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Antioxidants prolong life span and inhibit the senescence- dependent accumulation of fluorescent pigment (lipofuscin) in clones of Podospora anserina
Mechanisms of Ageing and Development, 7 (1978) 407-415 ©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
407
A N T I O X I D A N T S P R O L O N G L I F E SPAN A N D I N H I B I T T H E S E N E S CENCE-DEPENDENT ACCUMULATION OF FLUORESCENT PIGMENT ( L I P O F U S C I N ) IN C L O N E S O F P O D O S P O R A A N S E R I N A ~ *
KENNETH D. MUNKRES and RAJENDRA S. RANA Laboratories of Molecular Biology and Genetics, University of Wisconsin, Madison, Wisconsin 53706 (U.S.A.) (Received March 30, 1977) SUMMARY Culture of clones of Podospora anserina s÷with either nordihydroguaiaretic acid or reduced glutathione (GSH) at concentrations that were not inhibitory to growth significantly prolonged the average time to onset of senescence. GSH also prolonged the average time to onset of clonal death. The specific concentration of chloroform--methanol soluble fluorescent pigment was larger in senescent than in pre-senescent cells. The pigment exhibited fluorescence excitation and emission spectra and fluorescence polarization numbers characteristic of lipofuscin, an end-product of lipid peroxidation. Analyses of the lipofuscin concentration in either sub-clonal fractions of different times of origin from senescent clones, or in' sub-clonal fractions of identical age in time of origin from parent clones of different age, revealed a similar concentration distribution. Although pre-senescent cells contained rather large concentrations, a massive increase occurred during senescence prior to the time of onset of clonal death. Culture with GSH not only prolonged clonal life span but also inhibited the formation of lipofuscin by an average factor of 30. Furthermore, unlike untreated clones, the sub-clonal distribution of the pigment was not only low but was also independent of their age.
INTRODUCTION The growth of a mycelium ofPodospora anserina undergoes irreversible cessation of growth, /.e., senescence, after a period of time which is characteristic of a given race.
*Contribution no. 2130 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706 (U.S.A.).
408 Marcou [1] suggested that a spontaneous variant cytoplasmic factor was responsible for senescence. Subsequent studies by Smith and Rubenstein [2, 3] supported this hypothesis and demonstrated that the growth parameters of a given race are inherited cytoplasmically;/.e., the frequency of occurrence of a cytoplasmic variant factor and the rate of increase of the variant factor are under cytoplasmic genetic control. Additional evidence for the genetic control of senescence was recently reported by Esser and Keller [4]. Senescence can be completely prevented by the synergistic action of two linked nuclear genes incoloris and vivax. Whereas the wild-type strain s becomes senescent after 26 days and the mutants incoloris and vivax after 42 and 66 days, respectively, the double mutant showed no signs of senescence after culture for more than one year. Although the genetical basis of senescence of P. anserina has been thoroughly investigated, the nature of the "infective" senescence "agent" has remained obscure. All efforts to demonstrate viruses or virus-like particles in ageing hyphae ofP. anserina have failed [5]. Whatever the senescence agent or biochemical damage during senescence may be, P. anserina being homothallic can undergo sexual reproduction without transmission of senescence to the progeny, thus insuring survival of the species as in higher eukaryotes. Our investigations of the molecular biology of senescence with three model systems of Neurospora crassa, a close relative ofP. anserina, have revealed that membrane deterioration, lipid peroxidation and free radical reactions are interdependent processes that contribute to the time-dependent deterioration of clonal growth rate (senescence) and cellular death [7-13]. Continuous administration of drugs that act as free radical scavengers (antioxidants) or membrane stabilizers not only prolongs the life span in these systems, but also inhibits the occurrence of a number of biochemical and cytological abnormalities related to membrane deterioration, lipid peroxidation, and damaging free radical reactions. One of the oldest and best established molecular indices of ageing is the intracellular accumulation of fluorescent pigment, sometimes called lipofuscin or "age" pigment. This pigment accumulates with age in all organisms thus far examined, ranging from fungi to man [7]. Furthermore, the rate of accumulation of the pigment appears to be proportional to the rate of ageing [14]. In several organisms or cell types, administration of dietary antioxidants not only prolongs life span but also inhibits the accumulation of lipofuscin [7]. According to Tappel, it is not the accumulation of lipofuscin per se that is detrimental to cells. Rather, the occurrence of lipofuscin indicates the occurrence of much more extensive non-fluorescent damage to molecules by free radical reactions which accompany lipid peroxidation and the f°nnati°n of lipofuscin [ 14]. In general, fluorescent age pigment arises by the reaction of amino groups with an end product of lipid peroxidation, malondialdehyde: 2RNH2 + H O - C H = C H - C H O ~ RNH-HC=CH-CH-----N-R forming either an intra- or inter-molecular iminopropene bond with characteristic fluorescence spectra with activation maximum 340-370 nm and emission maximum of 420-
409 470 nm. The R-group may be protein, phospholipid, RNA or DNA. The nature of the Rgroup apparently does not greatly influence the fluorescence spectrum but does affect the solubility properties [14]. In the present paper, we describe the age-dependent accumulation of one class of lipofuscin pigments, soluble in chloroform-methanol, in sub-clonal fractions ofP. anserina. Since culture with antioxidants not only prolonged clonal life span, but also inhibited the accumulation of lipofuscin, we conclude that lipid peroxidation may be causallyrelated to clonal senescence.
