Biological aging is defined as the continuous loss of vitality and viability, with a concomitant increase in morbidity and mortality
[6]. Although there have been tremendous efforts to understand the aging process, there is still no consensus on how aging and its unwanted ‘side-effects’ can be reduced or countered. Rather, simple model systems have been utilized to foster our understanding of how processes, such as senescence and aging, mechanistically work. Fungi, for example, are attractive organisms on which to study the time-dependent changes that lead, ultimately, to organismal death
[7][8]. In addition to the unicellular baker’s yeast (
Saccharomyces cerevisiae), a considerable effort has been invested into characterizing aging at the molecular and cellular levels in the filamentous ascomycete,
P. anserina, which is a close relative of the more well-known
Neurospora crassa [9][10][11][12]. Among the studied pathways, the formation and detoxification of harmful ROS in
P. anserina are particularly well-characterized
[13][14]. According to the Free Radical Theory of Aging, ROS are causal agents of the aging process
[15][16]. Although
P. anserina is an obligate aerobe, mitochondrial defects are not necessarily lethal. In addition to the conventional complex IV (Cytochrome
c oxidase, COX), the fungus can use an alternative oxidase (AOX) for maintaining its viability in case COX activity becomes compromised
[17]. AOX-dependent respiration was found to lead to the decreased production of ROS in plants and fungi
[18][19]. Supporting this line of evidence is the observation that numerous long-lived mutants of
P. anserina utilize AOX, but not COX, as terminal oxidase
[17][20]. In this section, we will focus on the role of SODs on
P. anserina development (
Figure 2).
2.1.1. PaSOD1: A Differentially Regulated CuZnSOD in Certain Long-Lived Mutants
Two long-lived
P. anserina mutants, that display interesting differences regarding their SOD activity profiles, will be briefly discussed here. The findings show that, depending on the genetic context, these enzymes might be potentially linked to a lifespan extension. SOD activity was assessed in the mutants, grisea and ex1, and was compared to the wild type (WT), which exhibits a normal lifespan
[17][21]. In the WT, the activity of the mostly cytoplasmic CuZnSOD, PaSOD1, (
Figure 2) increased strongly with age, whereas the activity of PaSOD2, a MnSOD which is either ER-associated or secreted
[22], decreased
[17]. The mutant ex1, in which AOX was induced due to a deletion in the mtDNA that led to the loss of the
CoI gene (encoding the first subunit of COX), seemed to exclusively employ PaSOD1
[21]. In mutant grisea, a gene encoding a copper-modulated transcription factor (GRISEA) is functionally inactivated by mutation
[23]. This mutant experienced severely depleted cellular copper levels
[24]. Therefore, the activity of copper-containing proteins, such as COX and PaSOD1, is almost undetectable
[21][25]. Importantly, putative target genes of the transcription factor GRISEA are, at most, very weakly expressed. Among these is
PaCtr3 (encoding a high-affinity copper transporter of the plasma membrane), which contributes to the severely reduced copper levels in grisea. In the WT, the differential activity of PaSOD1 pointed to a redistribution of cellular copper during aging where the amount of available copper in the cytoplasm increased. This is an example of a clear developmental regulation of PaSOD1 activity at the protein level.
In addition, PaSOD1 seems to play a crucial role in extension of the lifespan of the
PaCox17::ble mutant
[26]. In this mutant, the putative copper chaperone, PaCOX17, was deleted by gene replacement. The mutant displays an increased lifespan compared to the WT; even after 320 days, 40 out of the 60 cultures were still alive, whereas WT isolates have a lifespan of two to three weeks. PaSOD1 levels are highly upregulated in
PaCox17::ble, suggesting an improved defense against superoxide anions
[26].
Zintel et al. determined that the size of PaSOD1, through an in silico analysis, was 15.8 kDa
[22]. Furthermore, PaSOD1 possesses two conserved CuZnSOD signatures. A predominantly cytoplasmic localization was revealed by the construction of a strain that synthesizes PaSOD1::GFP
[22].
2.1.3. PaSOD3: Increasing the Content of This Mitochondrial MnSOD, but Not Its Ablation, Has Effects on Aging
This SOD is, by far, the best studied of the three identified family members of P. anserina (Figure 2). Mutants in which PaSod3 levels were experimentally modulated revealed some unexpected findings, while cleary illustrating that PaSOD3 is linked to other important surveillance and quality control systems in P. anserina. PaSOD3 might also influence several processes by modulating ROS levels (superoxide anion and hydrogen peroxide) that are required for signaling, underscoring the role of ROS as cellular messengers.
PaSOD3 constitutes a mitochondrial SOD with a deduced molecular weight of 25.5 kDa. The enzyme contains a putative mitochondrial targeting sequence and was experimentally demonstrated to reside in mitochondria
[22]. The impact of PaSOD3 on aging was studied by utilizing mutants in which
PaSod3 was either constitutively overexpressed or deleted
[22]. ∆
PaSod3 strains were hypersensitive to compounds that generated superoxide anions, such as paraquat, which was expected. In contrast, the sensitivity to hydrogen peroxide was not altered in ∆
PaSod3. However, the overexpression of
PaSod3 led to a significant growth reduction compared to the WT control, even without any additional stressors. Unexpectedly, these strains were more sensitive to both paraquat and hydrogen peroxide when these compounds were added to the growth medium
[22]. Regarding the aging process, ∆
PaSod3 did not show any significant differences compared to the WT control. On the other hand,
PaSod3 overexpressors were short-lived when grown under standard conditions, reaching only around 75% of the median lifespan of the WT
[22]. This can be explained, firstly, by the protein levels of the peroxiredoxin PaPRX1, a mitochondrial peroxidase that detoxifies hydrogen peroxide which was strongly reduced in
PaSod3 overexpressors, suggesting a compromised defense against ROS. Secondly, several proteins responsible for the quality control of mitochondria, such as matrix proteases PaCLPP and PaLON, were almost absent, or they exhibited altered protein patterns, due to their incomplete processing or degradation, respectively
[22]. Thirdly, the mitochondrial heat shock protein PaHSP60 was clearly upregulated and proteolytically activated in the
PaSod3 overexpressing strains
[22]. The authors suggest that the elevated PaSOD3 levels led to the generation of high doses of hydrogen peroxide in mitochondria, which was subsequently converted to the highly reactive hydroxyl radical. The latter is formed by Fenton chemistry involving hydrogen peroxide and various metal ions and it has a highly destructive potential due to its extremely high reactivity with lipids, nucleic acids, and proteins
[28].
