubiquinone pool. The AOX, like complex IV, transfers four electrons to one molecule oxygen leading to the formation of water. It seems that the activity of the AOX reduces the formation of mitochondrial ROS significantly. This protein is considered to be part of the defense systems against oxidative stress.
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is not clear at present, is transmitted from impaired mitochondria leading to the induction of a set of nuclear genes. Among these, genes affecting lifespan seem to be induced. Currently, the nature of these genes is unclear. A gene encoding a SHAM sensitive alternative oxidase cannot be induced because in S. cerevisiae such an enzyme does not occur. In conclusion, investigations of about the last twenty years revealed a clear mitochondrial basis of aging in fungi. Mitochondria are very complex organelles. Their biogenesis is dependent on various well– ordered processes (e.g., coordinate expression of nuclear and mitochondrial genes, targeting of gene products, assembly of multienzyme complexes). Any defect in any process may lead to severe dysfunctions and may be responsible for the various degenerative syndromes (including aging) occurring in nature. Importantly, a mitochondrial etiology of aging is not restricted to filamentous fungi but also holds true for yeast and other species including humans (for review see: Osiewacz and Hermanns, 1992; Wallace, 1999). Although the molecular mechanisms involved in the control of aging may differ in their details from system to system it is clear that mitochondrial oxidative stress plays a major role in lifespan control in various systems. It is also clear that, in the future, experimental aging research performed with different species, including simple model systems, will be relevant to elucidating the molecular details of nuclear-mitochondrial interactions, ROS generating systems, and efficient cellular defense systems.
Acknowledgments We wish to thank Dr. M. Silliker (De Paul University, Chicago, USA) for carefully reading of the manuscript and the Deutsche Forschungsgemeinschaft (Bonn, Germany) for the continuous support of the experimental work.
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