MATERIAL AND METHODS
Stocks Homokaryotic stocks of P. anserina were provided by Dr. Karl Esser. They were grown on slants of minimal medium no. 2 [15] and stored at 4 °C. Growth and senescence A small piece of agar with mycelia and ascospores was taken from slants and centrally placed on a plate of minimal medium no. 2. The plates were incubated for 3 - 4 days at 30 °C. Cylindrical plugs of agar and mycelia (~5 mm dia.) were taken from the mycelial frontier and transferred to race tubes, 1.8 diam. × 100 cm length, containing minimal medium no. 2 with 2% sucrose and 1.5% agar. The tubes were incubated at 30 ~C in continuous fluorescent light. The position of the mycelial frontier was recorded at 24 h intervals. At least 10 replicate tubes of each treatment were measured. The time to onset of senescence (TOS) of a clone is def'med as that time when the growth rate began to decelerate. The time to onset of death (TOD) is defined as that time when the growth rate had decelerated to 10% or less of the maximum linear growth rate. Extraction and measurement of lipofuscin The methods for extraction and measurement of chloroform--methanol soluble fluorescent pigment (lipofuscin) were the same as those described in a previous paper in experiments with Neurospora crassa [ 13].
RESULTS Figures 1 and 6 illustrate the extensional growth of clones ofP. anserina s÷ at 30 °C. Growth proceeded at a linear rate of 0.36 + 0.02 mm/h for 580 +- 48 h. This time is defined as the average time to onset of senescence (TOS). At that time, some clones immediately ceased to grow and others decelerated in growth rate. The time when the growth rate was less than or equal to 10% of the maximal growth rate is defined as the time of onset of death (TOD).
410 3O0 TOS,TOD
/T TOS
200 A E
~
~,~-E ~o~o~ Oor
L 200
,oo
"
, 400 Time ( h )
TOO
, 600
8vO
Fig. 1. Typical extensional g r o w t h o f clones ofPodospora anserma s*. Clones were grown in race tubes
on minimal medium no. 2 at 30 °C as described in Methods.
,oo
g u
~ 5
o'5o
i
=
Z
I
%0
5be
600 Time (h)
~c;o
o ,00
5O0
6OO
7OO
8;0
Time (h)
Fig. 2. Prolongation of the time to onset of senescence of clones ofP. anserina s+ by nordihydroguaiarctic acid. Clones were grown as in Fig. 1 with 6.7 #M NDGA (X) and without NDGA (o). The % non-senescent clones in a population of 10 is noted. Fig. 3. Prolongation of the time to onset of senescence of clones ofP. anserina s+ by reduced glutathione. o, Control; x, with 1 mMGSH.
Culture with either nordihydroguaiaretic acid (Fig. 2) or reduced glutathione (Fig. 3) significantly prolonged the average time to onset o f senescence (Table I) by 25 and 14%, respectively. GSH, but not NDGA, also prolonged the average time to onset o f death by 14% (Table I). The concentrations o f NDGA (6.7 #M) and GSH (1 mM) neither stimulated nor inhibited the linear growth rates; however, a higher concentration o f NDGA ( 3 3 . 5 / a l / ) inhibited g r o w t h somewhat, and, therefore, was n o t tested for an effect upon senescence. Figure 4 illustrates the fluorescence excitation and emission spectra o f a chlorof o r m - m e t h a n o l soluble pigment that occurs in excess in senescent cells. The excitation
411 TABLE I PROLONGATION OF THE LIFE SPAN OF CLONES OF PODOSPORA ANSERIN.4 s+ BY NORDIHYDROGUAIARETIC ACID OR REDUCED GLUTATHIONE. Exp. No.*
Time (hi to onset o f Senescence
1
2
Death
Con~ol
+NGDA
P
Con~ol
+NDGA
P
430
538
0.16
645
645
1
Con~ol
+GSH
Control
+GSH
580
660
650
735
0.08
0.10
*Two independent experiments each with 10 clones per treatment. Values axe the average of 10 clones. NDGA, 6.7 Wl#;reduced glutathione, 1 raM. P is the probability that the mean of the differences of the two populations is not greater than zero. and emission maxima were 365 and 425 nm, respectively. In some preparations, an additional maximum was observed at 445 nm. The broad fluorescence peak around 500 nm was not found in all preparations and may be due to traces o f phytoene [16]. The fluorescence polarization number at the emission wavelength 425 nm was about 0.02-0.05. The fluorescence spectra and the high degree of fluorescence polarization are characteristic o f lipofuscin, an end product o f lipid peroxidation [ 13 ]. Analyses o f the specific concentrations of lipofuscin in sub-clonal fractions of a clone shortly after the time o f its death as a function of the age when they were formed revealed an age-dependent increase (Fig. 5). The youngest cells in time of origin contained the largest specific concentration. A five-fold increase of lipofuscin concentration occurred in the sub-clonal fractions which were