Summary:
Organismal ageing is a multifactorial process modulated by the interplay
between genetic and environmental factors, and it affects most, if not all,
tissues and organs of the body (Kirkwood, 2005; Kourtis and Tavernarakis,
2011). At the tissue level, ageing-related damage of biomolecules affects all
types of cells and the extracellular matrix. The cleareance of proteome damage
is achieved via the occurrence of a complex and highly regulated network of
effective surveillance, maintenance, and clearance systems that ensure
biomolecules’ functionality, thus preventing disruption of organism
homeostasis. Key players in preventing the breakdown of cellular proteostasis
(homeostasis of the proteome) are the molecular chaperones, the oxidized
protein repair enzymes MsrA and MsrB, the mitochondrial Lon protease, and the
two main proteolytic systems (i.e., the lysosome and the proteasome), along
with main antioxidant signaling pathway NFE2-related factor 2 (Nrf2)/Keapl
(Breusing & Grune, 2008; Sykiotis & Bohmann, 2008; Hubbard et al., 2012).
Proteasome is central to proteostasis maintenance, as it degrades both normal
and damaged proteins. Herein, we undertook a detailed analysis of proteasome
function and regulation in the in vivo setting of Drosophila melanogaster. We
report that a major hallmark of somatic tissues of ageing flies is the gradual
accumulation of ubiquitinated and carbonylated proteins; these effects
correlated with a 50% reduction of proteasome expression and catalytic
activity. In contrast, gonads of ageing flies were relatively free of proteome
oxidative damage and maintained substantial proteasome expression levels and
highly active proteasomes. Moreover, gonads of young flies were found to
possess more abundant and more active proteasomes than somatic tissues.
Exposure of flies to oxidants induced higher proteasome activities specifically
in the gonads, which were, independently of age, more resistant than soma to
oxidative challenge and, as analyses in reporter transgenic flies showed,
retained functional antioxidant responses. Finally, inducible Nrf2 activation
in transgenic flies promoted youthful proteasome expression levels in the aged
soma, suggesting that age-dependent Nrf2 dysfunction is causative to decreasing
somatic proteasome expression during ageing.
As the impact of in vivo proteasome dysfunction on the proteostasis networks
and ageing processes remain poorly understood, our study then was focused to
this issue. We found that RNAi-mediated knock down of 20S proteasome subunits
in Drosophila melanogaster resulted in larval lethality. We therefore studied
the molecular effects of proteasome dysfunction in adult flies by developing a
model of dose-dependent pharmacological proteasome inhibition. Impaired
proteasome function promoted several “old-age” phenotypes and markedly reduced
flies’ lifespan. In young somatic tissues and in gonads of all ages, loss of
proteasome activity induced higher expression levels and assembly rates of
proteasome subunits. Proteasome dysfunction was signaled to the proteostasis
network by reactive oxygen species (ROS) that originated from malfunctioning
mitochondria and triggered an Nrf2-dependent upregulation of the proteasome
subunits. RNAi-mediated Nrf2 knock down reduced proteasome activities, flies
resistance to stress, as well as longevity. Conversely, inducible activation of
Nrf2 in transgenic flies, upregulated basal proteasome expression and activity
independently of age, and conferred resistance to proteotoxic stress.
Interestingly, prolonged Nrf2 over-expression reduced longevity, indicating
that excessive activation of the proteostasis pathways can be detrimental.
We then studied transgenic flies were proteasomal subunits b1, β5, α4, α7,
Rpn11, Rpn6, Rpn10 and Rpt6 expression was suppressed by RNAi. We found that
reduced expression of the β5, α7, Rpn11 and Rpn6 subunits caused larval
lethality. Also, we noted the upregulation of proteasome subunits after
completely phenocopying the phenotypes observed with PS-341. Interestingly, the
finding that suppression of the subunits α4, β1, Rpn10 and Rpt6 allowed
hatching of the adult fly indicates that some proteasome subunits can be
substituted allow thus partial maintenance of proteasome function. For example,
the finding that the suppression of α4 subunit does not appear to cause
significant increase in mortality likely explained by a previous reports
showing that the α4 and α3 subunits have similar functions (Velichutina et al.,
2004). Nevertheless the reduced longevity of these flies clearly indicates the
absence of fully functional proteasomes.
Finally, considering that the advanced glycation end product (AGEs)-modified
proteins (a non-enzymatic glycation of free amino groups of proteins) along
with lipofuscin (a highly oxidized aggregate of covalently cross-linked
proteins, sugars and lipids) have been found to accumulate during ageing and in
several age-related diseases, we studied the impact of oral administration of
glucose (Glc)-, fructose (Frc)-, or ribose (Rib)-modified albumin or of
artificial lipofuscin (LF) in Drosophila. We report that continuous feeding of
young Drosophila flies with culture medium enriched in AGEs or in lipofuscin
resulted in reduced locomotor performance, in accelerated rates of
AGEs-modified proteins and carbonylated proteins accumulation in the somatic
tissues and the haemolymph of flies, as well as in a significant reduction of
flies healthspan and lifespan. These phenotypic effects were accompanied with
reduced proteasome peptidase activities in both the haemolymph and in somatic
tissues of flies and higher levels of oxidative stress and proteasome
expression levels; furthermore, oral administration of AGEs or lipofuscin in
flies triggered an upregulation of the lysosomal cathepsins B, L activities.
Finally, RNAi-mediated cathepsin D knockdown reduced flies longevity and
significantly augmented the deleterious effects of AGEs and lipofuscin
indicating that lysosomal cathepsins reduce the toxicity of diet-derived AGEs
or lipofuscin.
In conclusion, this work provides novel evidence supporting differential
regulation of proteostasis maintenance and proteome clearance in the young
organism, during ageing, or in response to oxidative challenge in the somatic
tissues and the gonads of Drosophila. Also, our findings support experimentally
the trade-off theories of ageing evolution, where ageing is considered a
consequence of increased energetic investment in germ line (that preserves
viability across generations) over soma (only needed to support survival of a
single generation) maintenance. Moreover, our in vivo studies add new knowledge
on the proteotoxic stress-related regulation of the proteostasis networks in
higher metazoans. Proteasome dysfunction triggers the activation of an
Nrf2-dependent tissue- and age-specific regulatory circuit aiming to adjust the
cellular proteasome activity according to temporal and/or spatial proteolytic
demands. Prolonged deregulation of this proteostasis circuit accelerates
ageing. Finally, our in vivo studies demonstrate that chronic ingestion of AGEs
or lipofuscin disrupt proteostasis and accelerate the functional decline that
occurs with normal ageing.
