Institutionen för neurovetenskap / Department of Neuroscience
Abstract
With advancing age, humans and rodents alike lose about one third of the
skeletal muscle mass. A process referred to as old-age muscle atrophy or
sarcopenia. Atrophy is a major contributor to disability and morbidity
among elderly adults hence the aim of this thesis is to shed light on the
molecular mechanisms underlying old-age associated muscle atrophy and
behavioral changes related to age in a rat model.
In Paper I, we characterized the growth patterns, survival and behavioral
alterations linked to advancing age in the rat. The median survival age
was, on average, between 29 30 months for both female and male
Sprague Dawley (SD) rats. There was a gradual decline in locomotor
activity and explorative behavior associated with age, while disturbances
in both coordination and balance did not become evident until later times
points. In old age, weight carrying capacity, limb movement and
temperature threshold were also impaired. While body weight continues to
increase over the better part of the life span of rats, the behavioral
changes in old age associated with a decrease in both total body weight
and, in particular, muscle mass. Dietary restriction (DR) was found to
increase median life span expectancy and impede the development of
sarcopenia, and to retard the pace of behavioral aging.
In Paper II, we used two-dimensional gel electrophoresis and mass
spectrometry techniques to determine changes in protein expression as
well as cDNA profiling to assess transcriptional regulations in skeletal
muscle of adult and aged male SD rats. Among the highly expressed
proteins, thirty-five were differentially expressed in aged muscle.
Proteins and mRNA transcripts involved in redox homeostasis and iron load
were increased, representing novel components previously not associated
with sarcopenia. Iron levels in tissue were elevated in senescence,
paralleling an increase in transferrin. Proteins involved in redox
homeostasis were found to display a complex pattern of changes involving
increases in SOD1 and decreases in SOD2. Together these results suggest
that an elevated iron load is a significant component of sarcopenia with
a potential to be exploited clinically and that the mitochondria of aged
striated muscle may be more vulnerable to radicals produced during cell
respiration.
Muscle atrophy, in many conditions, shares a common mechanism for
up-regulation of the muscle-specific ubiquitin E3-ligases Atrogin-1 and
MuRF1. E3-ligases are part of the ubiquitin proteasome system (UPS)
utilized for protein degradation during muscle atrophy. In Paper III, we
show that Atrogin-1 and MuRF1 are down-regulated in old age-associated
muscle atrophy. Our results suggest that this is mediated by AKT-induced
inactivation of FOXO4. DR impeded sarcopenia as well as both FOXO4
inactivation and up-regulation of Atrogin-1 and MuRF1 transcripts. Our
findings allow us to conclude that sarcopenia is mechanistically
different from acute atrophies induced by disuse, disease, and
denervation.
The 26S proteasome is responsible for most cytosolic proteolysis.
Molecules that inhibit or specially tag proteasomes are helpful tools for
analysis of the UPS. In Paper IV, we present a new class of proteasome
inhibitors, considerably extended in comparison to the commonly used
fluorescent substrates and peptide-based inhibitors. Modification of the
most active compound, Ada-Ahx3L3VS, capable of proteasome inhibition in
living cells, afforded a new set of radio- and affinity labels.
N-terminal extension of peptide vinyl sulfones was found to have a
profound influence on both their efficacy and selectivity as proteasome
inhibitors. Results demonstrated that such extensions greatly enhanced
inhibition and largely obliterated their selectivity towards individual
catalytic activities.
The role of the UPS in aging-related muscle atrophy is highly
controversial. In Paper V, we showed an accumulation of assembled
proteasome particles with a corresponding increase in both proteasomal
activity and protein degradation in old age muscle atrophy. This was
accompanied by a wide range of UPS enzyme-regulation, including an
increase in the activity of deubiquitylating enzymes. The accumulation of
proteasomes was found to correlate well with muscle wasting. Both the
accumulation of proteasome particles as well as the progression of muscle
atrophy, were impeded when the normal pattern of aging was challenged by
DR. In contrast to many conditions with UPS-associated muscle catabolism,
the accumulation of proteasomes during senile muscle atrophy is not
caused by transcriptional induction, but rather by decreases in their
degradation. The lysosomal pathway is a candidate for degrading
proteasomes. In Paper V, we demonstrated that impaired lysosomal
function, achieved through chloroquine treatment, induced accumulation of
proteasomes in adult rats. This emphasizes the existence of a functional
link between the lysosomal pathway and the UPS suggesting that a decline
in lysosomal function may contribute to increased proteasomal proteolysis
in old-age skeletal muscle atrophy