formed during the time when the parent clone was senescent. Culture with 1 mM GSH inhibited the accumulation o f lipofuscin by an average factor o f 30. Furthermore, unlike the control, the degree o f accumulation was independent o f sub-clonal age with GSH (Fig. 5). In the experiment illustrated in Figs. 6 and 7, new clones were started at 72 h intervals over a period of about 27 days. When the oldest clones became senescent, samples o f uniform size were taken from the mycelial frontiers of all o f the clones (Fig. 6) and lipofuscin concentrations were determined (Fig. 7). Although the cells were all o f identical age in terms o f time of origin, the specific concentration o f lipofuscin as a function o f age of the parent clones was quite similar to the concentration distribution in subclones o f different times of origin (Fig. 7 vs. Fig. 5). Here pre-senescent cells contained rather large concentrations of lipofuscin which were independent of the age o f the parent clones until the beginning o f senescence, during which time the concentration increased three-fold. Comparison o f the degree o f accumulation o f lipofuscin in senescent cells (samples no. 1 and 2, Fig. 6) with that o f their adjacent non-senescent mother cells (samples 1' and
412
/ TOS
TOD
I00 o c A .E u ...I
5o
x
/ 50
300
i
K
. x
t 350 400 450 Wavelength , nrn
,
500
~
550
0.01
,,60 Clonol
660 Age
8bo
(h)
Fig. 4. Excitation and emission spectra of chloroform-methanol soluble pigment from P. anserina s+. A concentrated solution of pigment in chloroform was analyzed with an Aminco-Bowman spectrophotometer with instrumental settings for high resolution. (The variable slit at the light source was at maximum position No. 5, polarizers were at positions 1 and 6 (Vii), slits at positions 2 and 5 were 0.5 mm and the slit at the photomultiplier turret position 7, was 0.1 mm.) The polarization number at the excitation maximum 425 nm was 0.05. Fig. 5. Concentration of lipofuscin in suboclonal fractions of P. anserma s÷ as a function of their time of origin and the inhibition of lipofuscin accumulation by growth with reduced glutathione. Clones were grown as in Fig. 3. Lipofuscin was extracted and measured as described in Methods with excitation and emission fluorescence at 370 and 420 nm, respectively. The lipofuscin concentration per cm of growth is expressed relative to the fluorescence of a solution of 1 pMquinine sulfate in 0.1N H2SO 4. o, Control; X, plus 1 mM GSH.
2 ' , Fig. 6) revealed t h a t t h e l a t t e r h a d a c c u m u l a t e d less t h a n t h e i r s e n e s c e n t d a u g h t e r s (Fig. 7), t h u s c o n f i r m i n g t h e t r e n d o b s e r v e d in t h e p r e v i o u s e x p e r i m e n t (Fig. 5).
DISCUSSION T h e results o f this i n v e s t i g a t i o n o f senescence o f P. a n s e r i n a are q u i t e a n a l o g o u s t o t h e results o f p r e v i o u s i n v e s t i g a t i o n s o f s e n e s c e n c e in t h r e e m o d e l s y s t e m s in N. crassa [7-13].
C o n t i n u o u s a d m i n i s t r a t i o n o f free radical scavengers ( a n t i o x i d a n t s ) n o t o n l y
413 300 Sornple No. Cionol Acje (h) I 720
I'
2
648 576
4
504
200 E
432
5
=_ o" I00
6
360
7
288
8
216 144
0~)
I00
200
~30
400
500
600
8~
700
Time (h)
Fig. 6. Parallel extensional growth of clones of P. anserina s+. Eighteen clones were grown as in Fig. 1 except pairs were started at successive 72 h intervals. The lines are the average of the duplicate tubes. The growth rate was 0.40 +- 0.04 mm/h. At 720 h, samples of mycelia and agar of uniform size were removed from the leading edge of the clones and analyzed for lipofuscin concentration (Fig. 7).
2
o2'
~ IO
Io
u
T
~6
g~
.[5
23
TOS TOO 1 1' Clone na2 TOS 1 0
i
i
200
i
i
i
400 Clonol Age ( h )
i
600
TOD 1 Clone no.I i
i
800
Fig. 7. Concentration of lipofuscin in apical hyphae of clones of P. anserina s* of different age. Samples of apical hyphae from clones of various age (Fig. 6) were analyzed for lipofuscin concentrations as described in the legend of Fig. 5. Points and bars are the average and variance, respectively, of determinations of duplicate clones.
prolongs life span in all o f these fungi, but also inhibits lipid peroxidation /n vivo as indicated by the inhibition o f lipofuscin accumulation. The root cause o f senescence in the Neurospora m o d e l s appears to be abnormal m e m b r a n e s w h o s e lipids are abnormally susceptible to n o n - e n z y m a t i c peroxidation. By analogy, excessive lipid peroxidation
in P. anserina m a y be either a cause or consequence o f m e m b r a n e abnormalities.
