Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Abstract

Autophagy is a well-conserved catabolic process, involving the degradation of a cell's own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.

Keywords: Podospora anserina; ageing; autophagy; carbon and nitrogen restriction; dietary restriction; lifespan extension.

Figure 1.

Effects of (a) d-glucose and (b) urea concentrations on the lifespan of P. anserina strain s. For the different d-glucose concentrations, the urea concentration was kept at 0.1% w/v; for the different urea concentrations the d-glucose level was kept at 2% w/v. The measurements of the five replicate lines growing on 0.02% glucose w/v and 0.1% urea w/v were truncated after 1 year of continuous growth, with all lines still alive.

Figure 2.

Phenotype of P. anserina strain s on carbon (d-glucose; left) and nitrogen (urea; right) restriction, respectively. Growth front in the 0.001% (w/v) urea conditions marked with triangles. (Online version in colour.)

Figure 3.

The lifespan of wild-type strains under glucose (a) and urea (b) restriction, respectively. The often small standard deviations are given for all columns, but when all strains lived longer than the cut-off point of 90 days, these are not visible in the graphs. The same letter above columns (a,b,c,d) indicates that these groups do not statistically differ in phenotype, while different letters indicate that these groups do.

Figure 4.

(a) Phenotype of a 5-day-old culture of wild-type collection strain s (left) and of the putative mitophagy-deficient ΔPaUth1 mutant (right) on PASM with 2% glucose and 0.1% urea. (b) Confocal laser microscopy picture of a hyphal tip of strain s with filamentous mitochondria expressing GFP on PASM with 2% glucose and 0.1% urea. (c) Part of an irregular formed hyphal tip of ΔPaUth1 mutant with diffuse mitochondria expressing GFP. (Online version in colour.)

Figure 5.

The lifespan of laboratory strains and derived autophagy-deficient mutants ΔPaAtg1, ΔPaAtg8, ΔPaPspA and ΔPaUth1 under glucose (a) and urea (b) restriction, respectively. All autophagy mutants are given to the right of the laboratory strain they originated from. The error bars indicate the standard deviation per experimental condition. Under experimental conditions in which all samples lived beyond 90 days there are no error bars, because the experiment was terminated at this point. The same letter above columns indicates that these groups do not statistically differ in phenotype.

Figure 6.

Fitted curves of the DR response at relative ages at onset of DR (PASM with 0.02% w/v glucose) for the autophagy-deficient mutants (ΔPaUth1, ΔPaAtg8, ΔPaPspA andΔPaAtg1), and the wild-type parental strain s (in order of appearance from left to right; growth experiments were stopped after 90 days, p-values fitted curves of less than 0.001). Refer to the electronic supplementary material, figure S1 for the individual replicate data points.

Figure 7.

Models of damage accumulation over time for wild-type (a) and autophagy mutant (b) strains growing under nutrient-rich (black line) and calorie-restricted (grey line) conditions. The shaded areas depict the damage levels in which a lifespan extending DR effect is (light shade) or is no longer (darker shade) possible. Normal accumulation of damage under nutrient-rich conditions will lead to a threshold level of damage after which cell death (cross symbol) becomes inevitable. Below such damage levels DR may postpone senescence phenomena and cell death for a long time. Mutants impaired in their autophagy pathway will show faster ageing processes under nutrient-rich conditions, resulting in a lack of response to DR conditions earlier in the life time of the mutants.