Alex K. Chen
Path 517 Review, Week 9
“Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila”. (Burnett et al. 2011)
While overexpression of the sirtuin Sir2 has been previously reported to increase lifespan in both C.
elegans and Drosophila, Burnett et al. show that when the when the genetic background is standardized
(and after isolating the effects of Sir2 by genetic outcrossing), then dSir2 overexpressed animals did not
live longer relative to their respective transgenic controls.
Their paper provides a major advance by decoupling the levels of dSir2 (as measured by Western
Blotting) with lifespan effects. They show that the specific strain of C. elegans used (LG100) had a
neuronal dye-filling (Dyf) defect that the previous papers failed to segregate from the dSir2 gene. This is
pertinent to lifespan research since Dyf mutants often show increased lifespan as a result of their
diminished ability to sense food. In a second independent experiment, they also compared the lifespans
of dSir2 deletion-mutant flies with wildtype flies and showed that all dSir2 genotypes responded
similarly to dietary restriction, showing that the effects of dietary restriction on lifespan did not require
the presence of dSir2, which casts doubt into the claim of dSir2 as a DR-mimic.
Major concerns:
1. Figure S4a-d shows very steep declines in survival (over very small windows of time) for all or
the animals exposed to SYA media. This does not seem to invalidate the final conclusion, as the
overexpressed dSir2 (the orange line) never appears to have a lifespan greater than the other
curves). Nonetheless, dSir2 deletion mutant flies do appear to have a lower lifespan than
wildtype flies (Figure S9b,c) so this demands further investigation.
2. There are a significant amount of censored deaths (Tables S1, S2, S3, and S5), and the numbers
of censored animals are not uniform from one experiment to another. For some animals,
especially those receiving sir-2.1 RNAi treatment, there seems to be an unusually high number
of censored animals (over half the value of the number of deaths). It is possible that some
animals are more susceptible to being censored from sir-2.1 RNAi than other animals, and that
the animals susceptible to censorship could potentially be a group of animals whose lifespans
could be affected by sir-2.1 RNAi differently than a group of control animals.
3. In Figure S3, it appears that sir-2.1 RNAi appears to increase lifespan in both wild type and
DR1786 (long-lived strain) flies. While this could be true and is consistent with some reported
results (especially those from an external paper shown by Figure S12), it goes against our
intuition, so this needs to be investigated further. It would be informative if they reported what
the animals died of, and whether or not the sir-2.1 overexpressing animals and the sir-2.1 RNAi
animals died from a different spectrum of diseases as compared to wildtype animals (or
whether or not they had different healthspans than wildtypes).
4. The authors only managed to outcross the animals to the 6th generation, even though standard
protocol suggests that they should outcross the animals to the 10th generation in order to truly
isolate the individual dSir2 gene from all surrounding genes (which the authors anticipate when
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they state “one remaining possibility is that the outcrossed sir-2.1 strains both contain secondsite mutations that suppress longevity effects). This may not be a concern, however, since 6
generations was sufficiently high enough to completely segregate the Sir2 gene from the Dyf
lifespan gene, although it is possible that there were other genes closer to Sir2 that were not
segregated from Sir2 and that could have negative effects on lifespan (although this is unlikely).
Most likely, further outcrossing would only make their conclusion stronger.
They were only able to compare the lifespans of the outcrossed lines with the other animals
who also experienced transgene insertions. The mutagenic effects of transgene insertions are
still not very well known, and we cannot be completely assured that conclusions applicable to
transgenic mutants should necessarily apply to normal animals, especially since all transgenic
lines were longer-lived than the Dahomey wild type controls, which could be the result of
heterosis (which is a process that could possibly be affected, and even possibly reduced, by
dSir2).
They also did not address the Rogina et al. 2004 and the Bauer et al. 2009 studies, which also
showed positive effects from dSir2, and which may not have had the same methodological flaws
as the Tissenbaum et al. 2001 study they addressed.
The Viswanathan and Guarente 2011 paper manages to address some of the problems from the
Tissenbaum and Guarente 2001 paper, acknowledge the existence of the unlinked mutation,
and show that the expression of sir-2.1 is dependent on two promoters – one of which, P0, was
not contained in the geIs3 transgenic line, and which is necessary for regulation of sir-2.1’s
expression in muscle cells and intestine. This P0 promoter, moreover, also acts on R11A8.5,
which could also interact with sir-2.1 (as nearby genes often do), and the association between
the promoter and both R11A9.5 and sir-2.1 could be disrupted by repeated outcrossings, like
those used by Burnett et al. 2011. R11A9.5 probably was not even expressed in the geIs3
transgenic line used in the Burnett et al. paper, because the geIs3 line failed to contain P0. So in
order to for the paper to be more convincing, they should run the same experiment again, with
P0 included.
While Burnett et al. 2011 do show that sir-2.1 levels can be fully decoupled from lifespan, we do
not know what tissues they measure the sir-2.1 levels from. In fact, the “Methods” section
shows that the sir-2.1 protein used in the Western blots was prepared from synchronous
nematode cultures, and we do not know if levels of protein from these cultures are
representative of the levels of protein from the individual tissues of C. elegans.
As is usual for most lifespan studies, they used heavily inbred strains of C. elegans and
Drosophila. It’s possible that what applies for heavily inbred strains may not necessarily apply to
wild (and genetically heterogeneous) populations. But this affects all papers, including the ones
whose conclusions they refute.
Minor concerns:
1. Figure S9 says that dSir2 mRNA levels in dSir24.5 are indistinguishable from wild type dSir2 levels.
The p-value there is 0.110, however, and the graph still shows a noticeable difference in the
relative quantity of dSir2 mRNA. While the differences are not statistically significant, statistical
insignificance does not necessarily imply indistinguishability. Western blotting also seems to
indicate that the products move slightly farther, though this is probably expected as the
products are slightly lighter.
2. We might not be fully aware of the effects of FUdR (used to prevent progeny production) on
lifespan, and if the effects could be dependent on variables relevant for this paper