414 The cellular control of the rate of lipid peroxidation is dependent upon many factors such as the relative concentrations of unsaturated lipids, pro-oxidants and natural antioxidants in membranes, the intracellular flux of oxygen-containing free radicals, and the relative activities of antioxygenic enzymes such as superoxide dismutase, catalase, glutathione peroxidases, and glutathione reductase. If the genes incoloris and vivax, which in combination confer immortality to clones of P. anserina [4], can be shown to act by inhibiting lipid peroxidation, it will still be a difficult task to determine the biochemical basis of such action. Marcou [1] and Esser and Tudzynski [5] emphasized the "infective" nature of senescence of P. anserina. Esser and Tudzynski [5, 17] recently showed that the onset of senescence may be prevented by supplying the growing mycelia with either the antimycoplasmal antibiotic tiamulin or streptomycin, or ethidium bromide, implicating 70S ribosomes and circular DNA in the origin, multiplication or propagation of the senescent agent [17]. Thus, bacterial or mitochondrial-like systems are implicated as senescence agents. Smith and Rubenstein [2] discussed the hypothesis that the senescence factor may be a mutant cytoplasmic or mitochondrial gene. The relationship between the results of this investigation and the hypothetical role of bacteria or mutant mitochondria in senescence is not clear. One might speculate that symbiotic bacterial infection and growth competes with the host for lipids in membrane synthesis and thereby leads to structurally abnormal host membranes whose lipids undergo more rapid peroxidation. On the other hand, mutation of mitochondrial genes in other fungi is known to lead to the synthesis of structurally and functionally abnormal inner mitochondrial membrane [18]. Such mutation(s) in P. anserina may lead to excessive lipid peroxidation. By either the bacterial or the mitochondrial model, administration of antioxidants would alleviate, but not prevent, senescence by inhibiting lipid peroxidation and membrane deterioration.
ACKNOWLEDGEMENTS The excellent technical assistance of P. Riese is acknowledged. We thank Dr. Karl Esser for providing P. anserina. This research was supported by the College of Agriculture and Life Sciences and a grant from the National Institutes of Health (GM 21205).
REFERENCES 1 D. Marcou, Notion de long6vit6 et nature cytoplasmique du d6terminant de la s6nescence chez quelques champignons, Ann. Sci. Nat. (Bot. Ser. 12e), 2 (1961) 653-764. 2 J. R. Smith and I. Rubenstein, The development of "senescence" in Podospora anserina, £ Gen. MicrobioL, 76 (1973) 283-296. 3 J. R. Smith and I. Rubenstein, Cytoplasmic inheritance of the timing of "senescence" in Podospora anserina, J. Gen. MicrobioL, 76 (1973) 297-304. 4 K. Esser and W. Keller, Genes inhibiting senescence in the ascomycete Podospora anserina, MoL Gen. Genet., 144 (1976) 107-110.
415
5 K. Esser and P. Tudzynski, Prevention of senescence in the ascomycete Podospora anserina by the antibiotic tiamulin,Nature, 265 (1977) 454-456. 6 I. K. Ross, J. C. Pammerville and D. L. Damm, A highly infectious 'mycoplasma' that inhibits meiosis in the fungus Coprinus, J. Cell Sci., 21 (1976) 175-191. 7 K. D. Munkrcs and M. Minssen, Ageing of Neurospora crassa. I. Evidence for the free radical theory of ageing from studies of a natural-death mutant, Mech. Ageing Develop., 5 (1976) 79-98. 8 K. D. Munkres and H. J. Colvin, Ageing of Neurospora crassa. II. Organic hydroperoxide toxicity and the protective role of antioxidant and the antioxygenic enzymes, Mech. Ageing Develop., 5 (1976) 99-107. 9 K. D. Munkres, Ageing ofNeurospora crassa. 11I. Induction of cellular death and clonal senescence of an inositol-less mutant by inositol starvation and the protective role of dietary antioxidant, Mech. Ageing Develop., 5 (1976) 163-169. 10 K. D. Munkres, Ageing ofNeurospora crassa. IV. Induction of senescence in wild type by dietary amino acid analogs and reversal by antioxidants and membrane stabilizers, Mech. Ageing Develop., 5 (1976) 171-191. 11 R. S. Rana and K. D. Munkres, Ageing ofNeurospora crassa. V. Lipid peroxidation and decay of respiratory enzymes in an inositoi auxotroph, Mech. Ageing Develop., 7 (1978) 241-272. 12 R. S. Rana and K. D. Munkres, Ageing ofNeurospora crassa. VI. Cytochemical and cytological correlates of senescence in three model systems, Mech. Ageing Develop., 7 (1978) 273-288. 13 K. D. Munkres and R. S. Rana, Ageing ofNeurospora crassa. VII. Age-dependent accumulation of fluorescent pigment (lipofuscin) and inh~ition of the accumulation by nordihydroguaiaretic acid, Mech. Ageing Develop., in press. 14 A. L. Tappel, Lipid peroxidation and fluorescent molecular damage to membranes, in B. F. Trump and A. U. Arstila (eds.), Pathobiology of Cell Membranes, Vol. I, Academic Press, New York, 1975, pp. 145-170. 15 K. Esser, Podospora anserina, in R. C. King (ed.), Handbook of Genetics, Vol. 1, Plenum Press, New York, 1974, pp. 531-551. 16 B. L. Fletcher, C. J. Dillard and A. L. Tappel, Measurement of fluorescent lipid peroxidation products in biological systems and tissues, Anal. Biochem., 52 (1973) 1-9. 17 P. Tudzynski and K. Esser, Inhibitors of mitochondrial function prevent senescence in the ascomycete Podospora anserina, Mot Gen. Genet., 153 (1977) 111-113. 18 D. Lloyd, The Mitochondria of Microorganisms, Academic Press, New York, 1974, p. 553.