To better understand the phenotypic effects of the increased
PaSod3 expression, a modeling strategy was employed that supported the hypothesis that excess hydrogen peroxide generated by PaSOD3 was responsible for the damaging effects
[29]. The computational studies suggested that the levels of the PaSOD3 cofactor, Mn
2+, were elevated by a factor of 80 in the
PaSod3 overexpressors. This result led to a study of the role of manganese supplementation of the growth medium of the WT and the
PaSod3 overexpressors on phenotypic parameters
[30]. Interestingly, the supplementation of the growth medium with MnSO
4, even in small amounts (20 µM), led to a reversion of the
PaSod3_OEx mutant phenotype. In addition to showing WT-like growth rates, the
PaSod3 overexpressors had restored the formation of aerial hyphae and fertility. Regarding aging, MnSO
4 concentrations of 80 µM allowed the
PaSod3_OEx strains to reach median lifespans indistinguishable from those of the WTs
[30].
Importantly, the quantification of the total SOD activity (PaSOD1, PaSOD2 and PaSOD3) demonstrated no significant differences in whole cell extracts or isolated mitochondria, regardless of whether Mn was added to the growth medium or not. The authors concluded that a general limitation of manganese on SOD activity in
PaSod3 overexpressors did not occur
[30].
However, in contrast to the WT, the Mn-supplemented
PaSod3 overexpressors demonstrated elevated levels of peroxidase and catalase activities in addition to the upregulated protein levels of the peroxiredoxin, PaPRX. Further results suggested that a hitherto unknown Mn-dependent protein contributed to an improved degradation rate of hydrogen peroxide
[30].
PaSOD3 protein levels were shown to be controlled by the protein, PaRCF1
[31]. PaRCF1 belongs to the HIG1 (‘hypoxia-inducible gene 1’) family of proteins, which play an important role in the assembly and organization of the mitochondrial respiratory chain
[32]. A deleted mutant of
PaRcf1, ∆
PaRcf1, contains only a fraction of PaSOD3 proteins (approximately 20%) compared to the WT, but its activity appears not to be affected
[31]. However, ∆
PaRcf1 is hypersensitive to the addition of the redox cycler, paraquat, into the medium in comparison to the WT. Additionally, this mutant fails to maintain a normal growth rate, is sterile, and is marked by a decreased median lifespan. It is important to note that in ∆
PaRcf1, PaSOD3, as well as other factors of maintenance and cellular quality control decreased, e.g., PaLON, PaPRX and PaCLP
[31]. Thus, the altered phenotype of ∆
PaRcf1 is likely the result of several deficiencies.
As mentioned above, the signaling function of ROS is altered in
PaSod3 deletion strains
[30]. It is known that ROS are modulators of various cellular processes; among these is the controlled cellular ’self-eating’, or autophagy
[33][34]. Usually, ROS are activators of autophagy, which acts as a system to assist in maintaining cellular homeostasis
[35]. It was shown that the ’unexpected healthy phenotype’ of ∆
PaSod3 was due to the induction of autophagy (
Figure 2)
[36]. The authors demonstrated this by analyzing the number of autophagosomes using the marker
Gfp-PaAtg8. These increased, even in juvenile stages. Increased autophagy (especially mitophagy) is important for ∆
PaSod3 survival because the ∆
PaSod3 ∆
Paatg1 double knockout strain reveals severe phenotypic deficiencies, such as reductions in both the growth rate and lifespan
[36].
SODs are enzymes that degrade superoxide anions. However, the generation of superoxide anions is an important process for growth and development in fungi.
P. anserina and many other organisms express genes that encode plasma membrane NADPH oxidases, which are sources of superoxide production
[37][38].
It was found that genes encoding NADPH oxidases (
Nox) are exclusively present in the genomes of multicellular organisms, regardless of their phylogenetic origin, pointing to a role of controlled superoxide production in differentiation processes
[39]. Deactivating the
P. anserina gene,
PaNox1, leads to several defects, such as the reduced pigmentation of the mycelium, the insufficient formation of aerial hyphae and, most importantly, the compromised formation of perithecia (fruiting bodies). PaNOX1 functions in cellular signaling upstream of the mitogen-activated protein kinase kinase kinase (MAPKKK), PaASK1
[40]. Meanwhile, the deletion of
PaNox2, a second NADPH oxidase isoform in
P. anserina, demonstrates that this gene is essential for the germination of ascospores
[40]. In summary, the regulated secretion of superoxide anions and peroxide during the life cycle is controlled by proteins, including PaNOX1, PaNOX2 and PaASK1
[40].
Enzymes such as PaSOD2 are potentially secreted, degrading superoxide anions, thereby modulating the O2−-mediated developmental signaling, although, for now, this is speculative and requires further investigation.
The work on the role of SODs in the developmental process of P. anserina firmly positions these proteins as crucial components of an elaborate network, controlling several vital processes, such as growth, fertility, the intracellular communication of biological quality control pathways and, ultimately, aging.