407
A N T I O X I D A N T S P R O L O N G L I F E SPAN A N D I N H I B I T T H E S E N E S CENCE-DEPENDENT ACCUMULATION OF FLUORESCENT PIGMENT ( L I P O F U S C I N ) IN C L O N E S O F P O D O S P O R A A N S E R I N A ~ *
KENNETH D. MUNKRES and RAJENDRA S. RANA Laboratories of Molecular Biology and Genetics, University of Wisconsin, Madison, Wisconsin 53706 (U.S.A.) (Received March 30, 1977) SUMMARY Culture of clones of Podospora anserina s÷with either nordihydroguaiaretic acid or reduced glutathione (GSH) at concentrations that were not inhibitory to growth significantly prolonged the average time to onset of senescence. GSH also prolonged the average time to onset of clonal death. The specific concentration of chloroform--methanol soluble fluorescent pigment was larger in senescent than in pre-senescent cells. The pigment exhibited fluorescence excitation and emission spectra and fluorescence polarization numbers characteristic of lipofuscin, an end-product of lipid peroxidation. Analyses of the lipofuscin concentration in either sub-clonal fractions of different times of origin from senescent clones, or in' sub-clonal fractions of identical age in time of origin from parent clones of different age, revealed a similar concentration distribution. Although pre-senescent cells contained rather large concentrations, a massive increase occurred during senescence prior to the time of onset of clonal death. Culture with GSH not only prolonged clonal life span but also inhibited the formation of lipofuscin by an average factor of 30. Furthermore, unlike untreated clones, the sub-clonal distribution of the pigment was not only low but was also independent of their age.
INTRODUCTION The growth of a mycelium ofPodospora anserina undergoes irreversible cessation of growth, /.e., senescence, after a period of time which is characteristic of a given race.
*Contribution no. 2130 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706 (U.S.A.).
408 Marcou [1] suggested that a spontaneous variant cytoplasmic factor was responsible for senescence. Subsequent studies by Smith and Rubenstein [2, 3] supported this hypothesis and demonstrated that the growth parameters of a given race are inherited cytoplasmically;/.e., the frequency of occurrence of a cytoplasmic variant factor and the rate of increase of the variant factor are under cytoplasmic genetic control. Additional evidence for the genetic control of senescence was recently reported by Esser and Keller [4]. Senescence can be completely prevented by the synergistic action of two linked nuclear genes incoloris and vivax. Whereas the wild-type strain s becomes senescent after 26 days and the mutants incoloris and vivax after 42 and 66 days, respectively, the double mutant showed no signs of senescence after culture for more than one year. Although the genetical basis of senescence of P. anserina has been thoroughly investigated, the nature of the "infective" senescence "agent" has remained obscure. All efforts to demonstrate viruses or virus-like particles in ageing hyphae ofP. anserina have failed [5]. Whatever the senescence agent or biochemical damage during senescence may be, P. anserina being homothallic can undergo sexual reproduction without transmission of senescence to the progeny, thus insuring survival of the species as in higher eukaryotes. Our investigations of the molecular biology of senescence with three model systems of Neurospora crassa, a close relative ofP. anserina, have revealed that membrane deterioration, lipid peroxidation and free radical reactions are interdependent processes that contribute to the time-dependent deterioration of clonal growth rate (senescence) and cellular death [7-13]. Continuous administration of drugs that act as free radical scavengers (antioxidants) or membrane stabilizers not only prolongs the life span in these systems, but also inhibits the occurrence of a number of biochemical and cytological abnormalities related to membrane deterioration, lipid peroxidation, and damaging free radical reactions. One of the oldest and best established molecular indices of ageing is the intracellular accumulation of fluorescent pigment, sometimes called lipofuscin or "age" pigment. This pigment accumulates with age in all organisms thus far examined, ranging from fungi to man [7]. Furthermore, the rate of accumulation of the pigment appears to be proportional to the rate of ageing [14]. In several organisms or cell types, administration of dietary antioxidants not only prolongs life span but also inhibits the accumulation of lipofuscin [7]. According to Tappel, it is not the accumulation of lipofuscin per se that is detrimental to cells. Rather, the occurrence of lipofuscin indicates the occurrence of much more extensive non-fluorescent damage to molecules by free radical reactions which accompany lipid peroxidation and the f°nnati°n of lipofuscin [ 14]. In general, fluorescent age pigment arises by the reaction of amino groups with an end product of lipid peroxidation, malondialdehyde: 2RNH2 + H O - C H = C H - C H O ~ RNH-HC=CH-CH-----N-R forming either an intra- or inter-molecular iminopropene bond with characteristic fluorescence spectra with activation maximum 340-370 nm and emission maximum of 420-
409 470 nm. The R-group may be protein, phospholipid, RNA or DNA. The nature of the Rgroup apparently does not greatly influence the fluorescence spectrum but does affect the solubility properties [14]. In the present paper, we describe the age-dependent accumulation of one class of lipofuscin pigments, soluble in chloroform-methanol, in sub-clonal fractions ofP. anserina. Since culture with antioxidants not only prolonged clonal life span, but also inhibited the accumulation of lipofuscin, we conclude that lipid peroxidation may be causallyrelated to clonal senescence.
MATERIAL AND METHODS
Stocks Homokaryotic stocks of P. anserina were provided by Dr. Karl Esser. They were grown on slants of minimal medium no. 2 [15] and stored at 4 °C. Growth and senescence A small piece of agar with mycelia and ascospores was taken from slants and centrally placed on a plate of minimal medium no. 2. The plates were incubated for 3 - 4 days at 30 °C. Cylindrical plugs of agar and mycelia (~5 mm dia.) were taken from the mycelial frontier and transferred to race tubes, 1.8 diam. × 100 cm length, containing minimal medium no. 2 with 2% sucrose and 1.5% agar. The tubes were incubated at 30 ~C in continuous fluorescent light. The position of the mycelial frontier was recorded at 24 h intervals. At least 10 replicate tubes of each treatment were measured. The time to onset of senescence (TOS) of a clone is def'med as that time when the growth rate began to decelerate. The time to onset of death (TOD) is defined as that time when the growth rate had decelerated to 10% or less of the maximum linear growth rate. Extraction and measurement of lipofuscin The methods for extraction and measurement of chloroform--methanol soluble fluorescent pigment (lipofuscin) were the same as those described in a previous paper in experiments with Neurospora crassa [ 13].
RESULTS Figures 1 and 6 illustrate the extensional growth of clones ofP. anserina s÷ at 30 °C. Growth proceeded at a linear rate of 0.36 + 0.02 mm/h for 580 +- 48 h. This time is defined as the average time to onset of senescence (TOS). At that time, some clones immediately ceased to grow and others decelerated in growth rate. The time when the growth rate was less than or equal to 10% of the maximal growth rate is defined as the time of onset of death (TOD).
410 3O0 TOS,TOD
/T TOS
200 A E
~
~,~-E ~o~o~ Oor
L 200
,oo
"
, 400 Time ( h )
TOO
, 600
8vO
Fig. 1. Typical extensional g r o w t h o f clones ofPodospora anserma s*. Clones were grown in race tubes
on minimal medium no. 2 at 30 °C as described in Methods.
,oo
g u
~ 5
o'5o
i
=
Z
I
%0
5be
600 Time (h)
~c;o
o ,00
5O0
6OO
7OO
8;0
Time (h)
Fig. 2. Prolongation of the time to onset of senescence of clones ofP. anserina s+ by nordihydroguaiarctic acid. Clones were grown as in Fig. 1 with 6.7 #M NDGA (X) and without NDGA (o). The % non-senescent clones in a population of 10 is noted. Fig. 3. Prolongation of the time to onset of senescence of clones ofP. anserina s+ by reduced glutathione. o, Control; x, with 1 mMGSH.
Culture with either nordihydroguaiaretic acid (Fig. 2) or reduced glutathione (Fig. 3) significantly prolonged the average time to onset o f senescence (Table I) by 25 and 14%, respectively. GSH, but not NDGA, also prolonged the average time to onset o f death by 14% (Table I). The concentrations o f NDGA (6.7 #M) and GSH (1 mM) neither stimulated nor inhibited the linear growth rates; however, a higher concentration o f NDGA ( 3 3 . 5 / a l / ) inhibited g r o w t h somewhat, and, therefore, was n o t tested for an effect upon senescence. Figure 4 illustrates the fluorescence excitation and emission spectra o f a chlorof o r m - m e t h a n o l soluble pigment that occurs in excess in senescent cells. The excitation
411 TABLE I PROLONGATION OF THE LIFE SPAN OF CLONES OF PODOSPORA ANSERIN.4 s+ BY NORDIHYDROGUAIARETIC ACID OR REDUCED GLUTATHIONE. Exp. No.*
Time (hi to onset o f Senescence
1
2
Death
Con~ol
+NGDA
P
Con~ol
+NDGA
P
430
538
0.16
645
645
1
Con~ol
+GSH
Control
+GSH
580
660
650
735
0.08
0.10
*Two independent experiments each with 10 clones per treatment. Values axe the average of 10 clones. NDGA, 6.7 Wl#;reduced glutathione, 1 raM. P is the probability that the mean of the differences of the two populations is not greater than zero. and emission maxima were 365 and 425 nm, respectively. In some preparations, an additional maximum was observed at 445 nm. The broad fluorescence peak around 500 nm was not found in all preparations and may be due to traces o f phytoene [16]. The fluorescence polarization number at the emission wavelength 425 nm was about 0.02-0.05. The fluorescence spectra and the high degree of fluorescence polarization are characteristic o f lipofuscin, an end product o f lipid peroxidation [ 13 ]. Analyses o f the specific concentrations of lipofuscin in sub-clonal fractions of a clone shortly after the time o f its death as a function of the age when they were formed revealed an age-dependent increase (Fig. 5). The youngest cells in time of origin contained the largest specific concentration. A five-fold increase of lipofuscin concentration occurred in the sub-clonal fractions which were formed during the time when the parent clone was senescent. Culture with 1 mM GSH inhibited the accumulation o f lipofuscin by an average factor o f 30. Furthermore, unlike the control, the degree o f accumulation was independent o f sub-clonal age with GSH (Fig. 5). In the experiment illustrated in Figs. 6 and 7, new clones were started at 72 h intervals over a period of about 27 days. When the oldest clones became senescent, samples o f uniform size were taken from the mycelial frontiers of all o f the clones (Fig. 6) and lipofuscin concentrations were determined (Fig. 7). Although the cells were all o f identical age in terms o f time of origin, the specific concentration o f lipofuscin as a function o f age of the parent clones was quite similar to the concentration distribution in subclones o f different times of origin (Fig. 7 vs. Fig. 5). Here pre-senescent cells contained rather large concentrations of lipofuscin which were independent of the age o f the parent clones until the beginning o f senescence, during which time the concentration increased three-fold. Comparison o f the degree o f accumulation o f lipofuscin in senescent cells (samples no. 1 and 2, Fig. 6) with that o f their adjacent non-senescent mother cells (samples 1' and
412
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i
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t 350 400 450 Wavelength , nrn
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(h)
Fig. 4. Excitation and emission spectra of chloroform-methanol soluble pigment from P. anserina s+. A concentrated solution of pigment in chloroform was analyzed with an Aminco-Bowman spectrophotometer with instrumental settings for high resolution. (The variable slit at the light source was at maximum position No. 5, polarizers were at positions 1 and 6 (Vii), slits at positions 2 and 5 were 0.5 mm and the slit at the photomultiplier turret position 7, was 0.1 mm.) The polarization number at the excitation maximum 425 nm was 0.05. Fig. 5. Concentration of lipofuscin in suboclonal fractions of P. anserma s÷ as a function of their time of origin and the inhibition of lipofuscin accumulation by growth with reduced glutathione. Clones were grown as in Fig. 3. Lipofuscin was extracted and measured as described in Methods with excitation and emission fluorescence at 370 and 420 nm, respectively. The lipofuscin concentration per cm of growth is expressed relative to the fluorescence of a solution of 1 pMquinine sulfate in 0.1N H2SO 4. o, Control; X, plus 1 mM GSH.
2 ' , Fig. 6) revealed t h a t t h e l a t t e r h a d a c c u m u l a t e d less t h a n t h e i r s e n e s c e n t d a u g h t e r s (Fig. 7), t h u s c o n f i r m i n g t h e t r e n d o b s e r v e d in t h e p r e v i o u s e x p e r i m e n t (Fig. 5).
DISCUSSION T h e results o f this i n v e s t i g a t i o n o f senescence o f P. a n s e r i n a are q u i t e a n a l o g o u s t o t h e results o f p r e v i o u s i n v e s t i g a t i o n s o f s e n e s c e n c e in t h r e e m o d e l s y s t e m s in N. crassa [7-13].
C o n t i n u o u s a d m i n i s t r a t i o n o f free radical scavengers ( a n t i o x i d a n t s ) n o t o n l y
413 300 Sornple No. Cionol Acje (h) I 720
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4
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6
360
7
288
8
216 144
0~)
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200
~30
400
500
600
8~
700
Time (h)
Fig. 6. Parallel extensional growth of clones of P. anserina s+. Eighteen clones were grown as in Fig. 1 except pairs were started at successive 72 h intervals. The lines are the average of the duplicate tubes. The growth rate was 0.40 +- 0.04 mm/h. At 720 h, samples of mycelia and agar of uniform size were removed from the leading edge of the clones and analyzed for lipofuscin concentration (Fig. 7).
2
o2'
~ IO
Io
u
T
~6
g~
.[5
23
TOS TOO 1 1' Clone na2 TOS 1 0
i
i
200
i
i
i
400 Clonol Age ( h )
i
600
TOD 1 Clone no.I i
i
800
Fig. 7. Concentration of lipofuscin in apical hyphae of clones of P. anserina s* of different age. Samples of apical hyphae from clones of various age (Fig. 6) were analyzed for lipofuscin concentrations as described in the legend of Fig. 5. Points and bars are the average and variance, respectively, of determinations of duplicate clones.
prolongs life span in all o f these fungi, but also inhibits lipid peroxidation /n vivo as indicated by the inhibition o f lipofuscin accumulation. The root cause o f senescence in the Neurospora m o d e l s appears to be abnormal m e m b r a n e s w h o s e lipids are abnormally susceptible to n o n - e n z y m a t i c peroxidation. By analogy, excessive lipid peroxidation
in P. anserina m a y be either a cause or consequence o f m e m b r a n e abnormalities.
414 The cellular control of the rate of lipid peroxidation is dependent upon many factors such as the relative concentrations of unsaturated lipids, pro-oxidants and natural antioxidants in membranes, the intracellular flux of oxygen-containing free radicals, and the relative activities of antioxygenic enzymes such as superoxide dismutase, catalase, glutathione peroxidases, and glutathione reductase. If the genes incoloris and vivax, which in combination confer immortality to clones of P. anserina [4], can be shown to act by inhibiting lipid peroxidation, it will still be a difficult task to determine the biochemical basis of such action. Marcou [1] and Esser and Tudzynski [5] emphasized the "infective" nature of senescence of P. anserina. Esser and Tudzynski [5, 17] recently showed that the onset of senescence may be prevented by supplying the growing mycelia with either the antimycoplasmal antibiotic tiamulin or streptomycin, or ethidium bromide, implicating 70S ribosomes and circular DNA in the origin, multiplication or propagation of the senescent agent [17]. Thus, bacterial or mitochondrial-like systems are implicated as senescence agents. Smith and Rubenstein [2] discussed the hypothesis that the senescence factor may be a mutant cytoplasmic or mitochondrial gene. The relationship between the results of this investigation and the hypothetical role of bacteria or mutant mitochondria in senescence is not clear. One might speculate that symbiotic bacterial infection and growth competes with the host for lipids in membrane synthesis and thereby leads to structurally abnormal host membranes whose lipids undergo more rapid peroxidation. On the other hand, mutation of mitochondrial genes in other fungi is known to lead to the synthesis of structurally and functionally abnormal inner mitochondrial membrane [18]. Such mutation(s) in P. anserina may lead to excessive lipid peroxidation. By either the bacterial or the mitochondrial model, administration of antioxidants would alleviate, but not prevent, senescence by inhibiting lipid peroxidation and membrane deterioration.
ACKNOWLEDGEMENTS The excellent technical assistance of P. Riese is acknowledged. We thank Dr. Karl Esser for providing P. anserina. This research was supported by the College of Agriculture and Life Sciences and a grant from the National Institutes of Health (GM 21205).
REFERENCES 1 D. Marcou, Notion de long6vit6 et nature cytoplasmique du d6terminant de la s6nescence chez quelques champignons, Ann. Sci. Nat. (Bot. Ser. 12e), 2 (1961) 653-764. 2 J. R. Smith and I. Rubenstein, The development of "senescence" in Podospora anserina, £ Gen. MicrobioL, 76 (1973) 283-296. 3 J. R. Smith and I. Rubenstein, Cytoplasmic inheritance of the timing of "senescence" in Podospora anserina, J. Gen. MicrobioL, 76 (1973) 297-304. 4 K. Esser and W. Keller, Genes inhibiting senescence in the ascomycete Podospora anserina, MoL Gen. Genet., 144 (1976) 107-110.
415
5 K. Esser and P. Tudzynski, Prevention of senescence in the ascomycete Podospora anserina by the antibiotic tiamulin,Nature, 265 (1977) 454-456. 6 I. K. Ross, J. C. Pammerville and D. L. Damm, A highly infectious 'mycoplasma' that inhibits meiosis in the fungus Coprinus, J. Cell Sci., 21 (1976) 175-191. 7 K. D. Munkrcs and M. Minssen, Ageing of Neurospora crassa. I. Evidence for the free radical theory of ageing from studies of a natural-death mutant, Mech. Ageing Develop., 5 (1976) 79-98. 8 K. D. Munkres and H. J. Colvin, Ageing of Neurospora crassa. II. Organic hydroperoxide toxicity and the protective role of antioxidant and the antioxygenic enzymes, Mech. Ageing Develop., 5 (1976) 99-107. 9 K. D. Munkres, Ageing ofNeurospora crassa. 11I. Induction of cellular death and clonal senescence of an inositol-less mutant by inositol starvation and the protective role of dietary antioxidant, Mech. Ageing Develop., 5 (1976) 163-169. 10 K. D. Munkres, Ageing ofNeurospora crassa. IV. Induction of senescence in wild type by dietary amino acid analogs and reversal by antioxidants and membrane stabilizers, Mech. Ageing Develop., 5 (1976) 171-191. 11 R. S. Rana and K. D. Munkres, Ageing ofNeurospora crassa. V. Lipid peroxidation and decay of respiratory enzymes in an inositoi auxotroph, Mech. Ageing Develop., 7 (1978) 241-272. 12 R. S. Rana and K. D. Munkres, Ageing ofNeurospora crassa. VI. Cytochemical and cytological correlates of senescence in three model systems, Mech. Ageing Develop., 7 (1978) 273-288. 13 K. D. Munkres and R. S. Rana, Ageing ofNeurospora crassa. VII. Age-dependent accumulation of fluorescent pigment (lipofuscin) and inh~ition of the accumulation by nordihydroguaiaretic acid, Mech. Ageing Develop., in press. 14 A. L. Tappel, Lipid peroxidation and fluorescent molecular damage to membranes, in B. F. Trump and A. U. Arstila (eds.), Pathobiology of Cell Membranes, Vol. I, Academic Press, New York, 1975, pp. 145-170. 15 K. Esser, Podospora anserina, in R. C. King (ed.), Handbook of Genetics, Vol. 1, Plenum Press, New York, 1974, pp. 531-551. 16 B. L. Fletcher, C. J. Dillard and A. L. Tappel, Measurement of fluorescent lipid peroxidation products in biological systems and tissues, Anal. Biochem., 52 (1973) 1-9. 17 P. Tudzynski and K. Esser, Inhibitors of mitochondrial function prevent senescence in the ascomycete Podospora anserina, Mot Gen. Genet., 153 (1977) 111-113. 18 D. Lloyd, The Mitochondria of Microorganisms, Academic Press, New York, 1974, p. 553.